Optically Characterized DNA Multilayered Assemblies and

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J. Phys. Chem. C 2009, 113, 21185–21195

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Optically Characterized DNA Multilayered Assemblies and Phenomenological Modeling of Layer-by-Layer Hybridization Noritaka Kato,*,†,‡ Lillian Lee,‡ Rona Chandrawati,‡ Angus P. R. Johnston,‡ and Frank Caruso*,‡ Department of Electronics and Bioinformatics, School of Science and Technology, Meiji UniVersity, Kawasaki 214-8571, Japan and Center for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The UniVersity of Melbourne, Victoria 3010, Australia ReceiVed: July 30, 2009; ReVised Manuscript ReceiVed: October 10, 2009

Layer-by-layer assemblies based on deoxyribonucleic acid (DNA) hybridization have potential for various bio- and nanotechnology applications because of their programmability, biodegradability, and ability to control the structure of the assemblies on the nanometer scale. Herein, we investigate the growth and salt stability of DNA films by the optical technique dual polarization interferometry and numerically model the film buildup. The DNA films were assembled by sequentially depositing pairs of oligonucleotides comprised of two different block sequences onto the surface. The oligonucleotides used in the assembly of the different films were as follows: a homopolymeric diblock pair of AxGx/TxCx (x ) 15, 20, or 30), a homopolymeric diblock pair of A15G15/C15T15 in which the orientation of the T15C15 diblock was reversed (C15T15), and a random diblock pair of X15Y15/X′15Y′15. The characteristics of the layer growth were highly dependent on the type of the oligonucleotide pair used: the mass of DNA deposited followed a linear, stepwise, or saturated growth with increasing layer deposition. The layer growth of each film was numerically modeled by taking into account the effective hybridization rate and the effective dissociation rate of the oligonucleotides. The proposed modeling offers a framework for molecularly designing oligonucleotide pairs to obtain DNA multilayer films with desired physicochemical properties (thickness, density, stability). Introduction Oligonucleotides exhibit high recognition and specificity to their complementary strands during hybridization. Furthermore, the DNA sequence can be easily programmed to form welldefined nanostructures.1 This makes them attractive building blocks for self-assembling nanostructured molecular architectures. It has been demonstrated that two-dimensional crystals,2 tile lattices,3 nanotubes,4 microspheres,5 hydrogels,6,7 and threedimensional nanopolyhedrons8,9 can be synthesized using programmable DNA assembly. As the assemblies consist solely of DNA, they are biodegradable and have wide potential for applications such as drug delivery, biosensing, and tissue engineering.10-13 Recently, we combined programmable DNA assembly with the layer-by-layer (LbL) technique14 to construct films comprising solely of DNA.15,16 The LbL approach was initially developed for the formation of thin polymer films by alternatively adsorbing positively and negatively charged polyelectrolytes on a charged surface. The use of the highly specific base pair interactions involved in DNA hybridization over the use of electrostatic interactions to drive the assembly of the film increases the versatility of the LbL technique. Analogous to the use of positively and negatively charged materials as building blocks in the assembly of the films,17-20 the DNA multilayer films are also constructed from complementary pairs of oligonucleotides. These oligonucleotides are designed to contain at least two blocks with different sequences for the LbL assembly. * To whom correspondence should be addressed. E-mail: nkato@ isc.meiji.ac.jp and [email protected]. † Meiji University. ‡ The University of Melbourne.

While one block has a base sequence complementary to the preadsorbed oligonucleotides on the surface, the other noncomplementary block remains in single-stranded form for the next hybridizing oligonucleotides.15 Using this technique, DNA films can be formed on planar supports and colloidal particles,15,21-24 whereupon dissolution of the colloidal template results in the formation of DNA capsules.15,21-23 The structural and physicochemical properties of the DNA film can also be further tailored by post-treating the LbL-assembled film. For example, cross-linking15,21,23 the DNA film improves the structural stability, while treatment with appropriate endonucleases degrades specific dsDNA sequences within the film, resulting in collapse of the film structure. Quartz crystal microgravimetry (QCM) has previously been used to investigate the assembly and structural properties of DNA films on planar substrates.15,21-24 The QCM is able to monitor the layer growth by changes in the resonant frequency of the piezoelectric oscillator. However, the swollen, hydrated, and mechanically soft DNA films induce large dissipation of the mechanical vibration energy, and the frequency changes cannot be accurately converted to mass changes using the Sauerbrey equation.25 Herein we optically determine the mass and thickness of the DNA films using dual-polarization interferometery (DPI). Because the DPI deduces the mass from the refractive index of the film, the mass obtained by DPI is free from the hydration and rheological property effects associated with the QCM. DPI has previously been used for analysis of the binding events of proteins and DNA.26-30 This technique enables us to gain a fundamental understanding of the buildup mechanism, which is crucial for advanced engineering of the DNA multilayers.

10.1021/jp907283k CCC: $40.75  2009 American Chemical Society Published on Web 11/18/2009

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TABLE 1: Oligonucleotide Sequences and Pairs

The DPI optical waveguide consists of a sensing and reference waveguide.31 The sensing waveguide is directly exposed to the solution, while the reference waveguide is embedded within the waveguide and isolated from any environmental change. The refractive index at the surface of the waveguide changes when material is adsorbed on the sensing waveguide. The evanescent field generated by the laser beam propagating through the sensing waveguide is affected by this refractive index change, resulting in a phase change of the beam. This phase change is seen as a change in the interference pattern, which is generated from the sum of the individual beams emitted from the sensing and reference waveguides. By monitoring the phase change of two perpendicularly polarized laser beams, a unique solution of the effective thickness and effective refractive index of the adsorbed layer can be obtained. Conventional ellipsometry or surface plasmon resonance spectroscopy typically requires knowledge of either the refractive index or the thickness of the layer to determine the thickness or refractive index of the layer, respectively. For DPI, the measured refractive index (neff) can be converted to the layer mass per area (m) using eq 132

m ) Teff(neff - ns)/

( dndc )

(1)

where Teff is the measured effective thickness, ns is the refractive index of the surrounding solution, and dn/dc is the refractive index increment of the material solution as a function of concentration (cm3 g-1). Table 1 shows the oligonucleotide sequences used in this study. The films, assembled using different oligonucleotide pairs, were examined using DPI to understand the influence of oligonucleotide composition on layer growth. Phenomenological modeling of the layer growth is proposed to evaluate the effective hybridization and dissociation rates of the oligonucleotides for each adsorption step. The proposed model successfully explains the difference in mass of the DNA films, which was found to depend on the composition of the oligonucleotides. The determined rates were used to elucidate the mechanism involved in the assembly of the DNA films. Experimental Section Materials. Poly(ethyleneimine) (PEI, Mw 25 000), sodium chloride (NaCl), sodium hydroxide (NaOH), absolute ethanol, and sodium citrate were obtained from Sigma-Aldrich. The oligonucleotides (Table 1) were custom synthesized by Geneworks (Adelaide, South Australia). All materials were used as received. Pure water used for all procedures was obtained from

Figure 1. Illustration of the expected film structures. The arrows indicate the 5′ to 3′ direction of the oligonucleotides. A layer of positively charged PEI was deposited on the negatively charged substrate, and the surface of the PEI layer was primed by a layer of negatively charged T30. (a) In the diblock film, the double strands formed by A15G15 and T15C15 are expected to hybridize in an extended configuration. The same structure is also expected for a random diblock film. (b) In the reverse diblock film, due to the reversed direction of the C15T15 compared to the T15C15, the direction of the double helix is forced to change upon hybridization, and the diblocks are expected to hybridize in a hairpin and/or stacked configuration.

an inline Millipore RiOs/Origin system and had a resistivity greater than 18 MΩ cm. A 1 mg mL-1 PEI solution was prepared using an aqueous solution of 0.5 M NaCl. The buffer solution (SSC buffer) for the DNA consisted of 50 mM sodium citrate with 500 mM NaCl, and its pH was adjusted using NaOH to 6.5. The concentration of the DNA for the film buildup was 5 µM (DNA solution in SSC buffer). NaCl solutions of various concentrations 500, 300, 200, 100, 75, 50, 25, and 0 mM (pure water) were used to probe the salt stability of the DNA films. The DPI waveguide chips, on which the DNA films were formed, were extensively cleaned with Piranha solution (70/30 v/v % sulfuric acid:hydrogen peroxide) followed by thorough rinsing in water and drying under a nitrogen stream prior to use. Caution! Piranha solution is highly corrosiVe. Extreme care should be taken when handling Piranha solution, and only small quantities should be prepared. This cleaning ensured the surface of the waveguide (silicon dioxide slightly doped by silicon nitride) is hydrophilic and negatively charged. Dual Polarization Interferometry. The growth of the DNA film was followed using a dual polarization interferometer (DPI, AnaLight Bio200, Farfield Scientific Ltd., Cheshire, U.K.). A detailed explanation on the instrumentation and technique can be found in the literature.26-31 (Brief details are given in the Supporting Information.) The obtained effective thickness (Teff) and effective refractive index (neff) can be subsequently converted to the mass per area (m) using eq 1. The refractive index increment (dn/dc) value of the oligonucleotide solutions and PEI used in the calculations was 0.175 cm3 g-1,33 because the dn/dc of PEI is almost identical to that of DNA.24 The refractive index of SSC buffer (ns) of 1.339 was obtained using the DPI. It was assumed that the refractive index increment does not change with the sequence and length of the oligonucleotides. LbL Assembly. To obtain the film structures shown in Figure 1, the following procedures were performed. A layer of PEI (50 µL of the PEI solution injected at a flow rate of 5 µL min-1) was first deposited to render the sensing waveguide surface positively charged. After 10 min incubation, the layer was washed with 160 µL of SSC buffer (flow rate of 20 µL min-1

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for 8 min). The surface was primed by depositing either a layer of T30 or X′ (100 µL of T30 or X′ solution injected at a flow rate of 5 µL min-1) via electrostatics onto the PEI layer. To assemble the DNA film, 100 µL of DNA solution was injected at a flow rate of 5 µL min-1, allowing an incubation time of 20 min. This was followed by washing in 160 µL of SSC buffer (flow rate of 20 µL min-1 for 8 min) to remove any unhybridized DNA. To assemble the homopolymeric diblock films, AxGx and TxCx were alternately deposited onto the T30 primer layer. Similarly, the reverse diblock film was assembled by sequentially depositing A15G15 and C15T15 onto the T30 primer layer. The random diblock film was assembled by depositing XY and X′Y′ on the X′ primer layer. A 5-bilayer film was assembled for the 30-mer diblock, reverse diblock, and random diblock systems, and a 4-bilayer film was assembled for the 40-mer and 60-mer diblock systems. The temperature of the solutions and the chip was maintained at 24 ( 0.005 °C throughout the measurements. Stability Experiments. To assess the salt stability of the films, the DNA films were exposed to a series of solutions with decreasing ionic strength. The DNA film (50 µL of NaCl solution injected at the flow rate of 5 µL min-1) was treated with the salt solutions for 10 min, followed by rinsing in SSC buffer (20 µL min-1 for 10 min) to compare the changes in the film before and after salt treatment. Results and Discussion Film Buildup Using Diblock, Random Diblock, and Reverse Diblock Pairs. The films listed in Table 1 have been studied using quartz crystal microgravimetry (QCM) in our previous studies.15,21,23,24 As mentioned above, a decrease in the resonant frequency of the QCM sensor (piezoelectric oscillator) corresponds to an increase in mass adsorbed on the sensor. The results showed that the decrease in frequency for the diblock (30-mer) film was more than double that obtained for the reverse diblock or random diblock films (both 30-mers) with the same number of layers. This suggests that the film formed by the diblock (30-mer) pair is thicker and consists of a larger amount of oligonucleotides. Nonetheless, an equally valid hypothesis could be that the diblock films are more swollen and contain more water rather than more DNA. To understand the sequence dependency on layer growth and the thickness and mass of the diblock, the random diblock and reverse films were investigated using DPI, which allows determination of the mass of the film without associated water. Figure 2 shows the typical temporal evaluation of the thickness (Teff) and the mass per area (m) (see eq 1) of the 30mer diblock (a), random diblock (30-mer) (b), and reverse diblock (30-mer) (c) films. When the PEI solution was first introduced over the chip, the thickness and mass values sharply increased before decreasing and stabilizing. When the SSC buffer was subsequently reintroduced over the chip, the thickness and mass of the PEI layer increased and stabilized at 3 nm and 2 ng mm-2, respectively, after 10 min. The PEI solution (1 mg mL-1 in 0.5 M NaCl solution) has a different composition compared to SSC buffer (50 mM citrate, 0.5 M NaCl), suggesting that there is a significant difference in the refractive index (RI). This RI difference results in invalid values of the thickness and mass while the PEI solution remained over the chip. There was no immediate significant change in the thickness when the T30 layer was first introduced over the chip (Figure 2a and 2c). However, after 20 min incubation followed by removal of the excess oligonucleotides, the film thickness and

Figure 2. Temporal evolution of thickness (solid lines) and mass per area (red dot-dash lines) of the multilayer buildup of the (a) diblock, (b) random diblock, and (c) reverse diblock films. Labeled bars indicate the injection of the oligonucleotides, and the unlabeled short arrows represent the injection of the SSC buffer rinse throughout the experiment.

mass increased by a very small amount (ca. 0.2 nm and 0.1 ng mm-2). This suggests that the T30 electrostatically adsorbs flat via the negatively charged phosphate backbone onto the PEI layer, resulting in a very thin layer as illustrated in Figure 1. The small layer growth of T30 is consistent with our previous QCM investigations.15 In contrast to the adsorption of PEI and T30, when A15G15 was introduced over the chip a large increase in thickness and mass was observed (Figure 2a). There was no loss of material after the SCC buffer wash, and the thickness and mass of the A15G15 layer were measured to be 4 nm and 1.6 ng mm-2, respectively. The T15C15 layer also followed a similar growth profile as the A15G15 layer (thickness of 4 nm and mass of 1.5 ng mm-2). Similar deposition profiles were observed for the following four bilayers. However, less DNA was deposited after the first bilayer, and the thickness and mass decreased to 2.4 nm and 0.6 ng mm-2 per layer (average values obtained from the second to the fifth bilayers), respectively. Figure 2b shows the layer growth of the random diblock film. In contrast to the regular buildup of the diblock (30-mer) pair at every layer, the layer growth was dependent on the number of layers deposited. Similar growth to the diblock (30-mer) film was initially observed up to the second layer (i.e., X′) due to the same electrostatic interactions involved in the deposition of the PEI and X′ onto the waveguide and PEI layer, respectively. X′ and T30 are expected to adsorb similarly via electrostatic interactions. When XY was first introduced over the chip, the increase in layer thickness and mass was similar to that of the diblock (30-mer) film (increased by 2.4 nm and 0.9 ng mm-2). However, a decrease in the thickness and mass

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was observed upon washing into SSC buffer (decreased by 0.8 nm and 0.4 ng mm-2), and thus, the overall increase in the layer thickness and mass was only 1.6 nm and 0.5 ng mm-2, respectively. This small thickness increment suggests that the hybridization rate of the XY to X′ is smaller than that of the A15G15 to T30, i.e., a lower affinity binding occurs in the former case. Similar adsorption behavior was also observed when X′Y′ was introduced over the chip where introduction of the X′Y′ solution resulted in an immediate increase in the thickness and mass of the film, followed by a small decrease in the thickness and mass. The overall increase in layer thickness was higher than that obtained during the first hybridization step (X′ and XY), and the obtained thickness and mass of the X′Y′ layer were 4.5 nm and 0.5 ng mm-2, respectively. With each subsequent layer deposition, a decrease in the thickness for each new layer was observed. After the sixth hybridized layer (X′Y′), there was no significant increase in the overall thickness and mass of the film. However, slight changes in the thickness and mass of the film during the hybridization of these layers results in a complex adsorption profile, which suggests that the doublestranded oligonucleotides are constantly rearranging. This could result in the swelling and shrinkage of the film during the entire adsorption process. The growth in the thickness and mass of the reverse diblock film is shown in Figure 2c. The reverse diblock pair showed a different layer growth to that observed in the diblock (30-mer) and random diblock film. The layer growth up to the fourth injection (PEI/T30/A15G15/C15T15) was identical to that observed in the diblock (30-mer) pair (Figure 2a). Beyond the fourth injection, the characteristic layer growth of the reverse diblock pair was distinct. A decrease in the thickness (maximum 0.5 nm) and mass (maximum 0.2 ng mm-2) was observed for every A15G15 hybridization step. Conversely, there was an increase in layer thickness and mass for every C15T15 hybridization step. The increase in thickness was as large as that observed in the diblock (30-mer) pair and was ca. 1 nm. Above the sixth layer, the increase in thickness for every subsequent C15T15 layer was less than the previous C15T15 layer. The thickness (Teff), mass (m, see eq 1), and density (m/Teff) of the films as a function of layer number are summarized in Figure 3. The values shown were taken after the film was rinsed in SSC buffer. Figure 3a shows the layer growth of the diblock (30-mer) film. Similarly, as observed in Figure 2a, there was little growth in thickness and mass for the T30 (as would be anticipated for electrostatic adsorption of a single layer of DNA flat onto the chip surface). The growth in thickness and mass increased almost linearly with layer number after the T30 layer and were 27.5 nm and 8.11 ng mm-2, respectively, after the deposition of 5 bilayers of A15G15 and T15C15. The average thickness and mass increments of the A15G15 and T15C15 layers were 2.6 nm and 0.74 ng mm-2 and 2.9 nm and 0.88 ng mm-2, respectively. Above the second layer, the density of the film decreased exponentially, indicating that the high density of the PEI layer gradually decreased by hybridization of the less dense DNA layers. The frequency change obtained by previous QCM measurements also exhibited a linear trend in the layer buildup,15,21 indicating that there is qualitative agreement between the DPI and QCM data. Figure 3b shows the layer growth using the random diblock pair. In contrast to the diblock (30-mer) film, the thickness and mass growth saturated around the seventh layer and the density was much lower compared to that of the diblock (30-mer) film. The densities of the diblock (30-mer) and random films after deposition of the 12th layer were 0.33 and 0.24 g cm-3,

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Figure 3. Film thickness, mass per area, and density as a function of layer number. Note that the values of the mass were multiplied by two. Data compiled for (a) the diblock (30-mer) film. The first and second layers are formed with PEI and T30, respectively, followed by the sequential hybridization of the A15G15 and T15C15. (b) Random diblock film. The first and second layers are formed with PEI and X′, respectively, followed by the sequential hybridization of the XY and X′Y′. (c) Reverse diblock film. The first and second layers are formed with PEI and T30, respectively, followed by the sequential hybridization of the A15G15 and C15T15.

respectively. As illustrated in Figure 1a, a similar film structure was expected in both the diblock (30-mer) and random diblock films. However, our results showed that the film growth was significantly less in the latter, indicating that the hybridization rate in the random diblock film is much lower than when compared to the diblock (30-mer) film. Earlier QCM work also showed that the decrease in resonant frequency of the random

DNA Multilayered Assemblies diblock film was less than one-half of that observed in the diblock (30-mer) film.21 However, the trend of the frequency change versus the layer number did not show saturation in growth as observed by the DPI (Figure 3b), indicating that DPI discloses a new aspect of layer growth for the DNA films. The reverse diblock film growth is shown in Figure 3c. As observed in Figure 2c, the thickness and mass growth did not increase past the ninth layer. There was also a fluctuating trend which appeared to be dependent on the depositing (A15G15 or C15T15) layer. The A15G15 layer was always associated with a decrease in thickness and mass. A similar trend was seen in QCM data,22 which showed a large frequency decrease with the adsorption of each layer, but when the A15G15 layers were adsorbed after the initial frequency decrease the frequency gradually increased (however the overall frequency change for each layer was negative). This suggests separation of the hybridized oligonucleotides on the film may be induced by the introduction of A15G15. In contrast, the density exhibited no such oscillation but rather decreased monotonically with increasing the number of layers. The density was higher than that of the diblock (30-mer) and the random diblock film, and the density of the reverse diblock film after deposition of the 12th layer was 0.44 g cm-3. This suggests that the oligonucleotide chains fold back and/or pack horizontally down on the surface (Figure 1b), resulting in a denser structure compared to the diblock film where the double-stranded chains are expected to extend vertically from the substrate (Figure 1a). Figure 3 shows that film structures with varying characteristics can be obtained by simply using pairs of diblock oligonucleotides with different sequences to construct the film. We showed that 3 types of film growth, linear (diblock 30-mer), saturated (random diblock), and stepwise (reverse diblock), can be obtained. Later, the mass increase of each film will be numerically analyzed, which will model the DNA layer growth using an effective hybridization rate and an effective dissociation (or separation) rate on each hybridization step. Structural Stability of Diblock, Random Diblock, and Reverse Diblock Films. The SSC buffer used for the film buildup contained a high Na+ concentration of approximately 650 mM. This was to limit the electrostatic repulsion among the oligonucleotides, thus increasing the hybridization rate to form denser DNA films. For DNA films and capsules to have biological relevance, they need to be stable under low salt (i.e., physiological) solutions. Thus, an understanding of the structural stability of the films upon exposure to low NaCl concentration solutions is required. The Na+ concentration of the solutions used in this study ranged from 500 to 0 mM (pure water). The thickness (Teff), mass (m), and density (m/Teff) of the film after treatment to various salt solutions as a function of the Na+ concentration are shown in Figure 4. The three films showed very small changes in their thickness, mass, and density above 100 mM, indicating that the films have a good tolerance to NaCl solutions as low as 100 mM, which is lower than physiological saline (150 mM). At concentrations below 100 mM Na+, the diblock (30-mer) film exhibited a significant decrease in thickness and mass as shown in Figure 4a. After exposure to the 25 mM Na+ solution, 56% of thickness and 46% of mass in the original film was lost. The random diblock and reverse diblock films also exhibited a decrease in thickness and mass below 100 mM Na+ concentration (Figure 4b and 4c), but the overall decrease was very small compared to the diblock (30mer) film. This is because the DNA mass on the PEI layer in the random diblock and reverse diblock films was very small compared to the diblock (30-mer) film: 1.84, 3.75, and 8.24 ng

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Figure 4. Change in thickness, mass, and density of a (a) 5-bilayer diblock (30-mer), (b) 5-bilayer random diblock, and (c) 5-bilayer reverse diblock film upon sequential exposure to saline solution of decreasing Na+ concentration. The 650 mM data point indicates the approximate Na+ concentration of SSC buffer used for film buildup.

mm-2 in the random, reverse, and normal diblock (30-mer) films, respectively. It is well known that the melting temperature (Tm) of the DNA duplex decreases as the salt concentration decreases.34 Correspondingly, a stable duplex can be separated by lowering the salt concentration at a constant temperature.35 There should be a dependence of Tm on salt concentration and hence the film stability on the salt concentration of the solution (Figure 4). In the diblock (30-mer) film, the sequential deposition of the A15G15 and T15C15 layers correspond to the hybridization between the A15 and T15 blocks (A15:T15) and the G15 and C15 blocks (G15:C15), respectively. The assembly sequence and hybridized blocks of each layer are the same in the reverse diblock film (see Table 1 and Figure 1). In the random diblock film, the hybridized blocks in the film are X:X′ and Y:Y′. The depen-

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Figure 5. Calculated melting temperature of the G15:C15, A15:T15, X:X′, and Y:Y′ duplexes at various salt concentrations. The Tm of X:X′ and Y:Y′ are calculated to be the same given the G:C contents in the X and Y are the same.

dency of the salt concentration on Tm was roughly calculated for the 15-base-pair duplexes, A15:T15, G15:C15, X:X′, and Y:Y′. A number of equations for predicting the duplex Tm have been developed for PCR and DNA microarray experiments.36,37 One of the most widely used equations is the salt-dependent formula, shown as follows:38

Tm ) 81.5 +

41(nG + nC) - 600 + 16.6 log([Na+]) nA + nT + nG + nC (2)

where nA, nT, nG, and nC are the number of adenine, thymine, guanine, and cytosine bases, respectively, and [Na+] is the Na+ concentration (M). In all present cases (A15:T15, G15:C15, X:X′, and Y:Y′), the denominator of the second term in eq 2 (nA + nT + nG + nC) is 15, as the number of base pairs is 15. Hence, according to this simple equation, Tm depends on nG + nC in the numerator of the second term and [Na+] of the third term. The calculated Tm values of a duplex under salt concentrations ranging from 0.01 to 0.6 M [Na+] are shown in Figure 5. As the X and Y blocks contain the same number of A, T, G, and C bases (see Table 1), Tm of X:X′ and Y:Y′ at each salt concentration is equal. G:C and A:T base pairs form three and two hydrogen bonds, respectively, and a higher content of G:C base pairs within a duplex results in higher Tm. Thus, the Tm of G15:C15 (15 G:C pairs, nG + nC ) 15) is higher than that of A15:T15 (no G:C pairs, nG + nC ) 0). The X:X′ or Y:Y′ sequences have 7 G:C pairs (nG + nC ) 7) and hence a Tm between G15:C15 and A15:T15. Due to the logarithmic dependence of Tm on [Na+], the Tm appears to decrease significantly below 100 mM of [Na+]. This corresponds well to the decrease in thickness and mass around 100 mM (shown in Figure 4). The calculated Tm of A15:T15 reaches 24 °C, while the Tm of G15:C15 at 100 mM is still higher than 24 °C. The stability measurements, performed at around 24 °C, indicate that the separation of the A15:T15 region in the alternately hybridized A15G15 and T15C15 leads to structural collapse (see Figure 4a) of the entire film. This breakdown mechanism was also expected in the reverse diblock film. However, there was no significant difference in thickness (< 100 mM) due to the very thin DNA layer in the reverse diblock film (see Figure 4c). As the Tm of X:X′ and Y:Y′ are identical, the breakdown of the random diblock film

Figure 6. Thickness, mass per area, and density of the (a) 30-mer, (b) 40-mer, and (c) 60-mer diblock films as a function of layer number. The first and second layers are PEI and T30, respectively, followed by the sequential hybridization of A15G15 and T15C15, A20G20 and T20C20, and A30G30 and T30C30 for the 30-mer, 40-mer, and 60-mer, respectively. Note that the values of the mass were multiplied by two.

is expected to be induced by the separation of both the X:X′ and Y:Y′ regions in the alternately hybridized XY and X′Y′. Film Buildup Using Longer Diblock Pairs. The dependence of oligonucleotide length on layer growth was also investigated using the DPI for the following diblock pairs: 30-mer (A15G15/ T15C15), 40-mer (A20G20/T20G20), and 60-mer (A30G30/T30G30). A 10-layer 30-mer (Figure 6a) and 40-mer (Figure 6b) film has a thickness of 25.4 and 29.1 nm, respectively. This indicates that longer diblock pairs give a thicker film. The 40-mer doublestranded oligonucleotides would be expected to extend away

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from the film surface longer than the 30-mer. As the mass of the 30-mer and 40-mer film was very similar (ca. 9.5-10 ng mm-2 for a 10-layer film), the 40-mer is a less dense film. The thickness and mass growth in both the 30-mer and 40-mer films were linear-like and similar. This resemblance indicates that the layer growth mechanism is the same in both cases. A thinner film was obtained with the 60-mer (Figure 6c), compared to the 30-mer and 40-mer films, and a relative increase in thickness was not observed as expected. Moreover, the trend in mass growth was not linear but rather similar to the trend observed in the reverse diblock film (Figure 3c), i.e., the mass increase on the A30G30 hybridization step is always less than the T30C30 hybridization step. These results indicate that the mechanism of the layer growth is sensitive not only to the base component and sequence but also the length of the diblock oligonucleotides (see Figures 3 and 6). The length-dependent layer growth observed in Figure 6 will also be analyzed using our phenomenological model in the next section. Phenomenological Modeling of Layer Growth The layer growth of the different oligonucleotide systems was numerically reproduced by a phenomenological model that takes into account the effective hybridization rate and effective denaturation rate at each hybridization step. To eliminate the effect of the electrostatically adsorbed precursor, the mass of the PEI and T30 or X′ layers was removed from the total mass of the film (i.e. layers above the second layer in Figure 3). To make the comparison between the experimental and calculated values simpler, the experimental mass of the first diblock layer was normalized to unity and subsequent layers were compared to this (Figure 7). For simplicity, the molecular weights of the A, G, C, and T bases were assumed to be the same; hence, the molecular weight of the oligonucleotide was determined by the number of bases only. Because each system is assembled using pairs of diblocks with the same number of bases, the normalized value of the mass is proportional to the number of oligonucleotide diblocks in the film. It was reported that the A15G15 oligonucleotides assemble into aggregates spontaneously in solutions containing Mg+ and Na+.39,40 Frayed wires of more than nine A15G15 and oligomers of less than seven A15G15 form in the presence of Mg+ and Na+, respectively.39 However, circular dichroism (CD) studies have shown that there are no frayed wires in our solutions. There was no change in the CD spectra of A15G15 in solution with a Na+ concentration of SSC buffer ranging from 0 to 2 M, and the spectrum was different from that of the frayed wires reported elsewhere.39,40 Thus, we do not model the A15G15 hybridization step differently from the other diblocks. Figure 1a shows an ideal scenario where there is 100% hybridization and no denaturation occurring at each hybridization step. In this case, the oligonucleotides extend perpendicular to the surface when the strands are hybridized, and the same number of free binding sites remains for each subsequent layer. Thus, if the hybridization rate is 100% and there is no denaturation and loss of DNA, the amount of DNA should increase linearly in 1 unit steps for each hybridized layer, e.g., 10 steps of hybridization should result in a film of 10 mass units (see Supporting Information, Figures S1 and S2). However, Figure 7 shows that the hybridization for all the films investigated had a film mass of less than 5, indicating that the actual growth mechanism deviates from the ideal case. To reproduce the empirical growth trends, two phenomenological parameters were introduced: the effective hybridization rate (HR) and the effective denaturation rate (DR) at each

Figure 7. Observed and calculated mass (DNA amount) of the (a) diblock (30-mer), (b) random diblock, and (c) reverse diblock film at each hybridization step. The mass growth shown in Figure 3 was normalized to 1 for the hybridization of the first layers on the T30 layer for the diblock and reverse diblock films and the X′ layer for the random diblock film. The calculated values in a and b were obtained by eq 3 and those in c by eq 4.

hybridization step. A HR less than 100% indicates that part of the binding sites remain unhybridized after the hybridization step. However, for simplicity, these remaining binding sites are assumed to be unavailable for subsequent hybridization. This assumption is reasonable as the remaining binding sites are unlikely to be available for subsequent hybridization because of the steric hindrance caused by the surrounding DNA strands. DR indicates the effective loss of DNA from the film that takes place after each hybridization step. This loss is considered to be stimulated by rinsing of the film into the SSC buffer containing no DNA and represents the separation and loss of some double-stranded DNA from the film. Although such a separation would create free binding sites for subsequent DNA hybridization steps, for simplicity, we assume that the subsequently introduced DNA cannot hybridize to these newly created binding sites. The denaturation or loss is considered to bring the system into an equilibrium state with lower free energy. Hence, hybridization on the newly created binding sites is unlikely to occur. Using these two parameters, HR and DR, the amount of DNA deposited after N (AN) is given by the following equation: N-1

AN ) (1 - DR)N-1

∑ HRi-1

for N g 2

(3)

i)1

where N represents the number of hybridization steps and AN is the total mass of DNA in the film, accumulated from the first

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Figure 8. Schematic of the possible hybridization configurations for more than 100% HR on the diblock pair. (a) Top diagram illustrates when two A15G15 strands hybridize to one T15C15 on introduction of A15G15, and the bottom diagram illustrates when two T15C15 strands hybridize to one A15G15 on introduction of T15C15. (b) Triple-stranded DNA structure formed by two polyTs and one polyA. The vertical lines indicate Watson-Crick hydrogen bonds, and the dots indicate Hoogsteen hydrogen bonds. The chain polarity rules of the triplex formations are based on ref 41. Bottom diagram illustrates the triplex formation upon T15C15 injection. The bound T15C15 strands upon injection are labeled with an asterisk, and dash circles highlight the regions of a TAT triad.

hybridization step (N ) 1) to the Nth step. (1 - DR) indicates the remaining rate of the number of the oligonucleotides in the film after the loss at DR (see Supporting Information, Figures S1-S3, for actual dependencies of AN on HR and DR). The experimental data obtained for the diblock (30-mer) and random diblock pairs were fitted using eq 3, and the results are shown in Figure 7a and 7b, respectively, together with the optimized values of HR and DR. There are deviations at N ) 2 and 3, but overall good agreement between experimental and calculated values was obtained in both cases. HR of the diblock (30-mer) pair (Figure 7a) was estimated to be about 200%, suggesting the presence of various multistranded structures. For instance, two T15C15 or A15G15 strands can be hybridized onto the freely extending G15 or T15 region on the surface, respectively. Multiple hybridization is possible due to the homopolymeric structure of the diblock pair as an introduced strand can hybridize anywhere along the complementary strand on the surface. Figure 8a illustrates configurations of two to one hybridization, but it can be more than two to one hybridization. Furthermore, recent work has suggested that the DNA film contains not only DNA duplexes but also triplexes.24 Specifically, the duplexes of the homopyrimidine and homopurine strands formed by Watson-Crick hydrogen bonding are able to bind another homopyrimidine or homopurine strand by Hoogsteen hydrogen bonding to form DNA triplexes.41 It is likely that formation of the base triad of TAT is possible when assembling our films at pH ) 6.5 with no divalent cations,41,42 as shown in Figure 8b. Hence, an HR larger than 100% is not surprising in the presence of these multistranded DNA structures. When HR is larger than 100% and DR is 0%, the DNA film grows exponentially with the number of hybridized steps (see Supporting Information, Figure S1), but this is not observed. To alter the exponential-like growth to linear, we used a DR of ca. 45% to represent the occurrence of a large loss of DNA at each hybridization step (see Supporting Information, Figure S3).

Kato et al.

Figure 9. Series of the A15G15 actions upon A15G15 injection. (i) Hybridization between the C15 block of C15T15 onto the G15 block of A15G15 on the film surface. (ii) Subsequent hybridization of A15G15 onto the C15T15 strands on the surface. (iii) Onset of the ‘peeling-off’ process where the G15 block of the introduced A15G15 strand further hybridizes with the double-stranded C15 block from the previous layer. (iv) Full dimerization between the A15G15 and the C15T15 strands that are easily removed by the rinsing process following layer adsorption.

This large loss suggests that there was separation of the diblock, loss of the branched strands from the multiple hybridization events, and reorganization of triplexes into regular duplexes (through loss of the extraneous single strand) to achieve a more stable structure at each hybridization step. In contrast to the diblock (30-mer) pair, the optimized values of HR and DR are about 85% and 3%, respectively, for the growth of the random diblock film, indicating that the growth is close to the ideal scenario illustrated in Figure 1a (100% HR and 0% DR). However, a lower effective hybridization rate of 85% and nonzero effective denaturation rate of 3% at each hybridization step deviate from the ideal linear growth behavior toward a saturated growth curve (Figure 7b). The obtained value of HR of 85% is reasonable because the random diblock pair has no homopolymeric pyrimidine or purine blocks in the oligonucleotides, which lowers the probability of obtaining multiple hybridization (Figure 8a) or the triplexes (Figure 8b). To explain the stepwise growth obtained by the reverse diblock pair (Figure 7c), another parameter was introduced. Although all hybridization steps of A15G15 except N ) 1 showed decreases in the amount of DNA within the film, the subsequent hybridization steps of C15T15 always showed increases in the amount of DNA within the film. This indicates that the binding sites formed by the separation of DNA from the film when A15G15 is injected are available for hybridization with the subsequent C15T15 strand. To explain this, we consider that, rather than the usual deposition seen in the diblock film when A15G15 is introduced, in this case, the injected A15G15 can strip the C15T15 from the surface of the film, forming a dimer of A15G15 and C15T15, which is washed away during the rinse step. We term this behavior the ‘peeling-off’ phenomenon, as summarized in Figure 9. To take into account this phenomenon, the rate of the ‘peeling-off’ (PR) is added only to the terms representing the hybridization steps of A15G15 in eq 3. The PR is defined as the ratio between the number of the hybridized A15G15 oligonucleotides (Figure 9ii) and the number of the A15G15 oligonucleotides that peeled C15T15 oligonucleotides off (Figure 9iv). As one A15G15 peels off one C15T15 due to dimerization, the rate of the amount of diblocks that come off

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is 2PR, and the rate of the amount remaining on the film is represented as (1 - 2PR). Using the hybridization rate HR, the net gain of DNA at the Nth A15G15 hybridization step is given by (1 - 2PR)HN-1 R , where N is an odd number. Thus, the formula for AN for N g 2 is as follows:

{

N/2-1

AN ) (1 - DR)N-1 (1 - 2PR)

AN ) (1 - DR)

{

N-1

∑

N/2

HR2i +

i)1

∑ HR2j-1 j)1

}

for even N g 2 (N-1)/2

(1 - 2PR)

∑ i)1

(N-1)/2

HR2i

+

∑ j)1

HR2j-1

}

for odd N g 3 (4) The only difference between eqs 3 and 4 is that the latter has an additional term (1 - 2PR)ΣHR2i, i.e., eq 4 is equal to eq 3, when PR ) 0 (see Supporting Information, Figures S4 and S5 for actual dependencies of AN on HR and DR). This is attributed to the phenomenon shown in Figure 9, where the number of the binding sites of the G15 blocks for the next C15T15 hybridization step does not change whether the A15G15 peels off the C15T15 or not (compare the number of G15 blocks on the surface in Figure 9ii as an example when PR ) 0% and Figure 9iv as an example for PR ) 100%). It should be noted that this modeling requires PR e 100%; otherwise, one A15G15 peels off more than one C15T15. However, this restriction (PR e 100%) holds only when one A15G15 hybridizes to the T15 block, as illustrated in Figure 9ii, and does not hold when multiple hybridization of A15G15 (Figure 10a (top) and 10b) takes place. A general (tighter) restriction for the PR value cannot be determined because the fraction of the one to one hybridization to the multiple hybridizations in the HR value and the denaturing process cannot be defined or evaluated precisely. The growth of the DNA film using the reverse diblock pair was fitted using eq 4, and both experimental and calculated plots are shown in Figure 7c. There was good agreement between the experimental and calculated data. The optimized values of HR, DR, and PR are 166%, 39%, and 12%, respectively. The HR is larger than the 100% obtained in the diblock (30-mer) pair. A HR greater than 100% suggests that the reverse diblock film also contains multiple hybridization and the formation of triplexes, as shown in Figure 10a and 10b, respectively. Due to the large HR, the reorganization, such as elimination of the weakly bound DNA and rehybridization to achieve a more stable structure, is expected to occur and a large loss of DR ) 39% was obtained. Such a large DR was also obtained on the diblock (30-mer) pair (see Figure 7a). Twelve percent (PR value) of the A15G15 which hybridized at the C15T15 terminated surface forms dimers with the C15T15 (Figure 9iv and Figure 11a) and peel off the previously hybridized C15T15 film. This PR value of 12% and the large DR value of 39% support the experimentally observed stepwise growth on the reverse diblock pair. The driving force of the dimerization is considered to be the free energy gain, i.e., the dimerization releases the strain on the DNA stands in the film (such as the hairpin structure shown in Figure 1b) and increases the degree of freedom of the system by releasing the bound DNA stands from the film. However, the reason why the dimerization induced ‘peeling-off’ phenomenon occurs only on the A15G15 injection is not clear. This is the subject of ongoing investigations. It is also pertinent to address the question of why the dimerization-induced ‘peeling-off’ phenomenon was not ob-

Figure 10. Possible hybridization configurations giving more than 100% HR on the reverse diblock pair. (a) Top scheme represents the situations where two A15G15 strands hybridize to one C15T15 upon A15G15 injection, and the bottom scheme represents the situation where two C15T15 strands hybridize to one A15G15 upon C15T15 injection. (b) Scheme represents the triplex formation upon C15T15 injection. The bound C15T15 strands are labeled with an asterisk, and dashed circles highlight the regions of a TAT triad.

Figure 11. Dimer structures of the (a) reverse diblock pair, (b) diblock (30-mer) pair, and (c) diblock (60-mer) pair.

served on the diblock (30-mer) pair because the diblock (30mer) pair is also able to form a dimer as shown in Figure 11b. To address this, the PR value is evaluated for the diblock pairs of 30-mer, 40-mer, and 60-mer using eq 4. The mass growth of the 40-mer and 60-mer films up to the eighth hybridization step was normalized using the same procedure as used for Figure 7. For the 30-mer film, the same data up to the eighth hybridization step in Figure 7a are used. The normalized data along with the fitted results using eq 4 are plotted in Figure 12. The optimized HR and DR values in Figure 12a for the 30-mer are almost the same compared those in Figure 7a, and a PR value of 2% is obtained for the 30-mer. This negligible PR value indicates that the ‘peeling-off’ phenomenon did not take place for the 30-mer pair. Thus, the optimized values reasonably correlate with those obtained by eq 3. When we compared the three different length diblock pairs, a specific length dependence of the optimized HR and DR values could not be recognized, and the HR and DR are around 200% and 45%, respectively. This indicates that the homopolymeric nature of the diblock pair, which leads to the formation of multistranded structures (200% HR, Figure 8) followed by a reorganization of the film to a more stable state through the denaturation of DNA (45% DR), does not change in the 30-mer to 60-mer range. In contrast to the HR and DR values, the optimized value of PR shows a dependence on the DNA length. The PR value increases with strand length. The 40-mer has a small PR (3.7%) value, indicating small losses of DNA due to the ‘peeling-off’ phenomenon, and corresponds well to the linear trend obtained experimentally, as shown in Figure 12b. A stepwise growth is observed as the PR value increases, as seen in Figures 7c and

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Figure 12. Observed and calculated mass (DNA amount) of the (a) 30-mer, (b) 40-mer, and (c) 60-mer film at each hybridization step. The mass growth shown in Figure 6 was normalized to the experimentally obtained DNA amounts. The calculated values are obtained from eq 4. (a) Film growth obtained by the diblock (30-mer) pair of A15G15 and T15C15. The odd and even numbers correspond to the hybridization steps of the A15G15 and T15C15, respectively. (b) Film growth obtained by the diblock (40-mer) pair of A20G20 and T20C20. The odd and even numbers correspond to the hybridization steps of the A20G20 and T20C20, respectively. (c) Film growth obtained by the diblock (60-mer) pair of A30G30 and T30C30. The odd and even numbers correspond to the hybridization steps of the A30G30 and T30C30, respectively.

12c for the reverse diblock pair (11.6%) and the 60-mer (9.3%), respectively, indicating that the dimerization-induced ‘peelingoff’ phenomenon takes place. When the dimer structures of A15T15:C15T15 (reverse diblock pair) and A15T15:T15C15 (30-mer pair) are compared (see Figure 11), it is clear that the latter is more unstable than the former, indicating the dimerization of the 30-mer needs more energy than that of the reverse diblock pair. Thus, the energy cost for dimerization overcomes the energy gain for stabilization of the system by the ‘peeling-off’ phenomenon, leading to suppression of the dimerization-induced ‘peeling-off’ phenomenon in the 30-mer film. However, the longer chain of the 60-mer decreases the energy cost for dimerization due to a longer radius of curvature of the loop dimer (lower strain), resulting in the dimerization-induced ‘peeling-off’ phenomenon as in the reverse diblock pair. This inhibits the growth of a thicker film using longer diblock oligonucleotides. Conclusion The thickness and mass of the data obtained using DPI show that the layer growth is highly dependent on the sequence of

Kato et al. oligonucleotides used in the assembly. The thicknesses of 5-bilayer diblock (30-mer), random diblock, and reverse diblock films were 27.5, 11.5, and 9.6 nm, respectively. The thickness and mass growth trends were close to linear for the diblock film, saturated for the random diblock film, and stepwise for the reverse diblock film. Suppression of the growth in the reverse diblock film was observed only on the A15G15 injection step. DNA LbL films were treated to saline solutions of decreasing concentrations. The films became unstable and disassembled at NaCl concentrations lower than 100 mM. This corresponds well to the calculated melting temperature, where a steep decrease in the melting temperature was obtained on decreasing the salt concentration lower than 100 mM. To interpret the growth mechanism, phenomenological modeling of the sequential hybridization showed a good correlation between the proposed formulas consisting of two or three parameters and the experimental data. The effective hybridization rate (HR) and the effective denaturation (or separation) rate (DR) on each hybridization step were introduced to explain the linear and saturated trends of growth observed in the diblock (30-mer) film and the random diblock film, respectively. The HR and DR of the diblock film were estimated to be about 195% and 43%, respectively, indicating that the effect of a high hybridization rate and large loss of DNA balance out, and a linear-like growth trend is observed. The HR of more than 100% was reflective of multistrand structures between A15G15 and T15C15 due to their homopolymeric nature, and the large DR mainly represented the separation of weakly bound DNA in the multi-DNA strands to stabilize the system. The HR and DR of the random diblock film were estimated to be about 83% and 3%, respectively. This implied that as the number of hybridization steps increased, the growth rate was suppressed by repeatedly multiplying the parameters less than 1, resulting in a saturated trend. To explain the stepwise growth observed in the reverse diblock film, an additional parameter, the rate of ‘peeling-off’ (PR), was introduced only to the terms representing the mass increment at the A15G15 hybridization step in the formula. The PR is the ratio of the A15G15 that separates the C15T15 from the assembled film, and the separation was considered to be driven by the stabilization due to dimerization of A15G15 and C15T15 in a linear form. The observed stepwise growth was successfully modeled using the HR, DR, and PR of 166%, 39%, and 12%, respectively. The 30-mer diblock film was also compared to films assembled using longer diblock pairs (40-mer and 60-mer). Although the layers in the A15G15/T15C15 film showed uniform increases at each layer, the layer growth on the A30G30 hybridization step was less than that on the T30C30 hybridization step in the A30G30/T30C30 film. This fluctuation in layer growth was reasonably explained by the PR of 9%. In contrast to the linear configuration of the A15G15:C15T15 dimer, the structure of the AxGx:TxCx dimer is a closed looped configuration. Accordingly, larger x diblock pairs form dimers more easily. This corresponds to a larger stabilization effect due to the dimerization, resulting in the fluctuating film growth in the A30G30 and T30C30 deposition steps. The present analysis shows that the DR and PR values have to be decreased to obtain thicker, denser, and more stable DNA LbL films. To do so, ligation and/or cross-linking of DNA after each hybridization step can be implemented. Modeling the assembly of LbL DNA films and gaining a greater understanding of the factors affecting the assembly of the films provides important insights into engineering stable and well-defined DNA films that may find application in diagnostics and drug delivery.

DNA Multilayered Assemblies Acknowledgment. This work was supported by the Australian Research Council under the Federation Fellowship and Discovery Project schemes and by the Victorian State Government under the STI Initiative. N.K. acknowledges the Support Program for the Internationalization of University Education provided for Meiji University by the Japanese Ministry of Education, Culture, Sports, Science and Technology. We also thank Farfield Scientific Ltd. for support with the DPI measurements. Supporting Information Available: Brief description of DPI is given in section 1; in section 2, calculated values of the DNA amount (AN) using eqs 3 and 4 as a function of number of hybridization steps (N) are given; dependence of AN on HR at DR ) 0% calculated using eq 3; AN on DR at HR ) 100% calculated using eq 3; AN on DR at HR ) 200% calculated using eq 3; AN on PR at HR ) 100% and DR ) 0% calculated using eq 4; and AN on PR at HR ) 200% and DR ) 45% calculated using eq 4. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) LaBean, T. H.; Li, H. Nano Today 2007, 2, 26. (2) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Nature 1998, 349, 539. (3) Park, S. H.; Finkelstein, G.; LaBean, T. H. J. Am. Chem. Soc. 2008, 130, 40. (4) O’Neill, P.; Rothemund, P. W. K.; Kumer, A.; Fygenson, D. K. Nano Lett. 2006, 6, 1379. (5) Matsuura, K.; Masumoto, K.; Igami, Y.; Fujioka, T.; Kimizuka, N. Biomacromolecules 2007, 8, 2726. (6) Um, S. H.; Lee, J. B.; Park, N.; Kwon, S. Y.; Umbach, C. C.; Luo, C. C. D. Nat. Mater. 2006, 5, 797. (7) Lee, C. K.; Shin, S. R.; Lee, S. H.; Jeon, J.-H.; So, I.; Kang, T. M.; Kim, S. I.; Mun, J. Y.; Han, S.-S.; Spinks, G. M.; Wallace, G. G.; Kim, S. J. Angew. Chem., Int. Ed. 2008, 47, 2470. (8) Goodman, R. P.; Schaap, I. A.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Science 2005, 310, 1661. (9) He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. Nature 2008, 452, 198. (10) Simmel, F. C. Nanomedicine 2007, 2, 817. (11) Liedl, T.; Dietz, H.; Yurke, B.; Simmel, F. Small 2007, 3, 1688. (12) Lee, J.; Cuddihy, M. J.; Kotov, N. A. Tissue Eng. B 2007, 14, 61. (13) Chan, G.; Mooney, D. J. Trends Biotechnol. 2008, 26, 382. (14) Decher, G. Science 1997, 277, 1232.

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