Article pubs.acs.org/Langmuir
DNA-Functionalized Quantum Dots: Fabrication, Structural, and Physicochemical Properties Dazhi Sun and Oleg Gang* Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, New York 11973, United States ABSTRACT: We have systematically investigated the effect of physicochemical conditions, such as pH, salt concentration, and DNA/ nanoparticle ratio, on the chemical conjugation process and structural and optical stability of carboxyl-functionalized quantum dots (QDs) functionalized with amino-modified DNA. We reveal the relationship between aqueous conditions and the amount of DNA conjugated on QDs, colloidal stability, and yield of the final QD-DNA conjugates. By carefully adjusting the environmental variables we have successfully achieved up to 20 DNA strands conjugated per QD, and demonstrated how this number can be tuned. The fabricated QD-DNA conjugates are dispersed and optically stable in salted solutions for over a month. We have also evaluated the involved interparticle interactions to explain the solution behavior of QD-DNA conjugates. Our results provide a basic understanding of the physiochemical processes governing a nanoparticle−biomolecule conjugation and the structural stability of the formed conjugates. Such fabricated QD-DNA conjugates might be of great benefit for programmable assemblies of optically active nanomaterials and for emerging biosensing methods based on nanomaterials. compared to organic dyes, the flexibility and precision of tuning interparticle distances by DNA strands, and the ease of assembling heterogeneous nanostructures via the DNAmediated approach.30,31 Owing to the above reasons discussed, there is a need for QD-DNA conjugates with defined structure, robust optical stability, and well-controlled recognition properties. The presented studies demonstrate practical ways for the realization of such conjugates using QD coated with a carboxylrich polymer and uncover the relationship between the physicochemical conditions and the efficiency of DNA-to-QD conjugation, as well as colloidal stability. The prerequisite for the assembly of hybrid nanostructures containing QDs through DNA-mediated assembly is the preparation of single-stranded DNA (ssDNA) conjugated QDs that have the following features: a strong end-attachment of ssDNA strands to QDs surface, a substantial number of ssDNA strands conjugated on each QD, and stable structural and optical properties in the aqueous dispersion. Several methods have been developed to conjugate QDs with ssDNA.32−39 For example, thiol-containing ssDNA can be directly attached to QD surface by ligand exchange.32,33 However, a drastic decrease of the quantum yield in water is often observed after QD conjugation and it is difficult to control the number of ssDNA strands conjugated to each QD. Positively charged protein, such as strepavidin, can be electrostatically absorbed onto QDs with a negatively charged
1. INTRODUCTION Self-assembly of nanoscale building blocks into designed architectures offers promising possibilities for rational and scalable fabrication of novel materials and devices with tailored functionalities. Numerous recent research efforts have been focused on the use of the recognition interactions provided by biomolecules, in particular, DNA, to direct the assembly of nanoparticles,1,2 which have yielded a great deal of nanoarchitectures such as well-defined nanoclusters,3−8 1-dimensional (1-D) and 2-D nanoparticle array,8−15 and 3-D superlattices.16−20 Among the rich family of inorganic nanoscale building blocks, semiconductor nanocrystals, also known as quantum dots (QDs), have attracted special attention for their potentials in optical, energy-related, and biosensing applications. The unique size-dependent optical properties, high fluorescence quantum yields, and excellent optical stability against photo bleaching are the most attractive attributes of QDs.21−24 In this respect, an application of the DNA-based approaches for the fabrication of optically active materials using QDs has recently emerged as one of the material applications of the DNA-programmable assembly. For example, some of the interesting questions on the collective optical properties of QDs near plasmonic nanostructures (e.g., gold and silver nanoparticles) in distinct or extended hybrid assemblies can be addressed by utilizing the DNA-mediated assembly due to its feasibility in the preparation of materials with controlled size, geometry, and interparticle distance.25−29 On the other hand, in a sensing arena, various prototypes of biosensors using QDDNA conjugates have also been demonstrated by taking advantage of the excellent optical properties of QDs as © 2013 American Chemical Society
Received: January 3, 2013 Revised: March 9, 2013 Published: May 2, 2013 7038
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Figure 1. pH effect on the conjugation of carboxyl-QDs with amino-DNA. (A) Schematic on the pH effect on the conjugation chemistry. (B) Schematic on the pH effect on the colloidal stability of carboxyl-QDs. (C) The major reaction efficiency parameters, the amount of ssDNA conjugated per QD (left axis), and the yield of dispersed QD-DNA conjugation (right axis), are shown as a function of the pH value. The optimal pH value is ∼7. (D) Table illustrating the conjugation efficiency as a function of the salt concentration.
fluorescence over several months. The comparison of experimental results with colloidal models allows understanding of how the major solution parameters determine the interactions of DNA-functionalized QDs.
surface,23,34 followed by conjugation with biotin-functionalized ssDNA. The sensitivity of the electrostatic binding between strepavidin and QDs to ionic strengths limits the use of this approach. QDs often aggregate during the conjugation, especially at high ionic strength, leading to a low yield of QD-DNA conjugates. Moreover, the number of conjugated ssDNA strands is usually small due to the relatively large size of strepavidin molecules that occupy the surface of QDs. Watersoluble silanized QDs containing functional groups can be conjugated with DNA via bifunctional linkers.35,36 As-prepared QD-DNA conjugates are stable in a salted aqueous solution and exhibit a high fluorescence intensity. However, this method involves multiple chemical modification steps, and thus, its complexity and a low amount of ssDNA per QD are somewhat restrictive. QDs capped with mercapoacetic acid ligands can be conjugated with primary-amine modified ssDNA.37 It has been found that during such conjugation process ssDNA with mixed bases tend to lie across the surface of QDs, therefore affecting the further attachment of ssDNA and DNA hybridization. Here, we have utilized a simple conjugation method that couples the primary-amine modified ssDNA to the carboxyl groups on the polymer layer associated with QDs.38,39 Because the surface of QDs is well protected by the polymer shells and not directly interacted with DNA, the superior optical properties of QDs can be expected after conjugation. However, the conditions of polymer stability on a particle surface beyond physiological conditions, and, more importantly, the effect of DNA conjugation procedure on optical and structural stability of QDs are not well explored. We have systematically investigated the physicochemical effects affecting conjugation process and QD stability, including pH, ionic strength, and the initial DNA/QD ratio on the amount of ssDNA per QD. We have found that the conjugation chemistry and colloidal stability are strongly dependent on these parameters and that the amount of ssDNA conjugated on each QD can be tuned via these experimental parameters. The conjugation process has been optimized to achieve around 20 ssDNA per QD with a yield of over 70%. These QD-DNA conjugates are structurally stable at high ionic strengths and maintain their optical
2. EXPERIMENTAL SECTION 2.1. Materials. Water-soluble polymer-coated CdSe/ZnS QDs containing carboxyl groups (carboxyl-QDs) with a fluorescence peak at 605 nm were purchased from Invitrogen. DNA oligonucleotides were purchased from Integrated DNA Technologies, Inc., as lyophilized powders with HPLC purification. Sequences for the DNA strands were as follows: Amino-modified DNA: 5′-NH2-C6-T15-TAA CCT AAC CTT CAT3′ (amino-DNA). Cy3-modified DNA: 5′-Cy3-ATG AAG GTT AGG TTA-3′. DNA oligonucleotides were dissolved in D.I. water with desired concentrations before conjugation. N-Ethyl-N′-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) and Nhydroxysulfosuccinimide sodium salt (NHS) were purchased from Aldrich and used without any purification. 2.2. Conjugation of Carboxyl-QDs with Amino-DNA. Carboxyl-QDs (80−100 carboxyl groups per QD surface) were conjugated with amino-DNA through a classic liquid-phase peptide synthesis procedure. In a typical one-step conjugating experiment, 25 μL of carboxyl-QD solution (8 μM) was diluted with 200 μL borate buffer (50 mM) in a glass vial. The pH value and [NaCl] were varied in order to study the effect of pH and salt concentration on the conjugation. Subsequently, 8 μL of 50 mM EDC and 8 μL of 50 mM NHS aqueous solutions were mixed with the above QD solution, respectively. Amino-DNA solution was then added immediately with various DNA/QD ratios. After a mixing, the conjugation was allowed at room temperature with gentle stirring for 2 h. For the conjugating experiment with stepwise salting, the procedure was same as the above one-step conjugating experiment with the initial [NaCl] = 0 M. During the conjugation, the salt concentration was increased with a time interval of 15 min and the final [NaCl] = 0.1 M. After reaction, the QD-DNA conjugates were first spun in a 0.22 μm PDMF filter at 2000 rpm for 15 min to remove any large aggregates. The excess DNA was then removed by using an ultrafiltration unit with 100 000 molecular weight cutoff (MWCO) at 2500 rpm for 20 min and washing 4−5 times with borate buffer (50 mM, pH = 8.5). The purified QD-DNA conjugates were finally 7039
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Figure 2. Influence of salt concentration on the conjugation of carboxyl-QDs with amino-DNA at pH = 7.1. Illustration of the salt concentration effect on (A) the colloidal stability of carboxyl-QDs and (B) the conjugation chemistry. (C) The major reaction efficiency parameters, the amount of ssDNA conjugated per QD (left axis) and the yield of dispersed QD-DNA conjugation (right axis), are shown as a function of a salt concentration. The optimal concentration of [NaCl] is 0.02 M, but the yield of the dispersed QD-DNA conjugates significantly decreased to 100. 3.1.4. Conjugation with Stepwise Salting. To further increase the amount of amino-DNA conjugated on carboxylQDs and still to retain a relatively high yield of dispersed QDDNA conjugates, a stepwise-salting strategy was utilized during the conjugation reaction (Figure 4). The conjugation started at [NaCl] = 0 with pH = 7.1 and DNA/QD = 100, followed by increasing the salt concentration to 0.1 M in one hour with an interval of 15 min (0.025 M NaCl per interval). After adding the salt, the solution was allowed for reacting for another hour before purification and collecting well-dispersed QD-DNA conjugates (Figure 4A). Compared with the conjugation reaction without salting under the same condition (pH = 7.1 and DNA/QD = 100, Figure 2), the amount of DNA conjugated on QDs was dramatically increased from ∼9 to ∼20 DNA strands per QD without a significant decrease of the yield of well-dispersed ssDNA functionalized QDs (Figure 4B,C). 3.2. Stability of QD-DNA Conjugates. We investigated the colloidal and optical stability of the QD-DNA conjugates
3. RESULTS 3.1. Conjugation of Carboxyl-QDs with Amino-DNA. 3.1.1. pH Effect. We summarize in Figure 1 the effect of pH on the amount of amino-DNA conjugated on carboxyl-QDs and the yield of QD-DNA conjugates at [NaCl] = 0 and DNA/QD = 100. The pH values ranged from weakly acidic (pH = 6.0) to basic (pH = 8.5) environments. At pH = 6.0, almost no dispersed QD-DNA conjugates were obtained after purification due to a complete QD aggregation, therefore the yield of QDDNA conjugates is considered to be 0 and the amount of ssDNA conjugated on QDs was not determined. The increase of the pH value resulted in the increasingly larger amount of well-dispersed QD-DNA conjugates that were collected after purification with the conjugation yield up to ∼90%. However, this process was accompanied by a decrease of the amount of ssDNA conjugated on QDs. For example, at pH = 6.5, the dispersed conjugates had ∼13 ssDNA strands on each QD, but the yield was only ∼12%. In contrast, at pH = 8.5, the yield of dispersed QD-DNA conjugates reached as high as ∼90%; however, only about one ssDNA per QD was conjugated. Therefore, depending on the application of QDs functionalized with ssDNA, the number of strands can be tuned by varying the pH condition of the buffer solution during the conjugation process. The optimal value achieved in the current experiment, due to the trade-off between the number of DNA strands and the conjugation yield, is about pH = 7.1, which results in ∼9 ssDNA per QD and a yield of ∼75%. 3.1.2. Ionic Strength Effect. In Figure 2 we show the effect of salt concentration on the amount of amino-DNA conjugated 7041
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prepared by three different representative conjugation conditions, described below. These three types of QD-DNA conjugates have ∼5, ∼9, and ∼20 DNA strands per QD, and they were correspondingly prepared under (1) pH = 7.6, DNA/QD = 100, and [NaCl] = 0 (labeled as QD-05DNA), (2) pH = 7.1, DNA/QD = 100, and [NaCl] = 0 (labeled as QD09DNA), and (3) pH = 7.1, and DNA/QD = 100 prepared with stepwise salting (labeled as QD-20DNA). These systems were compared with the control sample of QDs without DNA conjugation, labeled as QD-00DNA. 3.2.1. Colloidal Stability of QD-DNA Conjugates. The hydrodynamic diameters (Dh) of various QD-DNA conjugates and free QDs measured by DLS as a function of [NaCl] are shown in Figure 5A. QD-00DNA maintained dispersed at
Figure 6. (A) Optical fluorescence of QDs before and after conjugation with ssDNA. Sample QD-09DNA is used without adding NaCl and with 0.1 M NaCl. (B) Fluorescence intensity of QDs before and after conjugation with ssDNs as a function of QD concentration. Sample QD-09DNA is used without adding NaCl and with 0.1 M NaCl. Sample QD-09DNA in 0.1 M NaCl is also measured 3 months after the preparation.
Figure 5. (A) Dh of carboxy-QDs and different QD-DNA conjugates as a function of [NaCl]. The measurements indicate that QDs are more stable when conjugated with more DNA. (B) Structural stability of QD-DNA conjugates with different amounts of conjugated ssDNA in salted solutions.
of QD-DNA conjugates can be determined by utilizing the fluorescence intensity−concentration standard curve obtained from known concentrations of free QDs. Figure 6B shows the fluorescence intensity of free QDs, QD-09DNA, and QD09DNA in 0.10 M NaCl solution, and QD-09DNA in 0.10 M NaCl solution after 3 months as a function of QD concentration. The fluorescence intensities of all the samples described above show a similar linear dependence on the QD concentration in the studied concentration range. These results demonstrate the excellent optical stability of QD-DNA conjugates with time in salted environments.
[NaCl] = 0.01 M, but became unstable at [NaCl] = 0.02 M. At higher salt concentration, DLS results indicated the formation of submicrometer aggregates from QD-00DNA. In contrast, QD-DNA conjugates not only were more stable against saltinduced aggregation, but their stability increased with the corresponding increase of the conjugated DNA strands per QD. The detailed studies indicate that salt concentration thresholds for the dispersion−aggregation of QD-05DNA, QD-09DNA, and QD-20DNA are 0.05 M, 0.15 M, and 0.20 M, respectively. The corresponding systems of QD-DNA conjugates aggregated rapidly above those thresholds. The temporal stability is an important factor for any practical use of QD-DNA conjugates. Therefore, we studied their stability in the above-discussed solutions with time, and summarize our results on the Figure 5B. No change for the DLS measured Dh of QD-05DNA in 0.05 M NaCl was observed over the period of a month, and a slight increase was detected in two months, indicating a very weak aggregation process. In contrast, QD-09DNA dispersed in 0.1 M salted solution underwent practically no signs of Dh increase in at least three months. QD-20DNA was stable in 0.10 and 0.20 M NaCl solutions for at least one month. 3.2.2. Optical Stability of QD-DNA Conjugates. The practical use of QD-DNA conjugates to a large degree relies on their long-term optical stability. We examined optical behavior before and after DNA functionalization and their stability over time. Figure 6A shows the fluorescence spectra of QDs before and after DNA conjugation, and in 0.1 M NaCl solution. These three data sets exhibit nearly identical fluorescence spectra, indicating that both DNA conjugation and ionic strength do not alter the fluorescence properties of QDs in our conjugation process. Therefore, the concentration
4. DISCUSSION An outline of the physicochemical interactions in the conjugation of carboxyl-QDs with amino-DNA discussed in the current study is summarized in Figure 7. 4.1. pH Effect. Our studies demonstrate that the efficiency of the EDC-mediated conjugation is strongly affected by the pH value of the reaction solution. The reaction mechanism involves the formation of the amide (peptide) bond between carboxylic acids and amines, where EDC interacts with the proton in the carboxyl group to form an active intermediate species and further reacts with the amine group resulting in the formation of the covalent bonding.40 However, under a high pH value, the carboxylic acid is deprotonated; thus, the reactivity of EDC is significantly prohibited (Figure 1A). In order to achieve an efficient covalent coupling, the pH value should be relatively low, as confirmed by our experiments. On the other hand, if the pH value is too low, the large amount of free protons in the solution could prevent the dissociation of the proton from the carboxylic acid, thus, potentially lowering the covalent coupling efficiency. In the current study, the chosen pH range, from 6.0 to 8.5, permitted the described effects to be probed. In addition to the DNA conjugation, the colloidal stability of carboxyl-QDs is also affected by the solution pH value. When the pH value is high, carboxyl-QDs are well dispersed in the aqueous solution as shown in Figure 1B, primarily due to their negatively charged surface, which comes from the deprotonated carboxyl groups. The presence of long-range electrostatic repulsion helps QDs overcome their short-range van de Waals (wdW) attractions. Therefore, in order to achieve high conjugation efficiency and colloidal stability, the pH value should be balanced during the conjugation experiment. In fact, as shown in Figure 1C,D, a high yield of the QD-DNA 7042
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Figure 7. Schematic on the physicochemical interactions for QDs conjugated with ssDNA.
dispersion, around 90%, was obtained at pH = 8.5. This is due to the charge stabilization at high pH value, but only ∼1 DNA strand per QD has been conjugated due to the lack of protons at such high pH value. On the contrary, when pH = 6.0, the surface charge vanished; therefore, QDs were fully aggregated and the yield of dispersed QD-DNA conjugates was close to 0. In all the pH values studied, we found that pH = 7.1 is the optimal condition for achieving both a high yield of welldispersed QD-DNA conjugates and a relatively large amount of ssDNA conjugated on each QD under [NaCl] = 0 M and the initial DNA/QD = 100. ζ-potential of the carboxyl-QD solution as a function of pH value at [NaCl] = 0 M, used as a characteristics of the QD surface charge, is shown in Figure 8A. The observed reduction of ζ-potential magnitude with the pH increase suggests that
carboxyl-QDs have more negative charges at a higher pH value, and thus are more stable, which is consistent with the analysis based on Figure 1B and the experimental results shown in Figure 1C,D. Moreover, the ζ-potential values of carboxyl-QDs at pH = 6.0 and 6.5 were not possible to measure at the given concentration due to the QD aggregation. This phenomenon also agrees with the fact that at a low pH value, the QD surface is associated with H+, causing the reduction of the surface charge and consequently lowering the QD stability. 4.2. Ionic Strength Effect. The effect of ionic strength on both colloidal stability and the conjugation efficiency is illustrated in Figure 2A,B, respectively. On one hand, a low ionic strengths favor the dispersion of carboxyl-QDs due to an electrostatic repulsion (Figure 2A); hence, the QD-DNA conjugation is facilitated. On the other hand, at a low ionic strength the neighboring DNA strands, when conjugated on QDs, electrostatically repel each other, which prevents the further attachment of free DNA strands to QDs and results in a low conjugation efficiency (Figure 2B). Therefore, a high salt concentration is needed to screen off the electrostatic repulsion between DNA strands. In the experimental data shown in Figure 2C,D, for increasing [NaCl] a larger amount of DNA strands is conjugated on QDs, but the yield of the welldispersed QD-DNA conjugates significantly decreases due to the QD aggregation at a high salt concentration, This conclusion is consistent with the analysis based on Figure 2A,B. However, the amount of DNA strands conjugated on each QD saturates at around 12 for [NaCl] > 0.05 M. This is likely due to the substantial aggregation of QDs that inhibits the conjugation reaction. In our study on the effect of the salt concentration at pH = 7.1 and DNA/QD = 100, the optimal conjugation is achieved at [NaCl] = 0 M with the highest yield of the well-dispersed QD-DNA conjugates and a relatively large amount of DNA strands (∼9) on each QD. A widely used DLVO theory41 is applied here to estimate the stability of carboxyl-QDs under different ionic strengths. According to this theory, the total interaction potential (VT) is the sum of two main contributions. The first term is electrostatic double-layer potential (Vedl), which is repulsive, as expected. The Vedl between two identical spherical particles of
Figure 8. (A) ζ-potential of carboxyl-QDs as a function of pH. (B) The DLVO-estimated total interaction potential of carboxyl-QDs at various [NaCl]. Note that the borate buffer strength is 50 mM in all solutions. (C) Calculated depletion potential of carboxyl-QDs at different DNA/QD ratios; see text for details. The DLVO-estimated total interaction potential of carboxyl-QDs under the same ionic strength is also added for comparison. (D) ζ-potential of QD-DNA conjugates with different amounts of ssDNA per QD. 7043
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radius, Rp, as a function of particle separation distance, h, can be expressed as41 Vedl = 2πεε0R p Ψ2 ln[1 + exp(κh)]
conjugates were collected after purification; therefore, the yield was ∼0 in this case. At a low salt concentration of [NaCl] < 0.05 M, on the other hand, the electrostatic screening effect is weak and, the high colloidal stability allows for the reaction of more DNA strands with QDs, thus resulting in a higher yield of well-dispersed QD-DNA conjugates. 4.3. DNA/QD Ratio Effect. The presence of nonabsorbing polymers in a colloidal dispersion may induce attractions between the particles through an unbalanced osmotic pressure, the magnitude of which depends on the concentration and molecular weight of the dissolving polymers. This phenomenon is referred as depletion.42 DNA can be considered a polymer; therefore, in the current study, the free DNA strands in the solution would inevitably bring attractive forces between QDs, thus causing them to aggregate. The depletion-induced attractions by dissolving polymers have been well understood both theoretically and experimentally.43,44 The length scale of depletion forces is about the size of the dissolving polymers and the magnitude depends on the concentration and the size of the depletants. In the current experiment, a significant QD aggregation was observed when the initial DNA/QD > 200 under pH = 7.1 and [NaCl] = 0 M (Figure 3C,D), suggesting that the electrostatic repulsions that keep QDs from aggregation have been overcome by the depletion attractions at a high DNA concentration. Meanwhile, the amount of ssDNA conjugated saturates at ∼9 DNA per QDs, which indicates the maximum loading of DNA strands on carboxyl-QDs used in this study under pH = 7.1 and [NaCl] = 0 M. The depletion potential (Vd) between two identical spherical particles of radius, Rp, in the presence of dissolving polymers of radius gyration, r, can be calculated using the following equation41
(1)
where the product εε0 is the relative permittivity of water, Ψ is the surface potential, which can be expressed as Ψ = ζ/ exp(−κRp), ζ is the ζ-potential measured in the experiments, and κ is the inverse of the Debye length, κ−1 = ((εε0kBT)/ (2NAe2I))1/2 where kB is the Boltzmann constant, T is the absolute temperature, NA is the Avogadro number, and e is the elemental charge. The second term is vdW attractive forces (VvdW). The VvdW between two identical spherical particles can be written as41 VvdW
⎡ ⎞ 2R p 2 AH ⎢ ⎛ h(4R p + h) ⎞ ⎛ ⎟ ⎜ ⎜ ⎟ ln⎜ =− + 6 ⎢⎣ ⎝ (2R p + h)2 ⎟⎠ ⎜⎝ h(4R p + h) ⎟⎠ ⎛ 2R 2 ⎞⎤ p ⎟⎥ + ⎜⎜ 2⎟ ⎝ (2R p + h) ⎠⎥⎦
(2)
where AH is the effective Hamaker constant. The VT between carboxyl-QDs at different [NaCl] under pH = 7.1 (the buffer strength is 50 mM), calculated from eqs 1 and 2 by using the ζ-potential in Figure 8A, is shown in Figure 8B. The AH considered for these calculations is 1 × 10−19 J.41 At [NaCl] = 0 M, a large repulsive energy barrier (∼7kBT) over a relatively long range is predicted to prevent QDs from aggregation. As the ionic strength increases, this energy barrier decreases. When [NaCl] = 0.10 M, the maximum repulsive potential is even less than kBT, suggesting the instability of carboxyl-QDs at high ionic strengths. In this case, the thermal excitation is sufficient to bring QDs together, leading to aggregation. The above behavior, in the frame of DLVO theory, agrees well with the experimental results shown in Figure 2C,D and Figure 5A. Moreover, at higher pH, it is expected that the repulsive energy barrier should become larger due to the higher absolute value of the ζ-potential, as shown in Figure 8A, thus resulting in a more stable QD solution, which agrees with our experimental data shown in Figure 1C. We note that for the condition when [NaCl] = 0.02 M, pH = 7.1, and DNA/QD = 100, the amount of ssDNA conjugated per QD is ∼11 and the yield of well-dispersed QD-DNA conjugates is about 57% (Figure 2D), also suggesting a high conjugation efficiency. However, as shown in Figure 5A and predicted in Figure 8B, the carboxyl-QDs are unstable and severe aggregation occurs at [NaCl] = 0.02 M. These experimental results suggest that the conjugation of ssDNA helps stabilize QDs in a salted solution. Actually, during the reaction, the colloidal stability relies on the competition between the QD aggregation and the QD-DNA conjugation. Once carboxyl-QDs are conjugated, the attached DNA strands provide steric and additional electrostatic forces to prevent aggregation. When the salt concentration is high, the aggregation process is accelerated due to the electrostatic screening effect and the formation of the large QD aggregates significantly inhibits the further DNA conjugation with QDs. In the current experiment, the aggregation of QDs at [NaCl] > 0.05 M was visually observed during the experiments, which also accounts for a sharp decrease of the yield of the welldispersed QD-DNA conjugates at [NaCl] = 0.02−0.05 M, as shown in Figure 2C,D (from ∼57% to ∼9%). When [NaCl] = 0.2 M, QDs completely aggregated and no dispersed QD-DNA
Vd = −
⎛ 3(R p + h) (R p + h)3 ⎞ 4π ⎟P (R p + r )3 ⎜⎜1 − + 4(R p + r ) 3 16(R p + r )3 ⎟⎠ ⎝ (3)
where h is the particle separation, and P is the osmotic pressure of the polymer solution. The osmotic pressure of a dilute DNA solution can be estimated by45,46
P=
RT (ϕCc)2 ϕCc + 4Cs
(4)
where R is the ideal gas constant, T is the absolute temperature, ϕ is the osmotic coefficient, which equals to 0.245, Cc is the concentration of the DNA counterion ion, and Cs is the salt concentration. The Vd between carboxyl-QDs at different initial DNA/QD ratio (the QD concentration is fixed to be 1 μM), calculated from eqs 3 and 4, is shown in Figure 8C. The radius of gyration of ssDNA used in the current experiment was determined by DLS. The measured hydrodynamic diameter is ∼5 nm. Therefore, r ≈ 2.5 nm. Cc for the DNA strand with 30 bases equals CDNA × 30, where CDNA is the molar concentration of ssDNA. The DLVO curve for carboxyl-QDs at pH = 7.1 and [NaCl] = 0 M is added to Figure 8C for comparison. The depletion potential decreases rapidly as the DNA/QD ratio increases. At DNA/QD > 150 the attractive potential induced by ssDNA is sufficient to overcome the repulsive potential barrier determined by a DLVO curve, thus causing QDs aggregation. The above-described estimations qualitatively 7044
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tions on the effects of ionic strength and the initial DNA/QD ratio also agree well with the theoretical models. By carefully tuning pH, ionic strength and DNA/QD ratio, the number of ssDNA strands conjugated per QD has been increased up to around 20 with a high yield of around 70% for well-dispersed QD-DNA conjugates. These QD-DNA conjugates are optically stable and maintain their colloidal stability in a salted solution up to 0.2 M NaCl. The QDs of this type with other emission wavelengths have similar hydrodynamic diameters; therefore, the discussed approaches can be readily adopted for applications where different spectral properties are required. As-prepared QD-DNA conjugates have been utilized to assemble QDs and Au nanoparticles into well-defined 3D superlattices.19 Our ongoing work utilizes QD-DNA conjugates for the fabrication of heterogeneous nanoclusters with precisely assembled structures for optically tunable materials.
agree with our experimental results shown in Figure 3C,D. More specifically designed studies might be required to explore the effects of the particle surface structure. For example, the surface charges and the attached ssDNA interplay, via electrostatic and steric interactions respectively, with depletion induced attractions (see also Figures 5A and 8B). 4.4. Conjugation with Stepwise Salting. The salting process utilized here to increase the amount of ssDNA conjugated on QDs is similar to the DNA loading on Au nanoparticles by salt.47 However, the maximum amount of ssDNA strands conjugated on QDs in the current study is ∼20, much less than that on Au nanoparticles with a similar particle size (∼10 nm in diameter with ∼50−60 ssDNA strands). This difference may be due to the relatively low coupling efficiency between carboxyl and amine groups in an aqueous environment40 as compared to the strong binding between thiol groups and gold.48 Moreover, the salting process in the conjugation of ssDNA with Au nanoparticles can be accelerated by a short exposure to a sonication; however, sonication had a negligible effect on the conjugation of amino-DNA with carboxyl-QDs. 4.5. Stability of QD-DNA Conjugates. Compared to carboxyl-QDs, the QD-DNA conjugates become much more stable due to the attachment of the DNA strands, which provides not only more surface charges, but also additional steric repulsion. The increase of the particle diameter as the increase of the amount of DNA strands conjugated on each QD, as shown in Figure 5B, is a demonstration of the increased steric layer repulsion. Moreover, the ζ-potentials of QDs conjugated with different amount of DNA strands are also displayed in Figure 8D. The ζ-potential decrease with the increase of the amount of DNA strands on QDs provides more evidence of the increased stability of the QD-DNA conjugates. The QDs remain optically stable after conjugated with ssDNA as shown in Figure 6. Normally the fluorescence intensity and quantum yield of QDs in solutions are determined by their surface characteristics and if the surface is well-passivated by ligands and surfactants, QDs will possess a high quantum yield.49 However, a direct attachment of DNA strands to QD surface by ligand exchange often results in a significant decrease of the quantum yield in an aqueous solution due to the creation of additional surface defects during the functionalization.1 Moreover, the optical stability of such QDs is typically limited. On the contrary, in our current experiment, the amino-DNA strands do not directly attach to the surface of QDs, but are covalently bonded with the carboxyl groups on the protective polymer shell, therefore, the initial quantum yield of the QDs in water can be retained. This indirect conjugation may also serve as a general strategy for preparation of highly luminescent water-soluble QDs for various energy conversion and biosensor applications.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address
Dahzi Sun, Department of Micro-Nano Materials and Devices, South University of Science and Technology of China (SUSTC), 1088 Xueyuan Blvd., Shenzhen, Guangdong, China 518055. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Research carried out at the Center for Functional Nanomaterials, Brookhaven National Laboratory, is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. We thank Dr. Andrea L. Stadler for helpful discussions and suggestions and Dr. Zhihua Xu for the assistance in the optical measurements. Sun DZ also acknowledges funding JCYJ20120830154526538 at SUSTC.
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REFERENCES
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5. CONCLUSION In summary, we show that structurally and optically stable QD functionalized with DNA can be successfully fabricated. Our systematic investigation of the physicochemical interactions between carboxyl-QDs and amino-DNA during their conjugation, including conjugation chemistry, van de Waals attraction, electrostatic repulsion, and depletion provide insight into the major experimental parameters determining QD functionalization. We found that the number of DNA strands conjugated on QDs and the yield of well-dispersed QD-DNA conjugates are strongly affected and can be tuned by pH, ionic strength, and DNA concentration. The experimental observa7045
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