1.4 Hydrogels

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1.4 Hydrogels. 2

1.4.1 DNA-Functionalized Hydrogels. 2

3.1 Effect of DNA concentration. 5

3.2 Effect of hydrogel percentage. 8

3.3 Effect of crosslinker density. 9

3.4 Effect of AuNP size. 10

3.5 Effect of gel charge. 12

3.6 Binding model 13

3.7 Summary. 14

1.4 Hydrogels

Hydrogels are crosslinked 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 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 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 transition 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’-dimethylacrylamide 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 showed 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.............

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