1.4 Hydrogels
Hydrogels are cross-linked hydrophilic polymer networks49-51 that have attracted many fields of recent research, including the making of sensors.52-57 Hydrogels also possess many physical properties.56,58-65 The majority of their gel volume is water, thus making hydrogels highly porous, and to have a large surface area. Hydrogels swelling when immersed in water and can absorb as well as retain water in large amounts. This can reach up to several hundred folds of dry gel mass.66 Many environmental parameters, including temperature, pH, ionic strength, solvent composition, and light and electric field, affect gel volume.67 This is why many of these stimuli-responsive smart hydrogels have been used for various applications, including controlled drug release systems, sensors, cell culture substances, and flow control.49,50,68-70 However, the choice of input stimuli for the design of responsive hydrogels is quite limited.66
Moreover, hydrogels are ideal for immobilization of biomolecules as their volume is mainly composed of water and biomolecules. The latter can maintain their native structure and function.71-74 Hydrogels are also ideal for optical sensor immobilization because they have a a strong biocompatibility, a large sensor loading capacity, and low optical background.75 Importantly, hydrogel backbone property can be controlled by mixing different monomers.75 To this point, no design for hydrogel-based optical sensors has been based on a tuning gel backbone charge.
1.4.1 DNA-Functionalized Hydrogels
In the past 15 years, a number of DNA-functionalized hydrogels have been used in making biosensors,54,57,66,75,76 controlled release systems,55,77-79 biocompatible matrix,80 and stimuli-responsive materials.53,75,81-83. Most of the research has been done on hydrogel-based sensors, focusing on the gel’s physical properties such as swelling or phase transition through making stimuli-responsive smart gels,84-86 and few colorimetric sensors have been demonstrated.54,87-89 Acrydite-modified DNA can be conveniently linked to hydrogel background through co-polymerization as shown in Figure (9).66
Figure (9): The two types of conjugate chemistry for covalent attaching of DNA to the hydrogel. (A) Amino-modified DNA reacts with a polymer containing a succinimidyl ester on the backbone. (B) Copolymerization of acrydite-modified DNA into polyacrylamide.
Figure (10): Three stimuli-responsive gel-to-sol transition in DNA-functionalized hydrogels. (A) Transition formed by heating. (B) Transition formed by the addition of the c-DNA. (C) Transition formed by adenosine where the DNA includes the aptamer sequence. (D) A photograph of the adenosine responsive hydrogel with entrapped AuNPs in the presence of adenosine (right tube) or in the absence of adenosine (left tube). (E) Addition of adenosine formed the gel-to-sol transition in ~15 min.53
Figure (10) shows that three stimuli-responsive gel-to-sol transitions in DNA-functionalized hydrogels are formed from a solution, and can be made into various shapes and sizes.66 Upon gel formation, the viscosity can be changed and be easily observed. Crosslinkers such as N, N’-methylene-bis-acrylamide are typically used to form acrylamide-based gels. Gels cannot be easily returned to the sol state to produce a permanent 3-D polymer network. However, if DNA hybridization is used to crosslink polymer chains instead of bis-acylamide, reversible responsive gels can be prepared.
The first study in this field was described by Nagahara and Matsuda in the year 1996.90 A block copolymer using N, N’-dimethylacetamide and N-acryloxysuccinimide was prepared, and amino-modified DNAs reacted with the latter monomer. See Figure (10). Two crosslink polymer chains were prepared by using various DNA sequences. As a result, a hydrogel was formed after adding a linker DNA to assemble the two DNA strands as shown in Figure (10A). Because of DNA melting transition, the gels reversibly transitioned into a sol state when the sample was heated. In principle, this method can be utilized to detect DNA. The work, however, did not have an attractive method of DNA detection whereby, the amount of linker DNA needed to form the sol-gel transition is at the mM level. DNA-linked gold nanoparticles and DNA-functionalized gels were also demonstrated in the same year, after experiments. 16,91 Since then, the optical property of gold nanoparticles has received more attention because of the development of ultrasensitive colorimetric DNA detection.5,41,47,92
The mechanical property of DNA-crosslinked hydrogels was then reported in the year 2004.93,94 the authors employed a system, shown in Figure (10B), where the linker DNA includes an overhang that appears in red. In the presence of the complementary DNA (c-DNA), the linker can easily be removed by this overhang. There was no need for heating in this reaction. Moreover, the viscosity of the system was measured as a function of both crosslinking density and temperature. Therefore, gels were formed only when the crosslink concentration was higher than 23%. The viscosity decreased due to the increased temperature. Adding more c-DNA to the gel solution produced a faster leakage of entrapped fluorophores. This system demonstrated that DNA crosslinked gels can be responsive to multiple stimuli, not only the temperature but also by c-DNA.
In the year 2007, fluorescent quantum dots (QDs) as probes and the diffusion of QDs inside the gels, were used by Simmel and his co-workers by monitoring the fluorescence microscopy and fluorescence correlation spectroscopy.95 The kinetics of their method using the gel-to-sol transition and the c-DNA was extremely slow. Moreover, many hours were required to observe improved diffusion kinetics of the entrapped QDs. At the end of their paper, they suggested that using aptamers in such hydrogels might be more useful for controlled drug release applications.
Since then, the preparation of stimuli-responsive inorganic materials using aptamers has been reported using gold nanoparticles,96-98 QDs,66 and magnetic nanoparticles.99 The first work done on a gel-to sol transition using aptamer-assembled hydrogels was reported by Tan and his co-workers.53 In Figure (10C), the aptamer for adenosine is represented by the green strand. In the absence of adenosine, the aptamer DNA behaved in the same manner as a normal DNA attached to the hydrogels, while adding more adenosine-induced aptamer folding to cleave the crosslink. Even though adenosine is a small molecule, its diffusion into the monolithic gel was faster than that of c-DNA, as shown in Figure (10B). In addition, an intense background color was observed at high concentrations of AuNPs, and in the presence of adenosine, due to gel dissolution, as shown in Figure (10D). The result makes the detection of small concentration of AuNPs very difficult. Therefore, this type of detection method is not analytically considered to be sensitive.
3.1 Effect of DNA concentration
The design of the proposed system is shown in Figure (16A). A 5’-acrydite modified DNA was co-polymerized into a 70 µL polyacrylamide monolithic hydrogel. The prepared gel was then mixed with AuNPs functionalized with a 3’-thiol modified DNA in the presence of linker DNA (as indicated in blue). The output gel appeared as a red color, and the reason is due to the high extinction coefficient of AuNPs. In order to probe the binding between the AuNPs and the gel, DNA thermal denaturation was used, whereas AuNPs dissociated from the gel surface at high-temperature levels. More information can be concluded from such experiments, which relates to the polyvalent binding.21 It is expected that one will observe high melting temperatures as well as sharp melting transition of more DNA linkages between AuNP and gel.113 The DNA sequences used in this work are shown in Figure (16B).
Since hydrogels were prepared from a solution, a large degree of flexibility was found in terms of gel formulation. The effect of the acrydite-modified DNA concentration was studied first. The polyvalent binding of AuNP was directly affected by the surface DNA density, which was related to the DNA concentration used for making the gel, and this was the basis for any subsequent studies.
Figure (16): (A) Schematic presentation of DNA-directed assembly of AuNPs on a hydrogel surface. This directed assembly process is reversible and can be controlled by temperature. (B) DNA linkages and sequences utilized in this work.
Four kinds of gels were prepared with DNA concentration, and they were 1, 2, 5 and 10 µM. It was found out that the amount of associated AuNPs increased with higher DNA concentration (refer to Figure 17A). To be able to obtain melting curves, the gels were loaded into a quarts micro-cuvette and immersed in 400 µL of buffer (50 mM, NaCI, 20 mM HEPES, PH 7.6). The buffer extinction at the 520 nm plasmon peak was monitored.76 The melting curves of these samples are shown in Figure (17C); with increasing temperature, AuNPs gradually melted into the buffer to increase the 520 nm peaks. The melting method occurred in ranges of temperature at ~ 10 °C. This sharpness of melting transition was similar to the reported one concerning AuNPs melted from glass surface.21 Free DNA melting usually occurs over a range of >20° C, while the AuNP aggregates’ melt occurs within 6° C.5,21 Therefore, the surface of the resulted gel reacted in a similar manner as the glass surface.
Figure (17): Effect of DNA concentration on hydrogel (4% gels). (A) A photograph of the four hydrogels linked with AuNPs. The amount of attached AuNPs increased with increasing DNA concentration. (B) Quantification of AuNP on the gel after complete thermal dissociation of AuNPs. (C) The melting curves of the four hydrogels. (D) The melting temperature as a function of DNA concentration.
The quantification method of the amount attached to AuNPs was made by measuring the final extinction and all the AuNPs were thermally desorbed. As it is shown on Figure (17B), while the attached AuNPs increased with increasing DNA concentration, the relationship was not linear. Attempt to increase the DNA by 10-fold, only resulted in 1.25 fold increase of AuNPs. Therefore, there were likely to be more DNA linkages to AuNPs between the gel sample with 10 µM DNA than 1 µM one. The melting temperature (Tm) increased with increasing DNA density on hydrogel (Figure 17C), and it supports the presence of more DNA linkages. The change of Tm was about 4° C for 10-fold change of DNA density (Figure 17D). All melting curves have shown similar sharpness. Therefore, the effect of the polyvalent binding was still present even for the sample of 1 µM DNA gel. Even though it was recently reported that two DNA linkages can still show the cooperative melting and polyvalent binding effect, it was not surprising to note that the melting sharpness was relatively independent of the gel surface DNA density.114
The highest DNA concentration that has been tested was only 10 µM. Considering a random distribution of the DNA in the gel matrix, DNA-to-DNA distance was estimated at 22.3 nm for 4% gel, with 1 µ DNA, and the estimated distance was ~ 48 nm. Therefore, if the surface was not porous, it would have been quite difficult for a 13 nm AuNP to bind to several DNAs. This experiment suggested that porosity was extremely vital to allow a 3D polyvalent binding of AuNPs in the hydrogel matrix.
3.2 Effect of hydrogel percentage
Hydrogels are porous networks of cross-linked hydrophilic polymers. Hydrogels have a much higher porosity as the water content can routinely reach > 90%, as compared with many other surfaces. Hydrogel porosity is easily made tunable, by changing the gel percentage, which is likely to affect the polyvalent binding interaction between the gel and the AuNPs. Four kinds of gels were prepared containing 4 to 16% of the 29:1 acrylamide/ bisacrylamide solution in the presence of 10 µM acrydite-DNA. It has been observed that the number of attached AuNPs decreased significantly with increasing gel percentage (Figure 18A) and the decrease roughly followed a linear relationship (Figure 18B). Since all gels had similar bulk of DNA concentration; it has been considered that the main effect is gel pore size, surface area, and surface DNA density. The higher percentage gels were less porous with a smaller surface area, and this accommodated less AuNPs. In a 4% gel, the surface DNA’s was estimated at 22nm, and therefore, it was difficult for any AuNP to bind onto DNA on a non-porous surface. Thus, when the pores are available, DNA’s become accessible. Reduced Tm was also observed with increasing gel percentage (Figure 18C, D), that also suggests the decreased number of DNA linkages.
Figure (18): Effect of gel percentage (acrydite-modified DNA concentration = 10µM). (A) A photograph of the four hydrogels linked with AuNPs. The amount of attached AuNPs decreased with increasing gel percentage. (B) Quantification of AuNP on the gel after complete thermal dissociation of AuNPs. (C) The melting curves of the four hydrogels. (D) The melting temperature as a function of gel percentage.
SEM micrographs of the 4% gel (Figure 21B) were taken and AuNPs appeared to occupy the entire surface. Similarly, the AuNP were not packed on the same plane, but rather, many appeared to be embedded into the gel matrix. This highlighted the importance of gel porosity. The 4% gel with 1 µM DNA and the 16% gel with 10 µM DNA represented two kinds of sub-optimal conditions for AuNP binding. The former possessed large number of sites with the right port size, but only a fraction of those sites had the number of DNA required from the attachment. In the latter case, even though the overall DNA concentration was high, the majority of the DNAs were not accessible for AuNP binding.
3.3 Effect of crosslinker density
Through this study, reducing gel pore size has been done by increasing gel percentages. However, it was difficult to increase pore size by a further reduction in the gel percentages, since it is below 4%, the gel becomes quite soft and difficult to handle. Another method of modulating the gel porosity was by changing the crosslinker concentration. In the previously mentioned experiments, a ratio of 29:1 of acrylamide:bisacrylamide was used. To increase gel pore size, the following was prepared: 19:1, 38:1, 76:1, and 114:1 gels (all at 6%). It has been observed that the gel size was larger with lower crosslinker percentages (Figure 19A, E), suggesting increased pore size. The increased gel size was also quantified through weighing (Figure 19B). As shown in Figure 4F and 4G, the Tm barely changed (within 1 °C) for all the samples, suggesting that all AuNPs had a similar number of linkages with the gels. Figure (19C) demonstrates that the amount of association with the AuNPs increased with reduced crosslinker concentration. This also indicated that the number of AuNP binding sites increased, with lower crosslinker density or larger pore size.
Figure (19): Effect of acrylamide:bisacrylamide ratio (gel percentage = 6%, acrydite DNA concentration = 10 mM). (A) A photograph of the four hydrogels linked with AuNPs. The change of gel size can be observed. (B) The gel mass as a function of crosslinker ratio. (C) Quantification of AuNP on the gel after complete thermal dissociation of AuNPs. (D) Without linker DNA, no AuNP was associated with the gels. (E) A photograph of the gels after the melting experiment; most of the AuNPs dissociated. (F) The melting curves of the four hydrogels. (G) The melting temperature as a function of crosslinker ratio.
Non-specific association of AuNPs has been studied, in the absence of linker DNA. As it appears in Figure (19D), no AuNPs were adsorbed by the gels even for the 114:1 ratio, which had the largest pore size. This experiment confirmed that all the AuNPs in the previous one were adsorbed by the DNA linker, and no non-specific adsorption occurred. The gels were also observed followed by the melting experiment. As it is shown in Figure (19E), the reddish color of the gel was not clearly shown or observed, suggesting that the AuNPs were completely dissociated and little entrapment of AuNPs occurred.
3.4 Effect of AuNP size
To further probe the gel pore size, three AuNP of sizes 13, 30, and 50 nm were tested. The extinction coefficient of AuNPs is shown as a strong function of particle size and it increases by ~ 50-fold from 13 to 50 nm. All gel samples showed a comparable optical density as shown in Figure (20A). Therefore, the surface density of the larger AuNPs must be much lower than that of 13 nm one. The larger AuNPs also showed a slightly higher melting temperature (Figure 20B, C), suggesting that they had more DNA linkages with the gel.
Figure (20): Effect of AuNP size (4% gels). (A) A photograph of the three hydrogels linked with AuNPs. (B) The melting curves of the three hydrogels. (C) The melting temperature as a function of AuNP size.
To further understand the distribution of AuNPs on the gel surface, SEM studies have been applied on the 13 and 50 nm AuNP samples. A thin slice of gel was dried on a conductive silicon wafer and imaged. The surface morphology of a 4% gel is shown in Figure (21A). The white dots in Figure (21B) were the 13 nm AuNPs, they mostly occupied the whole surface area. Quite a large number of AuNPs appeared to be smaller than 13 nm, suggesting that these particles were from the internal pores and part of the particles were covered by the dried gel matrix. The 50 nm AuNPs were well-separated from each other with lower surface density (Figure 21C), consistent with the optical density observation. Almost no AuNP in Figure (21C) appeared to be 50 nm, suggesting that all the particles were partially embedded by the gel matrix during the drying process. The size-dependent experiment further indicated the effect of gel pore on size: a large AuNP would require a large pore size, and a small AuNP can easily stay in both small and large pores.
Figure (21): SEM and SHIM micrographs of dried hydrogel samples. (A) A low magnification SEM image of the dried gel surface. Gel surfaces containing 13 nm AuNPs imaged using SEM (B) and SHIM (C). (D) Gel surfaces containing 50 nm AuNPs imaged using SHIM. Scale bar = 100 nm (A), 200 nm (B, C), and 500 nm (D).
The effect of changing hydrogel percentage of crosslinker concentration on the gel pore size has been well documented. Ugaz and his co-worker made a conclusion based on TEM studies that the pore size was smaller, and with a narrower distribution at higher gel percentage or crosslinker concentration.105 With a 6% polyacrylamide gel, the average pore size was 15 nm and the largest pores can reach up to 30 nm. For 12% gel, however, the average pore size was only 7 nm and the largest pores were about 15 nm. This result was consistent with what has been obtained using nanoparticle probes: the 13 nm AuNPs can be effectively attached to the 4-8% gels, but not to the 16% one. The surface density of 50 nm AuNPs was more than 50-fold lower, where the number of large pores was quite low.
3.5 Effect of gel charge
All the previous experiments were related to probing the effect of gel pore size. Moreover, DNA-functionalized AuNPs are negatively charged. To further understand, the effect of gel charge was studied next. Polyacrylamide gel has no charges, but the charge can be introduced by incorporating cationic (allylamine), or anionic (AMPS) monomers. 75 As a result, the highest AuNP density was still achieved on the neutral gel (Figure 22A). The negatively charged gel demonstrated reduced AuNP density, which might be related to the electrostatic repulsion. There was a large difference in the cationic gel; very few AuNPs were on the gel surface. Thus, this experiment confirmed that the binding was still specific in this case since in the absence of linker DNA, there was no red color observed on the gel even after 2 days incubation (Figure 22B). Furthermore, the negatively charged gel showed a slightly reduced Tm compared to the neutral gel (Figure 22C, D). However, the cationic gel did not show any melting in the tested range of temperature, which might be related to the strong electrostatic attraction, whereas the AuNPs were bound to the gel surface.
Figure (22): Effect of gel charge. (A) A photograph of the three hydrogels linked with AuNPs. Most effective binding of AuNPs is shown by the natural gel, followed by the negative gel. (B) Soaking a cationic gel with DNA-linked AuNPs in the absence of linker DNA (left panel) and after washing (right panel). No adsorbed AuNP was observed. (C) The melting curves of the three hydrogels. No melting transition for the cationic gel was observed (D) The melting temperature as a function of gel charge.
3.6 Binding model
It is known that many of the non-porous surfaces, such as, gold and silica can be used for effective DNA-directed binding of nanoparticles. In such cases, a drop of concentrated DNA solution was applied to a small surface area on the substrate to achieve a high DNA density. It can be conceived that only a tiny spot of the AuNP was attached to the substrate surface (Figure 23A, DNA linkages represented by the blue color). Due to the high DNA density, multivalent binding can be achieved at the spot.21 in the prepared hydrogel, the surface DNA density was very low and therefore, porosity became extremely valuable to use, not only by the surface DNA but also by the interior ones to form 3-D binding pockets. It has been observed that the AuNP binding capacity dropped significantly with reduced pore size and this is schematically shown in Figure (23B, C). The black lines represent the first layers of the hydrogel matrix and the gray lines represent the subsequent layers. The density of larger pores was much smaller, and thus binding to larger AuNP occurred less often (Figure 23D).
Figure (23): Schematic presentation of the binding between DNA-functionalized hydrogels and AuNPs. The DNA linkages are shown as the blue bars and dots. The AuNPs are shown as red dots. The gel matrix is shown in black or gray lines. (A) In a planar surface, polyvalent binding is achieved via a high local DNA concentration in a small contacting area. In a porous hydrogel, polyvalent binding is achieved via formation of 3D binding pocket (B-D). A high percentage gel has a small number of surface binding sites (B) while a low percentage gel is more porous to bind more AuNPs (C). The number of binding sites reduced with increasing AuNP size (D).
In addition, this model explains system dryness. As reported previously, such AuNP attached gels could be completely dried, and after rehydration, the DNA linkages were still maintained and further enhanced. This can be understood by the fact that most of the AuNPs were sitting in the pores and upon rehydration; DNA can be renatured before the AuNPs can leave the pores. Compared to other systems that study porous hydrogels, DNA-functionalized AuNP probes work in the native state of the gel. This system can be used as biosensor and a controlled release system through taking advantage of the molecular recognition function of DNA. Therefore, the information obtained in this study can guide the engineering of such hybrid soft/ non-materials for various applications.
3.7 Summary
In this study, the DNA-functionalized AuNPs has been employed as a probe to study the binding with hydrogel. The main tool of understanding the polyvalent binding was the DNA melting method. Hydrogels are a special substrate with many useful properties for nanoparticle immobilization and the gel porosity is one of the most fundamental features. The porosity effect was tested on AuNP binding by tuning the gel percentage, crosslinker concentration, gel charge, and AuNP size; it was found that the binding site is highly dependent on the gel porosity and the AuNP size. The number of binding sites reduced significantly by reducing the pore size. As it concerns the non-porous substrate, DNA-directed assembly of AuNPs occurs only through the small contacting area. However, in a hydrogel, the formation of a 3D binding pocket becomes extremely vital, thus representing a totally different polyvalent binding mode. As the AuNP-hydrogel system can be used for colorimetric detection of DNA also, as a stimuli-responsive releasing system, it is important to take advantage of the molecular recognition property of DNA. With the fundamental understanding obtained in this study, it is sought that such new hybrid material can be better engineered for different analytical and biomedical applications.
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