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UV-visible spectroscopy-based quantification of unlabeled DNA bound to gold nanoparticles Brandi L. Baldock, and James E. Hutchison Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02640 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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Analytical Chemistry

UV-visible spectroscopy-based quantification of unlabeled DNA bound to gold nanoparticles Brandi L. Baldock and James E. Hutchison* Department of Chemistry and Biochemistry, University of Oregon, 1253 University of Oregon, Eugene, OR, 97403-1253, USA ABSTRACT: DNA-functionalized gold nanoparticles have been increasingly applied as sensitive and selective analytical probes and biosensors. The DNA ligands bound to a nanoparticle dictate its reactivity, making it essential to know the type and number of DNA strands bound to the nanoparticle surface. Existing methods used to determine the number of DNA strands per gold nanoparticle (AuNP) require that the sequences be fluorophore labeled, which may affect the DNA surface coverage and reactivity of the nanoparticle, and/or require specialized equipment and other fluorophore-containing reagents. We report a UV-visible based method to conveniently and inexpensively determine the number of DNA strands attached to AuNPs of different core sizes. When this method is used in tandem with a fluorescence dye assay, it is possible to determine the ratio of two unlabeled sequences of different lengths bound to AuNPs. Two sizes of citrate-stabilized AuNPs (5nm and 12 nm) were functionalized with mixtures of short (5 base) and long (32 base) disulfide-terminated DNA sequences and the ratios of sequences bound to the AuNPs were determined using the new method. The long DNA sequence was present as a lower proportion of the ligand shell than in the ligand exchange mixture, suggesting it had a lower propensity to bind the AuNPs than the short DNA sequence. The ratio of DNA sequences bound to the AuNPs was not the same for the large and small AuNPs, which suggests that the radius of curvature had a significant influence on the assembly of DNA strands onto the AuNPs.

INTRODUCTION DNA-functionalized gold nanoparticles (DNA-NPs), recently coined spherical nucleic acids,1 have enormous potential as sensitive and selective analytical probes and biosensors due to their ability to recognize and specifically respond to target molecules, free DNA strands, and DNA strands bound to gold nanoparticles (AuNPs) or planar gold surfaces.1,2 Analyte binding by DNA recognition sequences can be used to direct DNA-NP assembly1,3,4 or disassembly5 in solution, triggering a colorimetric response based on nanoparticle plasmon resonance coupling. The binding specificity of DNA sequences allows DNANPs to detect DNA in vitro, differentiating between sequences containing single base imperfections.6 The biocompatibility and colloidal stability of DNA-NPs make them ideally suited for sensing in vivo,7,8 where they can detect cellular mRNA levels9 and regulate biological events.9,10 The myriad of potential DNA-NP sensing applications means it is critical to understand and control their properties. The properties of DNA-NPs depend on their ligand shell composition that typically consists of a single recognition sequence,3,11,12 a mixture of two different recognition strands,9,13,14 or a mixture of recognition strands and diluent (such as polyethylene-glycol,13,15–17 polyadenosine18,19 or polythymine16 ligands). The number of DNA recognition strands bound to AuNPs dictates the number of complementary sequences they bind,18,20 their cellular uptake mediated by protein binding,15 resistance to oxidative decomposition17 and melting temperature of assembled DNA-NPs.19

DNA-NPs functionalized with two different types of DNA strands, e.g., a recognition sequence (typically 12-72 bases) and a diluent sequence (typically ≤18 bases), or two different recognition sequences, are of growing importance due to their defined targeting, signaling13 and regulatory properties.9 DNA-NPs functionalized with limited numbers of recognition strands have been developed as building blocks for programmable materials.1,21–23 “Diluent” DNA sequences have also been used tune the number of DNA recognition sequences displayed on the surface of DNA-NPs.14,16 Diluting the number of recognition strands is advantageous because it promotes DNA hybridization.18,20,24 The charged nature of DNA-based diluents helps maintain the colloidal stability of DNA-NPs. To produce DNA-NPs with desired properties, it is critical to control and rigorously characterize the type and number of DNA strands bound to the surface of AuNPs. The number of DNA strands per AuNP is typically determined using fluorescence ‘turn-on’18 or ‘turn-off’25,26 methods. In the ‘turn-on’ method, fluorophore-labeled DNA is attached to AuNPs and displaced by small thiol ligands. The displaced DNA is quantified by fluorescence emission. In the ‘turn-off’ method, the fluorescence emission of a fluorophore labeled DNA solution is determined before and after incubation with AuNPs, and the DNA concentration is quantified from the decrease in fluorescence due to quenching by the AuNPs. In both cases, the concentration of AuNPs is determined from their UV-visible absorbance at 520 nm. The main drawbacks of the ‘turn-on’ and ‘turn-off’ fluorescence quantification methods are that each DNA sequence to be quantified must be labeled with a different fluorophore, and assumptions must be made about the interactions between

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ligands and the AuNP core. Fluorophore labels can affect DNA-NP ligand structure, reactivity and the number of recognition strands bound to each AuNP.14,27 They are also timeconsuming to synthesize, expensive to purchase and often bleach under light exposure.14 To determine the DNA concentration using the fluorescent turn-on method, it must be assumed that all DNA ligands are completely displaced from the surface of the DNA-NPs. This can be problematic because the rate and extent of thiol:thiol ligand exchange depends strongly on the ligand identity.28 To determine the DNA concentration using the fluorescent turn-off method, it must be assumed that the fluorophore is completely quenched upon interacting with the AuNPs.29 Label-free DNA sequences attached to AuNPs have been quantified using the Oligreen fluorescence assay15,30 and the toehold displacement assay.14 The Oligreen assay is suitable for quantifying DNA in solutions containing sequences longer than 6 nucleotides,30 but is restricted to quantifying one DNA sequence. The toehold displacement assay is suitable for simultaneous quantification of recognition and diluent sequences, but only for sequences longer than18 nucleotides.14 Both methods require specialized equipment and reagents and assume complete ligand displacement by small thiol molecules. To date, no method exists to determine the number of DNA strands per AuNP without using fluorophores, or to quantify recognition and diluent ligands in the same sample. New methods to determine the number of DNA strands per AuNP without using fluorophores or making assumptions regarding nanoparticle reactivity would be useful for quantifying both the recognition and diluent DNA strands within a given sample. A current barrier to label-free detection using UV-visible spectroscopy is that AuNPs and their common impurities absorb light at the wavelength typically used for DNA detection (260 nm). We found that the contribution from these species can be subtracted after KCN digestion of the nanoparticles. In this paper, we describe a convenient, inexpensive UVvisible based method to quantify the number of DNA strands bound to AuNPs. This method is suitable for determining the number of DNA strands bound to gold nanoparticles at typical nanoparticle working concentrations (e.g. 5-50 nM for 12 nm AuNPs). Using this method in conjunction with a commercially available dye assay, it is possible to determine the number of recognition and diluent DNA sequences bound to AuNPs, allowing us to determine their ratio as a function of the feed ratio during ligand exchange. We demonstrate that this method can be applied to large (12 nm) and small (5 nm) AuNPs. The results of determining the number of recognition and diluent strands bound to small vs. large AuNPs suggest that the AuNP radius of curvature has a large influence on DNA assembly onto the AuNPs.

EXPERIMENTAL SECTION Materials and Reagents Citrate-stabilized AuNPs (dcore = 5 nm) were purchased from Nanocomposix (San Diego, California). All DNA samples were purchased from Integrated DNA Technologies (Coralville, Iowa). DNA sequences were purified by either the standard desalting method or HPLC. “Quant-It” OliGreen ssDNA Assay kits were purchased from Thermo Fisher Scientific (Grand Island, NY). 50 kDa spin column purification membranes were purchased from Millipore (Darmstadt, Ger-

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many). Clear and amber 1.5 mL microcentrifuge tubes and opaque polypropylene black 96 well plates (Costar) were purchased from VWR (Radnor, PA). Sodium citrate dihydrate, hydrogen tetrachloroaurate hydrate and UV-transparent 96 well plates (Corning) were purchased from Sigma Aldrich (St. Louis, Missouri).

Instrumentation UV-visible absorbance spectra of AuNP and DNA solutions were obtained using either a BioTek Synergy 2 instrument or a Mikropack DH-2000 UV-vis-NIR light source equipped with an Ocean Optics USB2000 spectrophotometer. UV-visible spectra of DNA were obtained using these or a Thermo Scientific Nanodrop 2000 spectrophotometer path length (10mm) and baseline corrected at 340nm. The endpoint (after 5-15 minutes) fluorescent emission of the solutions were measured in 96-well opaque black well plates (Costar) using a Biotek Synergy 2 instrument equipped with a tungsten lamp and filters (EX 485/20 nm, EM 528/20 nm). The data collection time was autoscaled, so that 80,000 counts were emitted from the well containing the highest concentration of DNA. Calculation of UV-visible extinction coefficients AuNP extinction coefficients were calculated using their average core diameters (dcore = 12.3 nm: ε =1.98x108; dcore = 5.0 nm: ε = 9.96x106) and previously reported empirical data31. The error associated with these extinction coefficient values is 1-3%.31 DNA sequence extinction coefficients (ε) were calculated using Integrated DNA Technologies’ “Oligo Analyzer” tool, which calculates values from thermodynamic modeling according to DNA base composition and nearest neighbors (Table 1).32 DNA extinction coefficient values are accurate within 4% error (IDT-DNA). Concentrations of AuNP solutions were determined from A520 and DNA concentrations were determined from A260. Preparation of 12 nm DNA-NPs 12 nm citrate-stabilized AuNPs were synthesized using a modified literature method.33,34 Briefly, a 250 mL 3-neck round bottom flask, glass stopper, magnetic stir bar and condenser were cleaned using aqua regia and rinsed copiously with nanopure water. Sodium citrate dihydrate (408 mg, 1.39 mmoles) was dissolved in 200 mL nanopure water and brought to 100°C while stirring. HAuCl4 (1mL of 200 mM solution) was added using a micropipettor. The solution instantly turned dark blue, a color change previously attributed to nucleation.33 Within one minute, the solution turned a deep red color, indicating AuNPs were formed. AuNPs were stirred at 100°C for 20 minutes, then removed from heat and allowed to stir overnight before being characterized using Small Angle X-Ray Scattering (SAXS) and Transmission Electron Microscopy (TEM). The AuNP size determined by SAXS analysis was 12.3 ±1.9 nm (Figure S-1). TEM analysis confirmed that the AuNPs were spherical (Figure S-2). Descriptions of SAXS and TEM data acquisition methods are available in the Supporting Information. AuNPs were functionalized with DNA using a modified literature method.25,35 DNA and AuNP solutions were mixed together. Typically, AuNPs and DNA were mixed together to prepare reaction solutions containing 16 nM AuNPs and 16 uM DNA. 10x excess DNA was added to maximize DNA


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Table 1: Names, primary sequences and calculated extinction coefficients of DNA sequences usedα

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Name

DNA Primary Sequence

Extinction Coefficient (Lmol-1cm-1)

DNA1

5’-AGA GAA CCT GGG GGA GTA TTG CGG AGG AAG GT-3’

331 900

DNA2

5’-A5-3’

63 400

DNA3

5’-A12-3’

147 400

DNA4

5’-T5-3’

41 100

DNA5

5’-CCC AGG TTC TCT-3’

102 500

α

All sequences are labeled at their 5’ end with disulfide (HO(CH2)6S-S-5’-DNA-3’

loading on the AuNPs, because a small but measurable increase in DNA density during functionalization was reported when excess DNA was used during ligand exchange.35 After 5 minutes, pH 3 citric acid buffer was added (10 mM). After 10 minutes, NaCl was added (70 mM). DNA and AuNPs were incubated overnight before being purified using four rounds of centrifugation (15 min at 20 000g). DNA-NPs were redispersed in buffer containing 1mM pH 8.2 Tris acetate and 100 mM NaCl after each centrifugation step, and finally dissolved in 225 uL nanopure water. Fluorescence spectroscopy was used to determine that this method removed all excess DNA (Figure S-3a). After each purification, UV-visible spectroscopy was used to confirm that most excess DNA (>>99%) is removed.

Preparation and analysis of 5 nm DNA-NPs The same procedures (with modifications) were used to prepare DNA-NPs from purchased 5 nm AuNPs (Nanocomposix, San Diego) and to analyze their ligand shell composition. SAXS analysis confirmed the AuNPs were 5.0 ± 0.5 nm and TEM confirmed they were spherical. AuNPs and DNA were mixed together. The reaction solutions contained 90 nM AuNPs and 13.5 uM DNA. DNA-NPs were purified by centrifuging five times (9 min at 13 500g) above a spin filter membrane with a 50 kDa molecular weight cutoff, discarding each flow-through. Fluorescence spectroscopy was performed to determine that this method removed all excess DNA (Figure S-3b). DNA-NPs were eluted according to the manufacturer’s instructions and redispersed using 230 uL nanopure water. UV-visible spectroscopy determination of DNA strands per nanoparticle The number of DNA strands per AuNP was calculated by dividing the DNA concentration by the AuNP concentration. The concentration of DNA-NPs in each sample was determined using the A520 and calculated extinction coefficient of AuNPs of the same core size. The concentration of DNA in each sample was determined using the A260 and calculated extinction coefficient of the DNA sequence. KCN solution (100 mM) was prepared in nanopure water adjusted to pH 12 using NaOH. KCN solution was mixed with 12 nm AuNPs or DNA-NPs and allowed to react overnight before measuring the resultant UV-visible absorbance spectrum. At minimum, 8 moles KCN (4 equivalents) were added for every mole of Au atoms (15 mM, typically). The number of gold atoms in solution was determined by calculating the number of gold atoms per AuNP from the average AuNP volume and the density and molar mass of gold, and multiplying it by the number of nanoparticles in solution (calculated by multiplying the solution volume, AuNP concentration, and Avogadro’s number). The DNA A260 was determined by sub-

tracting the contribution of decomposed AuNPs from the A260 of the DNA-NP decomposition reaction solution. The concentration of DNA in each sample was determined based on its extinction coefficient and DNA A260. To validate our method, Quant-It’s ssDNA Oligreen quantification assay30 was used to determine the DNA in the decomposed AuNP solutions, using the supplier’s instructions. Briefly: a series of standard DNA solutions (80 nM, 40 nM, 20 nM and 8 nM) were prepared. The decomposed DNANP A260 was used to determine how much to dilute the decomposed DNA-NP samples for the Oligreen assay. Buffer (pH 7.5, 10 mM Tris-HCl 1 mM EDTA) and water were added to each sample, then Oligreen dye. They were incubated 510 minutes before measuring the final fluorescent emission of the dye. The decomposed AuNPs did not affect the assay results. To determine the ligand shell composition of the 5 nm DNA-NPs, the same procedure was followed, except 2.5 moles KCN (1.25 equivalents) were added for every mole of Au atoms (typically 2-3 mM).

UV-visible and fluorescence spectroscopy determination of two types of DNA sequences bound to gold nanoparticles The number of strands of each DNA per AuNP was determined by dividing the concentration of each DNA sequence by the concentration of AuNPs. The concentration of DNANPs was determined as described previously. The concentration of the longer DNA strand was determined using the Oligreen dye assay and used to calculate the number of DNA strands per AuNP. The extinction coefficient of the longer DNA strand was then used to determine its contribution to A260 decomposed DNA-NPs. The absorbance of the shorter DNA strand was then calculated by subtracting the contributions from the decomposed nanoparticles and the longer DNA strand from A260 decomposed DNA-NPs, and using the DNA’s extinction coefficient to calculate its concentration. The percentage of each strand in the ligand shell was then calculated.

RESULTS AND DISCUSSION A simple approach to determining the number of DNA strands per AuNP would be to measure the UV-visible spectrum of the DNA-NPs, and use the absorbances due to the gold cores and the DNA bases to determine their concentrations. The concentration of AuNPs can be determined using UV-visible spectroscopy, based on their absorbance at 520 nm (A520) and empirically determined extinction coefficients.31AuNP extinction coefficients do not significantly change upon functionalization with DNA.18 Therefore established extinction coefficient values could also be used to de-



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termine the concentration of DNA-NPs. DNA concentrations are conveniently determined from their absorbance at 260 nm (A260) and extinction coefficients calculated using thermodynamic modeling.32 The main reason UV-visible spectroscopy has not been used to determine the DNA bound to AuNPs is that AuNPs and other gold-containing impurities (gold salts and small gold clusters) absorb light at 260 nm, the wavelength where DNA absorbs most strongly.36 Therefore, the gold-based contributions cannot be independently determined and subtracted from the UV-visible spectra of intact DNA-NPs. In addition, gold-containing impurities present in the citrate-stabilized AuNPs cannot be removed without destabilizing the NPs, making it more difficult to determine the contribution of the AuNP cores to A260 (prior to functionalization). To eliminate the strong absorbance from the AuNP core, we thought it would be feasible to decompose purified DNANPs by treatment with cyanide prior to determining the DNA concentration. Cyanide etching has long been used to extract gold from ores and has previously been used to decompose gold nanoparticles for quantification of DNA ligand shells based on fluorescence18 and radioactivity.37 Our initial strategy to quantify the number of DNA strands per AuNP was to determine the concentration of the DNA-NPs from their A520, decompose the DNA-NPs using KCN (Figure 1) and quantify the DNA based on A260. DNANPs were prepared using an established method,35 and purified by centrifugation (Figure S-3) in dilute buffer before being characterized using UV-visible spectroscopy. Buffers commonly used to purify DNA-NPs absorb light at 260 nm (Figure S-4), and must be diluted for accurate DNA quantification using this method.

Figure 1. Overall strategy for quantifying DNA bound to AuNPs using UV-visible spectroscopy. (1) The concentration of DNANPs is determined from their absorbance at 520 nm. (2) DNANPs are decomposed using KCN, and (3) the concentration of DNA is determined from the absorbance of the resultant solution at 260 nm, as shown in the equation.

The solution of DNA-NPs (Figure 2a) exhibited a UVvisible absorbance peak characteristic of AuNPs at 520 nm and an absorbance peak characteristic of DNA at 260 nm. The latter absorbance was absent from the UV-visible spectrum of DNA-free AuNPs of the same core size, which was normalized to have the same A520 .

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To use A260 to determine the DNA concentration, we decomposed the DNA-NPs and AuNPs using KCN and then measured the UV-visible spectra of the resultant solutions (Figure 2b). The decomposed DNA-NPs absorbed strongly at 230, 240 and 260 nm. (Figure 2b). The peaks at 230 and 240 nm are characteristic of the KAu(CN)2 salt formed during nanoparticle decomposition.38 The peak at 260 nm corresponds to the DNA absorption. The baseline absorbance of the solution containing KCN and AuNPs or DNA-NPs approached zero at 350 nm and the spectrum did not change after 15 minutes or 12 hours respectively. Therefore, all nanoparticles were allowed to react with cyanide until their A350 approached zero. It is essential to evaluate the endpoint of the reaction using UV-visible spectroscopy because the reaction solutions appeared colorless by eye before the spectrum stopped changing.

Figure 2. UV-Visible absorbance spectra of solutions containing AuNPs (a) before and (b) after oxidative KCN decomposition (arbitrary units). (a) Intact 12 nm DNA-NPs (solid line) and citrate-stabilized AuNPs (dashed line) (b) Products from reaction between 12 nm DNA-NPs and KCN (solid line), AuNPs and KCN (dashed line).

To determine the DNA concentration from A260, the overlapping contribution from KAu(CN)2 is subtracted. Because the absorbance due to KAu(CN)2 at 260 nm results from the reaction of KCN and AuNPs, the absorbance can be directly related to the initial AuNP concentration. Solutions containing different concentrations of AuNPs were allowed to react with KCN and their UV-visible absorbance spectra measured. A260 of the resultant solutions directly correlated with the initial AuNP concentration (Figure 3a). A plot relating A260 of the


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decomposed AuNPs to the initial AuNP concentration (Figure 3b) was prepared. The fact that the plot was linear meant this could be used as a calibration curve to predict A260 for unknown solutions of DNA-NPs.

Figure 3. (a) Representative UV-visible spectra of solutions prepared by reacting various concentrations (3.5-14.2 nM) of 12 nm AuNPs with KCN. UV-visible spectroscopy can determine the DNA bound to AuNPs in solutions containing ≥1.5 nM 12 nm AuNPs. (b) Calibration curve used to determine the contribution of decomposed AuNPs to decomposed DNA-NP absorbance spectra (n = 24). The R2 value for the linear fit was 0.968.

UV-visible spectroscopy determination of DNA bound to AuNPs To evaluate whether UV-visible spectroscopy could be used to determine the number of DNA strands per AuNP, we prepared AuNPs functionalized with DNA1(5’-AGA GAA CCT GGG GGA GTA TTG CGG AGG AAG GT-3’), determined the DNA-NP concentration from A520 and used Equation 1 to determine A260 of DNA1, where dDNA-NPs denotes decomposed DNA-NPs and dAuNPs are decomposed citratestabilized nanoparticles. DNA1 was chosen as a representative sequence for this study because its length (32 bases) is comparable to recognition sequences typically used to functionalize AuNPs. Using DNA1’s extinction coefficient (Table 1) and Beer’s law, we determined that there were 58±7 DNA strands per 12 nm AuNP (n = 9). A260 DNA1 = A260 dDNA-NPs - A260 dAuNPs (Equation 1) If A260 of the decomposed AuNPs was not subtracted from A260 of the solution prior to calculating the DNA concentration, it would cause 28±4% determinate error.

Method Validation To validate this method, we also quantified the number of DNA1 strands per AuNP using an established Oligreen fluorescent dye assay.14,15,30,39,40 The value calculated using this method (59±4) agreed with our method within 1% error, which is within the experimental error introduced by determining the AuNP concentration using UV-visible spectroscopy. This suggests that UV-visible spectroscopy can be used to easily determine DNA bound per AuNP. This method is advantageous because it is convenient, inexpensive and does not require using fluorophore-containing reagents or making assumptions about ligand displacement rates. Because extinction coefficients can be determined for any DNA sequence, our method can be used to quantify shorter DNA sequences than those analyzed using the Oligreen fluorescence assay30 or toehold displacement assay.41 The method is sufficiently sensitive to determine the number of DNA strands bound to gold nanoparticles at typical DNA-NP working concentrations (e.g. 5-50 nM). The number of DNA1 (a 32 base sequence) strands per 12 nm AuNP (59 per AuNP) was lower than the number of DNA (a 12 base sequence) strands adsorbed to similarly sized (13 nm) AuNPs (85 per AuNP) under similar conditions.35 After taking into account the difference in surface areas between 12 nm and 13 nm AuNPs, the surface density of bound DNA1 strands was 27% lower than the density reported for the shorter sequence.35 The differences in density suggest the two DNA sequences interact differently with the AuNP surface. Based on these observations and those reported in the literature, it seems likely that part of DNA1 lies flat on the surface of the AuNPs during functionalization. Hurst et al.42 observed that the final surface coverage of thiolated DNA on AuNPs is inversely related to the adenosine content of the sequence near the thiol anchoring group, suggesting that bases near the thiol anchoring group continue to lie flat and interact strongly with the gold surface after the AuNPs are saturated with DNA. Additional DNA binding took place when interactions with the surface were disrupted by sonication.42 If DNA1 initially lies flat on the surface of our AuNPs, it may electrostatically or sterically hinder the adsorption of additional DNA, leading to the lower number of strands per AuNP. UV-visible and fluorescence spectroscopy determination of two types of DNA sequences bound to gold nanoparticles Given the importance of using mixed and diluted DNA ligand shells to control DNA-NP reactivity, we wanted to extend our technique to determine the number of recognition and diluent DNA sequences bound to the surface of AuNPs. We prepared DNA-NPs functionalized with two DNA sequences, DNA1 and DNA2 (5’-A5-3’), by performing ligand exchanges on 12 nm AuNPs using solutions containing DNA1 and DNA2. DNA2 was selected as a representative diluent sequence because sequences of this length (≤5 bases) do not interact appreciably with the Oligreen dye reagent.30 Typically, such short sequences must be labeled with a fluorophore for quantification. Determining the concentration of each sequence and dividing it by the AuNP concentration (Figure 4) allowed the percentage of DNA1 in the ligand shell to be determined. The concentration of DNA-NPs was determined using UV-visible spectroscopy and the longer sequence, DNA1, was determined using the Oligreen dye assay. The DNA1 concen-



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tration and its extinction coefficient were used to determine its A260. The DNA2 concentration was then determined from the absorbance of the decomposed DNA-NP solution at 260 nm, after subtracting the contributions from KAu(CN)2 and DNA1. The indeterminate error associated with determining the percentage of DNA1 in the ligand shell was 2.6%, which reflects the variance between A260 DNA1 determined from UV-visible vs. fluorescence spectroscopy. To use the Oligreen dye assay to measure DNA1 in solutions containing DNA2, it must be assumed that DNA2 does not appreciably affect the fluorescent emission of the assay solution. To test this assumption, DNA1 was quantified using the Oligreen dye assay with and without other DNA sequences (DNA2, DNA3 or DNA4) present. In all cases, the shorter DNA sequence did not affect the fluorescent emission of DNA1 (Figure S-5).

Figure 4. UV-visible spectroscopy and fluorescent spectroscopy determination of two different DNA strands (DNA1 and DNA2) bound to AuNPs. (1) The concentration of DNA-NPs is determined from A520, and used to determine A260 KAu(CN)2. (2) DNA-NPs are decomposed using KCN (3) A260 of the resultant solution is measured. (4) The concentration of DNA1 is determined from a linear (typical R2= 0.999) calibration curve relating DNA1 concentration to Em528 and used to calculate A260 DNA1. A260 DNA2 is determined by subtracting A260 DNA1 and A260 KAuCN2 from A260 solution. The DNA2 concentration is then calculated using its extinction coefficient.

To use this method to quantify two different DNA sequences bound to the nanoparticles, the sequences must exhibit greatly different reactivity towards a commercially available fluorescent dye, e.g. vary significantly in length or thymine composition.To quantify two sequences of similar length, our method could be used concurrently with a different technique. For mixtures of sequences that are longer than 18 nucleotides, one ligand can be quantified using our method, and the other using sequential strand displacement by DNA “toehold sequences”41 From the results obtained by quantifying the number of DNA1 and DNA2 strands bound per AuNP, it was apparent that DNA1 was under-represented in the AuNP ligand shell after functionalization (Figure 5). When equimolar amounts of DNA1 and DNA2 were added during ligand exchange, only 3.5 ± 0.1 % of the DNA bound to the AuNP surface was DNA1. This was intriguing because previous studies found a proportional relationship between the feed ratio and bound DNA ratio.18,25 To further investigate the impact of the ligand exchange feed ratio on the type and number of DNA strands bound per nanoparticle, we varied the ratio of recognition:diluent strands during ligand exchange, and used this method to quantify both

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strands. In all cases, a lower percentage of DNA1 was present in the AuNP ligand shell than was present in the ligand exchange mixtures (Figure 5). There was a non-linear relationship between the percentage of DNA1 during ligand exchange and the percentage of DNA1 bound to the AuNPs (Figure 5). The reason that DNA1 is under-represented in the ligand shell is likely because the DNA1 sequence is substantially longer and less adenosine rich. It has been shown that adsorption rate of unthiolated43 and thiolated44 DNA sequences to AuNPs is inversely related to the chain length of the sequence, and that the initial rate of DNA adsorption is directly related to the base content of the sequence, with polyadenosine sequences exhibiting the highest adsorption rate.45The sequences used in studies where the bound ratio was proportional to the feed ratio were the same25 or similar length,18 with similar base content near the anchoring thiol group, which explains why they observed a linear proportional relationship between the feed ratio and the bound DNA ratio.

Figure 5. Ligand shell composition of DNA-NPs prepared by mixing 12 nm AuNPs with various amounts of DNA1 and DNA2 sequences. The percentage of DNA1 in the ligand shell was determined using UV-visible and fluorescence spectroscopy. Error bars represent the standard deviation for the mean % DNA1 in ligands bound to 12 nm AuNPs (error bars do not overlap).

Having investigated the effect of the feed ratio upon the bound DNA ratio, we proceeded to investigate the effect of the nanoparticle’s radius of curvature on the bound DNA ratio. While the total number of thiolated DNA strands bound to an AuNP increases as a function of its core size,42 the density of bound DNA strands is inversely related to the nanoparticle’s radius of curvature, with DNA strands forming a smaller effective footprint on smaller AuNPs.39 We hypothesized that the smaller effective footprint of the DNA ligands on a small nanoparticle would allow the bulkier DNA1 ligand to make up a larger proportion of the ligand shell.

Determination of label-free DNA sequences bound to 5 nm AuNPs To evaluate the effect of the nanoparticle radius of curvature upon the bound DNA ratio, we prepared 5 nm nanoparticles functionalized with a mixture of recognition (DNA1) and diluent (DNA2) sequences, and used this analytical method to determine the type and number of DNA strands bound. 5 nm DNA-NPs were selected as a representative size for this study because they are often used for fundamental and applied studies.46–48 They are convenient for studying assembly of DNA-NPs in solution, because their assemblies are less prone to precipitation and therefore produce more uniform


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SAXS patterns than DNA-NPs with larger core sizes.46 Small AuNPs are advantageous for in vivo bioimaging and drug delivery applications involving negatively charged nanoparticles because of their increased propensity to enter tumor cells.47,48 Solutions containing different concentrations of 5 nm citrate-stabilized AuNPs were prepared and decomposed, and their UV-visible spectrum was measured. 5 nm AuNPs contain fewer gold atoms than 12 nm AuNPs, and they exhibited a lower A260 when decomposed. A260 of the decomposed 5 nm DNA-NP solutions varied linearly with the original NP concentration (Figure 6a).

Figure 6. (a) Calibration curves for determining A260 of decomposed citrate-stabilized NPs. UV-visible spectroscopy can determine the DNA bound to AuNPs in solutions containing ≥30 nM 5 nm AuNPs. The linear fit for the decomposed 5nm AuNPs had an R2 value of 0.990. (b) Percentage DNA1 in ligand shell of 5nm AuNPs functionalized from different feed ratios of DNA1:DNA2 sequences. Error bars represent the standard deviation for the mean % DNA1 in ligands bound to 5 nm AuNPs.

5 nm DNA-NPs were prepared by incubating AuNPs with DNA1 and the number of DNA1 strands per 5 nm AuNP (n = 9) was analyzed using spectroscopy (18±2) and the fluorescent dye assay (15.1±0.8). The calculated ranges of DNA per AuNP agreed reasonably well, suggesting that the UVvisible based method is suitable for determining the DNA bound to smaller, as well as larger, core sizes. Although the primary focus of this work is smaller AuNPs that exhibit desirable optical properties and high colloidal stability, this method could also be extended towards quantifiying ligands bound to larger AuNPs. Based on the

extinction coefficient of DNA1, and the average number of DNA strands that larger AuNPs bind,35,42 this method could be used to determine the DNA bound to AuNPs in solutions containing ≥0.5 nM 50 nm AuNPs. For AuNPs functionalized with mixtures of DNA1 and DNA2, DNA1 was under-represented in the ligand shell of the 5nm DNA-NPs, similar to what was observed for the larger DNA-NPs. However, the ratio of DNA sequences bound to the AuNPs was different for the large and small AuNPs. For example, when AuNPs are incubated with solutions containing 75% DNA1 during ligand exchange, the ligand shell of the 12 nm nanoparticles contains 15 ± 3 % DNA1 and the ligand shell of the 5 nm nanoparticles contains 23 ± 3 % DNA1. This evidence that the ratio of DNA sequences bound to the AuNPs was different for the large and small AuNPs suggested that the radius of curvature influenced the assembly of DNA strands onto the AuNPs. If chain length and adenosine content were the only factors influencing DNA adsorption, AuNPs of different core sizes functionalized using the same feed ratios of DNA sequences would produce DNA-NPs with the same ligand shell composition. Instead, the adsorption of longer DNA sequences is promoted by increasing the AuNP radius of curvature, which suggests a more complex reaction mechanism. The influence of the nanoparticle’s radius of curvature on the ratio of bound active vs. diluent strands can be explained based on the model describing how DNA strands interact with AuNPs during ligand exchange. To maintain nanoparticle stability, DNA (unthiolated, disulfide-terminated or thiolated) must be added prior to adding salt, suggesting that DNA strands adsorb rapidly and non-specifically (via DNA bases) to the AuNPs, preventing their aggregation. 26,35,42,49 After adding salt, non-thiolated DNA strands form sparse monolayers on AuNPs, whereas thiolated DNA strands rearrange to permit additional binding and form dense monolayers.44 This suggests that non-thiolated DNA strands maintain a horizontal orientation with respect to the AuNP surface, whereas thiolated DNA strands initially adsorb in a horizontal orientation, then adopt a vertical orientation after specific binding.44 When DNA-NPs are prepared by incubating disulfideterminated DNA strands with AuNPs at pH 3, an initial sparse monolayer is rapidly attained,35 and our results suggest that the added NaCl allows the bound DNA strands to rearrange and form thiol bonds in an orientation that permits additional DNA adsorption. At pH 3, the adenosine residues are protonated,50 thereby reducing their binding affinity for the gold. Adding salt further reduces electrostatic repulsion between DNA strands on the AuNPs and DNA in solution.26 DNA can thus rearrange and additional binding can occur after adding the buffer and salt to the ligand exchange reaction mixtures. During this rearrangement and additional binding step, adsorption of the bulky DNA1 ligand is hindered, resulting in an increase in the DNA2 content on the surface of the AuNPs even when it is a minor component of the ligand exchange mixture. This effect is less for AuNPs with a larger radius of curvature, because the gold surface is more accessible for binding. The fact that the radius of curvature influenced the assembly of DNA strands on the AuNPs therefore leads us to conclude that disulfide-terminated DNA strands nonspecifically adsorb to the AuNPs and rearrange to form specific bonds with the AuNPs, following an adsorption mechanism similar to the two-step model followed by thiolated DNA,



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rather than the one-step adsorption model followed by nonthiolated DNA.44

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

CONCLUSION We developed a rapid, convenient and inexpensive method to quantify the number of label-free DNA strands attached to AuNPs of large or small core sizes. The number of strands per nanoparticle can easily be determined from solutions of DNA-NPs at concentrations typically used in sensing assays. The UV-visible spectroscopy assay was used in concert with a conventional Oligreen dye assay to determine two different DNA sequences bound to AuNPs, without the need for labeled DNA. The results of our mixed ligand shell analysis support a model for disulfide-terminated DNA adsorption in which there is fast non-specific adsorption of DNA to the gold surface dictated by chain length and base composition, followed by rearrangement and additional specific binding to the gold surface. The generality of our approach means that in principle, this method can be extended to determine the number of DNA, complementary DNA, RNA or synthetic peptide strands (whose UV-visible signatures overlap with that of decomposed gold nanoparticles)51–53 bound to gold or silver nanoparticles.These materials are of interest due to their ability to form versatile nanoparticle assemblies,54,55 specifically induce apoptosis in tumor cells,56 and act as sensitive analytical probes in single molecule experiments.55 The concentration of silver nanoparticles can be determined from their UVvisible Aλmax and empirically determined extinction coefficients.57 Solutions of silver nanoparticles, at the concentration used for in vivo toxicity assays,58 undergo oxidative decomposition by KCN59 to form salts that absorb light at 260 nm.38 Using this method in concert with a dye that specifically binds double-stranded DNA would allow the number of bound complementary DNA strands to be determined. The simplicity and wide applicability of this method makes it well suited for determining the number of recognition and diluent DNA strands bound to gold nanoparticles. This information is essential to understanding the relationship between the structure of a nanoparticle’s ligand shell and its analytical and biosensing properties. We anticipate that information gained using this method will lead to design of nanomaterials with enhanced properties.

ASSOCIATED CONTENT Supporting Information TEM and SAXS characterization of AuNPs, fluorescent spectroscopy analysis of DNA-NP purification, UV-visible absorbance of commonly used DNA-NP purification buffers, fluorescent emission of mixtures of DNA sequences and Oligreen dye. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author *Corresponding author: Professor James E. Hutchison Phone: (541) 346-4228 Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

We thank the Center for Advanced Materials Characterization in Oregon and the Institute of Molecular Biology for use of their facilities and technical support. We thank the Materials Science Institute and the University of Oregon Department of Chemistry and Biochemistry for financial support. We thank Andy Berglund for helpful discussions. We acknowledge the Air Force Research Laboratory (under agreement FA 8650-05-1-5041) for financial support.

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