Rapid Immobilization of Oligonucleotides at High Density on

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Rapid immobilization of oligonucleotides at high density on semiconductor quantum dots and gold nanoparticles Abootaleb Sedighi, and Ulrich J. Krull Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03840 • Publication Date (Web): 30 Nov 2016 Downloaded from http://pubs.acs.org on November 30, 2016

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Rapid immobilization of oligonucleotides at high density on semiconductor quantum dots and gold nanoparticles Abootaleb Sedighi, Ulrich. J. Krull Univeristy of Toronto Mississauga, Mississauga, Ontario, Canada

Abstract Oligonucleotide-coated nanoparticles (NPs) have been used in numerous applications such as bioassays, as intracellular probes and for drug delivery. One challenge that is confronted in the preparation of oligonucleotide-NP conjugates derives from surface charge, as nanoparticles are often stabilized and made water soluble with a coating of negatively charged capping ligands. Therefore, an electrostatic repulsion is present when attempting to conjugate oligonucleotides. The result is that the conjugation can be a slow process, sometimes requiring 1-2 days to equilibrate at the highest surface density. The effect is compounded by electrostatic repulsion between neighboring oligonucleotide strands on the NP surfaces, which tends to lower the surface density. Herein, we report a novel method that enables conjugation in less than 1 min with a surface density of oligonucleotides up to the theoretical physical limit of occupancy. Negatively charged NPs are first adsorbed onto the surface of positively charged magnetic beads (MBs) to create MB-NP conjugates. Oligonucleotides are subsequently electrostatically adsorbed onto the MB surfaces when added to a suspension of MBNP conjugates. This creates an oligonucleotide concentration 105 to 106 greater than in bulk solution in the vicinity of the nanoparticles, resulting in the promotion of the

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kinetics by over 1000 fold, and achieving the maximum density possible for the conjugation reaction. Keywords: Quantum dots, gold nanoparticles, DNA immobilization, oligonucleotide surface density

Introduction Oligonucleotides are widely used in the preparation of nanoparticle-DNA conjugates, comprising a nanoparticle (NP) core and a shell composed of oligonucleotides. Such assemblies can exhibit functional properties that have been used in a variety of bio-applications including in-vitro diagnostics,1 intracellular assays,2 drug delivery,3 and DNA-programmed nanoparticle assembly.4 Many of the desirable properties of the conjugates such as sharp oligonucleotide melting transitions, enhanced hybrid binding affinities, high cellular uptake of NPs, minimal immune response and high stability in both in vitro and in vivo environments, are dependent on achieving a high packing density and structural organization of the oligonucleotides in the shell that is around the NP core.5 The effectiveness of the NPDNA conjugation step is fundamental to many applications.11,

21, 22

One of the first

NP-DNA conjugates was reported in 1996, based on self-assembly (i.e. direct dative binding) of thiolated DNA to gold nanoparticles (AuNPs). However, the early NPDNA conjugates suffered from low stability because the electrostatic repulsion between the neighboring DNA oligonucleotides on the NP surfaces, and this limited the DNA packing density. This issue was later ameliorated using a method known as “salt aging”, in which salt was incrementally added to the solution containing the

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AuNP-DNA conjugates to screen the charge repulsion and allow for the formation of a densely-packed DNA shell.6 Salt aging remains the most common method for promoting the immobilization density of DNA on AuNPs, and has also been applied for DNA immobilization to other nanoparticles such as silver nanoparticles,7 and quantum dots.8 However the method is slow and tedious in that incremental salt addition typically is done over 1-2 days. Several methods have been reported to introduce faster immobilization of DNA on AuNPs (Table 1).9, 10, 11, 12, 13 The main focus of these methods is on enhancing the immobilization kinetics by alleviating the NP-DNA repulsion. For instance, surfactant pretreatment of AuNP buffer solution, 11 or AuNP surfaces 13 was used to shorten the immobilization process to several hours12. More recently, Zhang et al. developed a 3-min method for immobilization of DNA to citrate-capped AuNPs using citrate buffer at pH 3.12 The extension of the method for conjugation of DNA to other nanoparticles that are known to be unstable at acidic pH, such as QDs,14 has not been reported. Various approaches have been used to immobilize DNA strands onto QDs, including self-assembly, ligand secondary binding, polymer encapsulation and silica coating.15 The self-assembly approach, operating by direct binding of DNA to NP surface, tends to produce DNA shells of high density. High density DNA coatings are particularly useful to promote cellular uptake of coated nanoparticles, and also for in vitro assays that use transduction based on Förster Resonance Energy Transfer (FRET).16, 17

Similar to AuNPs, self-assembly of DNA to QDs has also been exploited. For

example, thiol groups placed at the termini of oligonucleotides have high binding

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affinity to Zn that is associated with the widely used protective ZnS shell that is grown over a luminescent QD core.15 However, the thiol-ZnS binding interaction is more labile than that of the interactions between thiol and gold surfaces, which contributes to lower conjugation efficiency and poor stability of coatings.18 The replacement of the monothiol linker with a dithiol linker enhances the binding strength and stability through cooperativity. However, the kinetics remain an issue as the self-assembly typically requires a 2 day procedure.8 A faster method (1 h) for self-assembly of DNA to QDs makes use of polyhistidine linkers in place of thiols to enhance the kinetics using multiple surface anchoring points .19 Table 1: Various methods developed for faster immobilization of oligonucleotides on nanoparticles. Method Salt aging

Nanoparticles

Immobilization time AuNP, silver nanoparticles 1-2 days [AgNP], QDs AuNP 2h

Surfactant stabilization Polyhistidine QDs linker Low pH AuNP, AgNP immobilization Positively charged AuNP tail linker

References [6], [7], [8] [13]

1h

[19]

3-5 min

[12]

5 min

[10]

Herein we report a novel approach for preparation of high densities of DNA shells on QDs and AuNPs in less than a minute. In this method, negatively charged NPs are first electrostatically adsorbed onto the surface of positively charged magnetic beads (MBs) to create MB-NP conjugates. Addition of DNA oligonucleotides to a suspension of MB-NP conjugates results in further electrostatic adsorption, where

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the negatively charged oligonucleotides associate with the MB surfaces. The accumulation and concentrating of oligonucleotides in the vicinity of the nanoparticles promotes the NP-DNA conjugation reaction.

The magnetic bead

loading (MBL) method provides for a combination of rapid electrostatic attraction and oligonucleotide preconcentration at the MB interface, enabling oligonucleotide conjugation with the maximum theoretical surface density to be achieved within seconds.

Results and discussion. An overview of the MBL method used for DNA immobilization onto QDs is schematically provided in Figure 1. Negatively charged glutathione (GSH)-coated QDs are first adsorbed onto positively charged diethylaminoethyl (DEAE)functionalized magnetic beads to form MB-QD conjugates. Next, dithiol-modified single-stranded DNA oligonucleotides (capture oligonucleotides) are added and the oligonucleotides are allowed to immobilize on the QD surfaces for 30 s. Finally, the QD-DNA conjugates are released from the MB surfaces by incubation of the conjugates in a buffer solution with high ionic strength (1 M NaCl) for 30 s. Magnetic isolation provides a convenient and efficient method for purification and recovery of the conjugates. A FRET-based assay was used to monitor the packing density of oligonucleotides conjugated on the QD surfaces. Details about the FRET assay are provided in the supporting information. To form the FRET pair, the released QD-DNA conjugates

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were allowed to undergo DNA hybridization with fully complementary DNA strands modified with Cy3 dyes. The QD-FRET pair operates with the QD as the donor, having a photoluminescence (PL) peak maximum at 520 nm (QD peak), and Cy3 molecular dye acts as the acceptor, with PL peak maximum at 560 nm (FRET peak). The FRET ratio, defined as the ratio of the FRET peak intensity divided by the QD PL peak intensity, is known to linearly increase with the number of Cy3 acceptors on each QD. 8, 20, 21 The use of a ratio provided for internal normalization that accounted for any drift in excitation intensity or detector sensitivity. The FRET ratio was monitored as a relative measure of oligonucleotide surface density and the relative number of oligonucleotides conjugated on each QD (DNA/QD).

Figure 1. Schematic representation of DNA immobilization on QDs using the MBL method, and subsequent determination of relative DNA packing density using FRET assay. Figure 2 presents typical FRET spectra and the corresponding kinetics of the immobilization of oligonucleotide CP1 (described in Table 2) to QDs using the Bulk 6


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method (Fig. 2A & 2B) and the MBL method (Fig. 2C and 2D). The Bulk method begins with DNA immobilization at low salt conditions, and requires 18-20 h for the FRET ratio signal to saturate with a final value of 1.1 ± 0.2, which gives a rate constant of 1.2 × 10-3 min-1 as obtained using curve fitting (exponential fit). This slow process is consistent with electrostatic repulsion between DNA and the surface ligands associated with the QDs.6, 8, 20, 22 A zeta potential value of -23.9 mV obtained from dynamic light scattering measurement on GSH-QDs at pH 7.4 confirms the negative surface charges of the nanoparticles (Figure S7). A subsequent stepwise addition of salt to achieve salt aging,8 was used to screen charges and increase the oligonucleotide density on QD surfaces. The salt aging process enhanced the FRET ratio to a value of 4.2 ± 0.5.

Figure 2. DNA immobilization on QDs using the Bulk and MBL methods. A and B show the FRET spectra and the corresponding kinetic curves obtained using the Bulk method, respectively. C and D shows the corresponding graphs obtained from the MBL method. For each measurement, 140 pmol of capture oligonucleotides (CP1) were added to 3 pmol QDs. For MBL experiments QDs were previously adsorbed on 0.1 mg of MBs. DNA hybridization made use of 140 pmol of CS-1 oligonucleotide target. The control experiments were carried out at the same condition as the experimental ones, except that CP-5 were used as the capture oligonucleotides, i.e.,

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no modification with dithiol phosphoramidite (DTPA), a lipoic acid-like structure with a disulfide bond in the pentane ring. TCEP reduction breaks the dithiol bond and provides the bi-dentate attachment of DNA to QD surface.

Figure 2D shows the corresponding kinetic curve obtained from the MBL method. The graph illustrates two remarkable improvements in comparison with the Bulk method. First, the kinetics are greatly improved with the FRET ratio reaching equilibrium in ≥ 0.25 min, which corresponds to a rate constants of ≥ 5.8 min-1. This rate constant is ≥ 4.8 × 103 times greater than the value obtained by the Bulk method. Second, the FRET ratio obtained from the MBL method reached 18 ± 4, which is 4.3 times greater than obtained from assembly that made use of the Bulk method. This enhancement in the FRET ratio suggests a higher density of oligonucleotide achieved in the MBL method. We observed that the FRET ratio in the MBL method was not affected by the order in which QDs and DNAs were conjugated to the MB surfaces. As shown in Figure S16, the FRET ratio remained unchanged when the order was QD first/DNA next, DNA first/QD next or when both reagents were added simultaneously. Also, our investigation showed that immobilization of monothiol-modified oligonucleotide on the GSH-QDs was only achieved using the MBL method but not the bulk method (See Figure S9).

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Table 2: The nucleotide sequences of capture oligonucleotides and complementary strands Name

Sequence

CP-1

5’-DTPA-AATATCATCTTTGGTGTT-3’

CP-2

5’-SH-AATATCATCTTTGGTGTT-3’

CP-3

5’-SH-TTTTTTTTTTTTTTTTTT-Cy5-3’

CP-4

5’-Cy5-AATATCATCTTTGGTGTT-DTPA-3’

CP-5

5’-AATATCATCTTTGGTGTT-3’

CP-6

5’-Cy5-AATATCATCTTTGGTGTT-3’

CS-1

5’-AACACCAAAGATGATATT-Cy3-3’

CS-2

5’-AACACCAAAGATGATATT-Cy5-3’

IS-1

5’-ACAGGGTTTTAGACAAAT-Alexa 647-3’

IS-2

5’-ACAGGGTTTTAGACAAAT-3’

The FRET assay has been used as a convenient method to monitor the relative oligonucleotide packing density on QDs. A corroborating method has been used to directly determine the average total number of oligonucleotides conjugated on a QD as well as to determine the fraction of those conjugated oligonucleotides that hybridized to the complementary strands and hence contributed to the enhanced FRET ratio. A dual-labeled oligonucleotide (CP-4, dithiol and Cy5-modified) was used as the capture oligonucleotide to determine the total number of oligonucleotides conjugated on each QD. Figure 3A shows the QD packing density as a function DNA concentration from 0.12 to 3.5 μM. The packing density is represented by the FRET ratio (the blue curve) and also DNA/QD (the red curve). To obtain the DNA/QD ratios, the concentration of QDs and oligonucleotide in the purified conjugate solutions were independently measured (Refer to SI). The

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packing density increases with DNA concentration and reaches a plateau at 2 μM of DNA. Interestingly, over a certain range, the FRET ratio correlates well with the QD/DNA values determined using an independent method. The DNA/QD value was used to compare the packing densities achieved in the Bulk and MBL method (See Figure S14). Note that both curves display trends that conform to the Langmuir adsorption isotherm model. A total of 4.0 ± 0.6 and 18.9 ± 4.2 oligonucleotides were conjugated on each QD using the Bulk and MBL methods, respectively. These results indicate a 4.7 fold increase in the oligonucleotide loading density achieved by the MBL method, which is consistent with the data from the FRET-based method. Also, the fraction of conjugated capture oligonucleotides that hybridized with the complementary strands was 67% (2.7 ± 0.3 of 4.0 ± 0.6) and 38% (7.3 ± 2.7 of 18.9 ± 4.2) for the QD-DNA conjugates prepared using the Bulk and MBL methods, respectively. The reduced fraction of hybrids formed for coatings prepared by the MBL method is consistent with electrostatic and steric constraints observed when using high oligonucleotide densities at a solid interface.13 The QD-DNA conjugates have been characterized using dynamic light scattering (DLS) and gel electrophoresis (See Figure S8). DLS measurements show the hydrodynamic diameter (dh) of QDs increased from 5.6 ± 0.4 nm for GSH-QDs to 6.7 ± 0.5 nm when conjugated with CS-1 oligonucleotides using the Bulk method. This small increase in the QD-DNA size is consistent with a low packing density of oligonucleotides, and is expected to result in a configuration where the flexible strands tend to associate with the NP surface.5 The size of QD-DNA constructs produced by the MBL method was significantly larger with a dh value of 10.2 ± 0.5,

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which suggests a high packing density of oligonucleotides that tends to adopt a physical configuration of an outstretched molecular brush, increasing the size of the conjugates.23 Gel electrophoresis images of the QD-DNA conjugates show a single narrow band, which demonstrates that magnetic bead-loading of QDs results in uniform coatings of DNA.

Theory and examination of control of density and speed of reaction We propose a model for immobilization of DNA on QDs using the MBL method, and contrast this to the immobilization using the Bulk method. DNA immobilization on the QDs in the Bulk model is based on the following one-step reaction: + ⇌

(1)

Where DNAb represents the DNA strands in the bulk solution, s represents the binding sites on the QD surfaces, and DNAim represents the DNAs immobilized on the QD surfaces. The rate of the above equation is given by the Langmuir adsorption equation (Eq. 2),

= −

(2)

Where kon and koff are the rate constants for the forward and reverse reactions, respectively. The packing density of DNA on a QD surface is represented as a surface area concentration, [DNAim], given by the Langmuir adsorption isotherm (Eq. 3)

= [s]

(3)

According to Eq. 2 and Eq. 3, at a constant concentration of QDs both the rate of immobilization reaction and also the DNA packing density would be directly 11


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proportional to the bulk concentration of DNAb. This model serves as a basis for interpretation of the kinetics and packing densities achieved by the Bulk method. The slow kinetics observed in the Bulk method were likely due to depletion of the DNAb in the vicinity of QD surfaces due to the electrostatic repulsion between QDs and DNAs. This is consistent with low immobilization density using the Bulk method in low salt condition, and with the improvement of density when the electrostatic repulsion was screened using the salt aging method (Fig. 2B). The MBL method offers a different set of conditions for interfacial interactions and we propose that the immobilization of DNA in the MBL method occurs in two steps: + ∗ ⇌ ∗

(4)

∗ + ⇌

(5)

Where, DNA* represents DNA oligonucleotides adsorbed on the MB surfaces and * represents the cationic sites on MB surfaces. The interactions designated as Eq. 4 and 5 are processes in equilibrium, and the oligonucleotides on the magnetic beads are exchangeable. The high local concentration achieved by preconcentrating of oligonucleotides represented by Eq. 4 pushes the equilibrium to the right for Eq. 5. In the first step, the solution-phase DNAb is adsorbed onto the positively charged surfaces of magnetic beads (Eq. 4), and the second step is the conjugation of the adsorbed DNA* strands on the QD surfaces (also loaded on MB surfaces) (Eq. 5). The adsorption process is facilitated by the electrostatic attraction between negatively charged DNAs and the positively charged MB surface, and is expected to occur at a faster rate than the assembly of DNAs on QD surfaces, which is hindered by the electrostatic repulsion between DNA strands and QDs. It is therefore the second

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step that is likely the rate-determining step in the MBL method. The reaction rate and DNA packing density achieved in the MBL method are given by Equation 6 and Equation 7, respectively.

!" = ∗ −

!" = ∗ [s]

(6) (7)

Assuming that the rate constants of QD immobilization (kon & koff) remain similar for the Bulk and the MBL method, it can be seen from Eqs. 2, 3, 6 and 7 that the ratios of reaction rates (

$%&'()* $%&')+,-

/0123

), and the ratio of packing densities (/0123 ()* ) in the two )+,-

methods are directly proportional to a preconcentration factor (ΦMBL/Bulk), where 4!"/ =

∗ (8)

The basis of our assumption is that both thiolated DNA and GSH-QD remain unchanged between the Bulk and MBL method. Since kon primarily depends on the affinity, the kon is not expected to change significantly. The rate of forward reaction, given by Eqs. 2 & 6, depends on the concentrations of the reactants. Thus, the rate in the MBL method may be larger than in Bulk method, even with a constant kon, due to the increased local DNA concentration close to QD surface. While the kon for surfaceadsorbed oligonucleotides could be different than that in bulk solution, the assumption helps to highlight the larger effect of preconcentration on the reaction rate (5-6 orders of magnitude). In order to provide an estimated range of preconcentration factor, we consider the ratio of MB-adsorbed DNA (DNA*) over the total bulk DNA (DNAb), and also estimate the ratio of the volumes that surface-adsorbed vs. solution-phase 13


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oligonucleotides occupy (V*/Vb). First, DNA*/DNAb is obtained by monitoring the adsorption of fluorescently-labeled oligonucleotides on the MB-QD conjugates. The red curve in Figure 3C shows the amount of DNA* when MB-QD conjugates (3 pmol QD conjugated on 0.1 mg MB (~28 attomol) were suspended in solutions of IS-1 oligonucleotide. The oligonucleotides were non-thiolated and were only modified with Alexa Fluor 647 (ex. 650, em. 665), which allowed for fluorescence measurement of DNA that was independent of emission from QDs. The curve shows that the amount of DNA* increased by up to ~500 pmol of DNAb, which is estimated to be the maximum capacity of MB-QDs. The data suggest a range of 0.80-0.97 for DNA*/DNAb in linear range of the curve. Next, the range for V*/Vb can be established by estimating the volumes that the DNA* layer occupies. In a typical MBL experiment, 0.1 mg MB with a total surface area of 53 mm2 is used in 100 μL buffer solutions. The thickness of DNA* layer depends on the configuration of adsorbed DNAs with regard to MB surfaces and the configuration may vary from completely collapsed to completely upright (Figure 3D). We assume that at low concentration of DNAb, the adsorbed DNAs are more likely to adopt a collapsed configuration to maximize the number of ionic bonds with the MB surfaces. The collapsed configuration results in a thickness of 1 nm for DNA* layer (thickness of ssDNA). At high DNAb concentration, a higher number of DNA* oligonucleotides adopt the upright configuration to compensate for the electrostatic repulsion and accommodate more oligonucleotide on the MB surfaces. The upright configuration results in a thickness of ~6 nm for DNA* layer (i.e. the length of an 18-mer DNA strands). Also, a fraction of DNA* oligonucleotides are

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expected to adopt a mix of configurations between the two extremes, which results in a thickness of between 1-6 nm. These calculations provide an estimate of 5.3 × 10-5 to 32 × 10-5 mm3 for the volume of DNA* layer. On the basis of the above calculations, a range of 3.1 × 105 to 1.9 × 106 is obtained for

the

preconcentration

factor,

which

indicates

the

enormous

DNA

preconcentrating effect that occurs in the MBL method. The magnitude of preconcentration explains both the fast kinetics (cf. Eq. 6) and also the high DNA surface density (cf. Eq. 7) that is achieved using the MBL method.

Figure 3. The effect of DNA preconcentration at MB surfaces. (a) QD packing density as a function of DNA solution concentration (0.12-3.5 μM). The blue and red curves show the packing density as represented by FRET ratio and DNA/QD, respectively. Both curves demonstrate a trend that conforms to a Langmuir adsorption isotherm. (b) Schematic showing DNA preconcentration on MB-QD conjugates. (c) The MB15


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adsorbed DNA (DNA*) and FRET ratio as a function of the amount of solution phase DNA (DNAb). To obtain DNA*, MB-QD conjugates (3 pmol QD conjugated to 0.1 mg of MBs) were dispersed in 100 μL of TB buffer pH 7.4 containing different amounts of Alexa Fluor 647-labelled DNA oligonucleotides (IS-1, 50-800 pmols). The MBadsorbed DNAs were released from the MB and fluorescence measurements were done using excitation at 640 nm and an emission range of 650-700 nm, respectively. For other conditions see Figure 2. (d) Schematic representation of different configurations that DNA* can adopt with regards to the surface of MBs. (i), (ii) and (iii) show the upright, collapsed and mixture of configurations, respectively.

Figure 3C shows the correlation between the amounts of DNA that electrostatically adsorbed on the MB surfaces (the red curve) with the packing density of DNA immobilized on the QDs, as indicated by the FRET ratio (the blue curve). In Figure 3C, three distinct trends of DNA* and packing density are observed at different ranges of DNAb concentration. At the low range of DNAb (50-175 pmols) both DNA* and the packing density increase with DNAb; at the medium range of DNAb (175-500 pmol), DNA* increases with DNAb but packing density saturates; and at the high range (500-800 pmol), DNA* also reaches saturation. On the basis of these results, we infer that the packing density increases with DNA* up to a point where the maximum limit of packing density is reached, which is in close correspondence to the theoretical maximum possible based on steric and electrostatic effects. In 2009 Hill et al. systematically studied the maximum packing density of thiolated oligonucleotides on AuNPs of different sizes,26 and reported that this maximum limit increases with the radius of curvature of nanoparticles. To test our hypothesis, in a subsequent section we compare the packing density achieved in the MBL method with the ones reported as the maximum limit by Hill et al. (vide infra).

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The surface of MBs that have adsorbed both QDs and DNA is a complex mixture primarily held in place via electrostatics. Counter ions are expected play an important role in such a polyelectrolyte complex environment, and the FRET ratios have been examined at increasing concentrations of Na+ (10-300 mM). Figure 4A shows that the FRET ratio increased with the cation concentration up to a [Na+] of 150 mM. The cation is known to screen the repulsion between DNA strands and facilitates a dense packing of DNAs on the MB surfaces.24 An opposite trend is observed at the higher range of [Na+] (150-300 mM), where the FRET ratio decreases with increasing [Na+]. We reason that another effect of the cation prevails at higher range of [Na+], which is weakening of the electrostatic charge attraction between DNA and MB. This effect impedes the DNA preconcentration and causes a reduction in the packing density on QDs. To further understand the results of immobilization using the MBL model, we have examined DNA immobilization in the presence of a polyanion competitor. The preconcentrating of DNA (a polyanion) at the surface of a MB is driven by electrostatic adsorption.

Thus, other polyanions may compete with DNA for

electrostatic adsorption on MB surfaces and impede DNA preconcentration. We chose polyacrylic acid (PAA, average MW ~1800) as a competitor because it is a linear polymer with one negative charge per monomer unit, similar to that spatial charge pattern of an oligonucleotide. The histogram in Figure 4B shows the FRET ratios obtained from the MBL method applied in a pH range of 4-7.4. The capture oligonucleotides were mixed with either 0X or 30X of PAA molecules. The histogram shows that the FRET ratio is almost completely suppressed, from ~17 in absence of

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PAA to >1 in presence of 30X PAA at pH 7.4 and pH 6, which indicates a strong competition from PAA in impeding DNA preconcentration, and hence DNA conjugation. The FRET ratio slightly increased at pH 5 (~2.3), and jumped to 15.6 at pH 4. We attribute the reemergence of the high FRET ratio at pH 4 to the protonation of carboxylate groups of PAA. The pKa of PAA is 4.5, 25 and at pH 4 most of the PAA carboxylic groups are in undissociated form, resulting in little polyanion charge and low affinity to MB surfaces. At the same pH, DNA maintains its negative charge, which leads to an efficient conjugation through DNA preconcentration effect.

Figure 4. DNA self-assembly on QDs prepared using the MBL method. A The dependence of FRET ratios on [Na+] for the range of 10-300 mM. [Na+] of 10-100 mM was adjusted by changing the borate buffer concentration from 5-50 mM (no NaCl). [Na+] in the range of 100-300 mM was adjusted by addition of 0-200 mM NaCl to 50 mM borate buffer. B Histogram showing the FRET ratio obtained from conjugates prepared using the MBL method in phosphate buffer at different pH. The capture oligonucleotides were either mixed with 0 or 30X of PAA. C The graph

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shows FRET ratios vs. MB surface area per QD (S.A./QD) with or without the presence IS-2 as the DNA competitor. In each experiment 2 pmol of QDs were adsorbed onto varying quantities of MBs (0.012 to 0.28 mg), and DNA immobilization was done using 140 pmol of the capture oligonucleotides (CP-1) with or without the presence of 140 pmol of the DNA competitor (IS-2). For other conditions see Figure 2. Each MB of 1000 nm diameter can accommodate approximately 112,000 to 5,200 QDs in the range of S.A./QD values of 28-600 nm2. To understand how MB surface area per QD (S.A./QD) affects the DNA packing density an investigation was done to determine the influence of a non-thiolated oligonucleotide of equal size and concentration of the capture oligonucleotide. The blue and red curves in Figure 4C were obtained from DNA immobilization using the MBL method at S.A./QD values of 28-600 nm2, in the presence and absence of DNA competitor, respectively. Two interesting trends can be observed in the graphs. First, the FRET ratio in both curves increases with S.A./QD at the lower range and then reaches a plateau. Second, even at the plateau range, where FRET ratio is independent of the surface area, the FRET ratios from the red curve (1X DNA competitor) remain lower than the corresponding values of the blue curve (no competitor). On the basis of these observations, it is the number of adsorbed DNAs interfacing QDs, but not the total number of DNAs on the MB surfaces, that limits the DNA packing density. In this model, DNA competitor gets an equal chance as the capture oligonucleotide to engage in adsorption at the interface. This results in a dilution in the number of capture oligonucleotides at the interface, and ultimately manifests as lower FRET ratios observed in presence of the competitor. The electrostatic adsorption of DNA onto the MB surfaces is responsible for the rapid and high density immobilization of DNA that is achieved by the MBL method.

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The affinity of MB surfaces for DNA adsorption can be further adjusted by varying pH, and has impact on the density of conjugated DNA on QDs. DNA adsorption was monitored by incubating MB-QD conjugates with IS-1 oligonucleotide (nonthiolated, labeled with Alexa Fluor 647) in borate buffers at pH 7.4, 8.4 and 9.4. The data presented in Figure 5A indicates that the quantity of adsorbed DNA decreases as pH is raised. This data is consistent with a reduction of the positive charge on the MBs (bulk solution pKa ~10 for the surface ligands), leading to a lower electrostatic adsorption capacity of MBs. FRET ratios were used as an indication of the DNA packing density at the corresponding pH values (Figure 5A). The extent of DNA adsorption did not directly correlate with the extent of conjugation, as was also suggested by the data in Figure 4. Further work was done to visualize DNA adsorption on the MB-QD surfaces using fluorescence microscopy. The microscope images in Figure 5B show the IS-1 oligonucleotides adsorbed on the surfaces of MB-QD conjugates at pH 7.4, 8.4 and 9.4. The images were taken at two different conditions: 1- the MBs with adsorbed QDs and DNA remained immersed in solution that contained IS-1 (top), and 2 - the MBs with adsorbed QDs and DNA were separated from the IS-1 solution and were washed twice (bottom) before re-suspension in buffer solution. The images in the second row show that the quantity of adsorbed DNA is more dependent on pH in a circumstance where dissociation of adsorbed molecules into bulk solution is not compensated by a concentration of oligonucleotides in solution. The data of Figures 4 and 5 DNA confirm that the DNA adsorption on MB surfaces is labile, and this

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explains the how various extents of preconcentration result in similarly high density of conjugated DNA. The MBL method for DNA conjugation has been further examined for effectiveness in conjugation of oligonucleotides to gold nanoparticles (AuNPs).

AuNP-DNA

conjugates were selected as these systems have been well studied and widely used in biological applications,5 and comparison data reflecting immobilization density by various methods is available . The MBL method was applied to citrate-capped AuNPs of 5, 10 and 15 nm diameter. Table 3 presents the number of oligonucleotides immobilized on the surfaces of QDs, and on the three sizes of AuNPs, using the Bulk method with salt-aging to maximize density, and the MBL method. To quantify the number of oligonucleotides immobilized on each AuNP, the concentration of AuNP and immobilized DNAs (fluorescently-labelled) in the purified AuNP-DNA conjugate solutions were independently determined using absorption and fluorescence spectroscopy, respectively (refer to SI for detailed procedure). An average of 26.4 ± 3.9 oligonucleotides per 5 nm AuNP was achieved using the Bulk method, which is much higher than the corresponding value of 4.0 ± 0.6 oligonucleotide obtained per QD (~5 nm in size). We believe that this observation may in part be due to the strong thiol-gold affinity compared to thiolzinc ion interaction, which can overcome electrostatic repulsion to form a high density of DNA on the AuNP surfaces. An average of 65 ± 6 and 106 ± 16 oligonucleotides per AuNP were achieved for 10 nm and 15 nm AuNPs, respectively, and this value is similar to that reported elsewhere.26 These packing densities correspond to surface densities of 3.4 ×1013, 2.1 ×1013 and 1.5 ×1013 DNA/cm2 for 5

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nm, 10 nm and 15 nm AuNPs, respectively. The higher surface density of oligonucleotides for smaller nanoparticles was reported previously and is attributed to their higher radius of curvature that reduces the repulsion between the immobilized oligonucleotides.26 In contrast, the MBL method resulted in the loading densities of 18.9 ± 4.2, 18.1 ± 3.3, 50 ± 5 and 95 ± 14 DNA molecules for QDs and AuNPs of 5 nm, 10 nm and 15 nm in diameter, respectively. It is important to note that in the MBL method, only the solution-facing side of the NPs are expected to conjugate with oligonucleotides, and the face of NP that is attached to the bead is assumed to remain unconjugated due to the geometrical constraints. To conjugate DNAs on the bead-facing side of nanoparticles prepared by MBL method, a solution-phase conjugation was done after the release of nanoparticles from the beads. This process is referred to herein as capping. The MBL procedure followed by capping resulted in DNA/NP of 25.3 ± 5.1, 26.2 ± 3.9, 66 ± 6 and 116 ± 18 for QDs, and AuNPs of 5 nm, 10 nm and 15 nm in diameter, respectively, which correspond to a surface density of 3.2 ×1013, 3.3 ×1013, 2.1 ×1013 and 1.6 ×1013 cm-2 for QDs, and AuNPs of 5 nm, 10 nm and 15 nm in diameter, respectively. These results indicate that the MBL method, in comparison with the Bulk method, provides packing densities of 6 times higher for QDs and approximately equal for AuNPs. The levels of packing densities observed for AuNPs corroborate with the previous reports,

6, 12, 13, 26, 27

which were attributed as the

highest level of DNA densities possible for each AuNP depending on the radius of curvature of NPs. For instance, maximum surface density values of 2.2 ×1013 and 1.6 ×1013 cm-2 were reported by Hill et al. for 10 and 15 nm AuNPs, 26 which correlates

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well with the values obtained using the MBL meth (2.1 ×1013 and 1.6 ×1013 cm-2 for the 10 nm and 15 nm AuNPs, repectively). This corroboration confirms our hypothesis that the MBL method provides for the maximum possible oligonucleotide packing density on nanoparticles, with salt aging providing comparable results to earlier work using AuNPs, and much improved packing densities for QDs. The maximum packing density also explains the observation in Figure 3C that at above a threshold of DNA*, the packing density is not increasing with DNA*. Now, we now understand that the threshold is based on the physical limit caused by the electrostatic and steric hinderace. In conclusion, we present herein a novel method for creating a high density shell of DNA oligonucleotides on quantum dots and gold nanoparticles within seconds. By electrostatic adsorption, the Magnetic Bead Loading creates a local concentration of mobile oligonucleotides in the vicinity of NPs that drives conjugation forward. The oligonucleotides were immobilized on QDs and AuNPs to levels that represent the physical limit of occupancy for the surface area and geometries of packing that are available. The MBL method offers rapid preparation of coatings of nanoparticles that may be useful in various biological applications.28

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Figure 5. A Histogram showing the correlation between the DNA adsorption and the FRET ratio. The bars indicate the amount of IS-1 oligonucleotide adsorbed on MBQD conjugates at pH 7.4, 8.4 and 9.4, and the data points indicate the corresponding FRET ratios. B Fluorescence images show the IS-1 oligonucleotides adsorbed on MBQD conjugates. The images were collected using excitation and emission wavelengths of 637 and 660 nm, respectively. All the experiments were performed in borate buffer 50 mM at pH 7.4, 8.4 and 9.4. For other conditions see Figure 2.

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Table 3. The average number of DNA molecules conjugated and the calculated surface densities on each QD, or on AuNPs of 5 nm 15 nm in diameter. Oligonucleot ide surface density (cm2) a 3.9± 0.6 Bulk 3.9 ± 0.6 -0.51 ×1013 QD MBL 18.9 ± 4.2 6.4 ± 0.9 25.3 ± 5.1 3.2 ×1013 26.4± 3.9 Bulk 26.4 ± 3.9 -3.4 ×1013 AuNP (5 nm) MBL 18.1 ± 3.3 8.1 ± 0.6 26.2 ± 3.9 3.3 ×1013 65 ± 6 Bulk 65 ± 6 -2.1 ×1013 AuNP (10 nm) MBL 50 ± 5 16 ± 3 66 ± 6 2.1 ×1013 106± 16 Bulk 106± 16 -1.5 ×1013 AuNP (15 nm) MBL 95 ± 14 21 ± 4.2 116 ± 18 1.6 ×1013 a The surface densities were calculated based on the total DNA/NP b The maximum surface densities were obtained from Ref. [26]. DNA/NP Before capping

DNA/NP After capping

DNA/NP Total

Maximum surface density (cm-2)b --2.2 ×1013 1.6 ×1013

Experimental section: Reagents: Diethylaminoethyl (DEAE)-functionalized magnetic beads (MB, 1 um) were from Bioclone Inc. (San Diego, CA). Green-emitting CdSe/ZnS core/shell quantum dots (PL= 518 nm) were from Cytodiagnostics (Burlington, ON, Canada). Amicon Ultra0.5, 10 and 100 kDa centrifugal filters were from Millipore Corporation (Billerica, MA). AuNPs of 5 nm and 15 in dimeter, sodium tetraborate, tris(2carboxyethyl)phosphine hydrochloride (TCEP), sodium dodecyl sulfate (SDS), Lglutathione (GSH, reduced, ≥98%), DTT, polyacrylic acid (PAA, MW ~1800) and tetramethylammonium hydroxide (TMAH) were from Sigma-Aldrich (Burlington, ON, Canada). CP-4 oligonucleotide was from ACGT Corp (Toronto, ON, Canada). All other oligonucleotides were synthesized and purified by Integrated DNA Technologies (Coralville, IA). All buffer solutions were prepared using a deionized water purification system (Milli-Q, 18 MΩcm-1) and were autoclaved prior to use.

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The buffer solutions included 100 mM tris-borate buffer (TB, pH 7.4), 50 mM borate buffer (BB, pH 7.4, 8.4, 9.4 and 10) and phosphate buffer (PB, pH 4, 5, 6, 7.4 and 8).

Preparation of QD-DNA conjugates using Bulk method The glutathione-capped quantum dots (GSH-QDs) were prepared using a previously reported procedure,21 which is briefly described in the SI. The immobilization of DTPA-modified oligonucleotides in the bulk solution was done using a 2-day procedure established previously.8 During day one, 1.4 nmol of oligonucleotides were first incubated with 140 eq. of TECP for 15 min. Then 30 pmol of GSH-QDs and 5 μL of 1M NaOH was added to the solution and the volume was adjusted to 450 μL using BB 9.2 containing 100 mM NaCl. The solution was agitated overnight on an orbital mixer. During day 2, anther 140 nmol TCEP was added to the solution. The salt aging procedure was applied by the addition of 15 μL of 2.5 M NaCl in 15 min intervals up to a total volume of 100 μL. Thereafter, the solution was agitated overnight on the orbital shaker. For kinetic measurements, 50 μL aliquots of the solution were taken at different incubation times from 30 to 2000 min. For FRET measurements, 140 pmol of Cy3-modified complementary DNA was previously added to 50 μL of QD-DNA conjugate and the solution was agitated for 10 min.

Preparation of QD-DNA conjugates using MBL method 0.10 mg of DEAE-functionalized magnetic beads (MB, 1 μm in diameter) was transferred to a 2 ml centrifuge tube and the tube was transferred to a magnetic stand. The MBs were isolated and the supernatant was removed. Then, MBs were

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washed twice with TB buffer pH 7.4 containing 1M NaCl and twice with TB buffer pH 7.4 containing 20 mM NaCl (TBS). The wash procedure included addition of wash buffer, vortex mixing for 30 s, isolation of the MBs and removal of the supernatant. The MBs were then re-dispersed in 100 μL of TBS buffer, and 3 pmol of GSH-QDs was added to the tube. The mixture was agitated on a vortex shaker for 30 s and the MB-QDs were isolated and re-dispersed in capture buffer solution. Unless otherwise noted, BB pH 7.4 (50 mM) containing 50 mM NaCl was used as the capture buffer. 175 pmol of capture oligonucleotides, previously incubated with a 100 eq. of TCEP, was added to the mix and the tube was agitated for 30 s (unless otherwise noted). For kinetic measurements, MB-QD conjugates were agitated with 50X capture oligonucleotides for various times from 0.25-20 min. Thereafter, the beads were washed twice with TBS. Finally, the QD-DNA conjugates were freed by adding 50 μL of release buffer (BB pH 10 (50 mM) containing 1 M NaCl) to the isolated beads with agitation for 30 s. For capping of NPs, 40 pmol of capture oligonucleotides were added to QD-DNA solution in the release buffer and the solution was allowed to agitate for 30 min. FRET measurements were done after addition of 140 pmol Cy3-labeled complementary DNA (CS-1) to the QD-DNA conjugate, followed by 5 min of agitation.

Preparation of AuNP-DNA using Bulk method Self-assembly of DNA to 5 nm and 15 nm AuNPs was achieved using the previously reported salt aging method.6 Briefly, CP-3 was first reduced using 100 eq. of DTT in phosphate buffer (PB) pH 8 for 1 h and DTT was extracted out using ethyl acetate.

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Then, 175, 100 and 40 pmol of the reduced oligonucleotides were added to AuNPs of 5 nm (2.5 pmol), 10 nm (0.4 pmol) and 15 nm (0.1 pmol) in diameter, respectively. Thereafter, the concentration of PB and sodium dodecyl sulfate (SDS) was adjusted to 10 mM and 0.01%, respectively, and the mixture was agitated for 20 min. After agitation, the concentration of NaCl concentration was adjusted to 50 mM, and then adjusted to 100 mM after another 20 min agitation period. This process was repeated for every 100 mM of salt until there was 1M of NaCl. The concentration of PB and SDS was maintained throughout the process at 10 mM and 0.01%, respectively. The salt aging process was followed by agitation overnight at room temperature.

Preparation of AuNP-DNA conjugates using MBL method The MBL method used for self-assembly of DNA on the 5 nm and 15 nm AuNPs was similar to that used for QDs except that 175, 100 and 40 pmol of the CP-3 oligonucleotide were added to AuNPs of 5 nm (2.5 pmol), 10 nm (0.4 pmol) and 15 nm (0.1 pmol) in diameter, respectively. For capping, 50, 20 and 11 pmol of the CP-3 oligonucleotide were added to AuNPs of 5 nm, 10 and 15 nm in diameter in the release buffer, and the solutions were allowed to agitate for 30 min.

Quantification of the loading of oligonucleotides on nanoparticles To determine the loading of oligonucleotides on nanoparticles, the concentration of nanoparticles and oligonucleotides in the purified conjugate solutions were quantified

using

absorption

spectroscopy

28

and

fluorescence


spectroscopy,

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respectively. CP-2 oligonucleotide (modified with DTPA and Cy5) and CP-3 (modified with SH and Cy5) was used to determine the number of oligonucleotides on QDs and AuNPs, respectively. Refer to SI for the detailed procedure.

Fluorescence microscopy An in-house multicolor fluorescence microscope was used to collect the fluorescence images of oligonucleotides (Alexa Fluor 647-modified) adsorbed on the MB-QD conjugates. A solution chamber was prepared by attaching a glass coverslip on a microscope slide using two-sided tape. Two small holes had been drilled into the microscope slide and were used as the solution inlet and outlet. The MB-QD conjugate solutions were agitated and 40 uL of the solutions were pipetted into the chamber inlet and allowed to fill the chamber. The fluorescence microscopy images of the Alexa Fluor 647-modified oligonucleotides adsorbed on MB-QD conjugates were taken at excitation and emission wavelengths of 637 and 660 nm, respectively.

Acknowledegements: We gratefully acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada (STPGP 479222). We thank Dr. U. Uddayasankar for his useful suggestions, and Dr. A. Mazouchi for his assistance in acquiring the fluorescence microscopy images.

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Rapid Immobilization of Oligonucleotides at High Density on

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