Stimuli-Responsive DNA-Based Hydrogels on Surfaces for Switchable Bioelectrocatalysis and Controlled Release of Loads

The assembly of enzyme [glucose oxidase (GOx)]-loaded stimuli-responsive DNA-based hydrogels on electrode surfaces, and the triggered control over the stiffness of the hydrogels, provides a means to switch the bioelectrocatalytic functions of the hydrogels. One system includes the assembly of GOx-loaded, pH-responsive, hydrogel matrices cross-linked by two cooperative nucleic acid motives comprising permanent duplex nucleic acids and “caged” i-motif pH-responsive duplexes. Bioelectrocatalyzed oxidation of glucose leads to the formation of gluconic acid that acidifies the hydrogel resulting in the separation of the i-motif constituents and lowering the hydrogel stiffness. Loading of the hydrogel matrices with insulin results in the potential-triggered, glucose concentration-controlled, switchable release of insulin from the hydrogel-modified electrodes. The switchable bioelectrocatalyzed release of insulin is demonstrated in the presence of ferrocenemethanol as a diffusional electron mediator or by applying an electrically wired integrated matrix that includes ferrocenyl-modified GOx embedded in the hydrogel. The second GOx-loaded, stimuli-responsive, DNA-based hydrogel matrix associated with the electrode includes a polyacrylamide hydrogel cooperatively cross-linked by duplex nucleic acids and “caged” G-quadruplex-responsive duplexes. The hydrogel matrix undergoes K+-ions/crown ether-triggered stiffness changes by the cyclic K+-ion-stimulated formation of G-quadruplexes (lower stiffness) and the crown ether-induced separation of the G-quadruplexes (higher stiffness). The hydrogel matrices demonstrate switchable bioelectrocatalytic functions guided by the stiffness properties of the hydrogels.


Instruments
UV/vis absorption spectra were recorded with a temperature-controlled UV-2401PC spectrophotometer (Shimadzu, Japan).
Fluorescence spectra were recorded with a Cary Eclipse Fluorometer (Varian Inc.).
SEM images were taken with the Extra High Resolution Scanning Electron Microscope Magellan (TM) 400L with the following microscope settings: 2 kV, 13 pA. The hydrogel sample on Au-coated glass slide was frozen by immersing it in liquid nitrogen, followed by sublimation under high vacuum.
The 1 H-NMR and the diffusion-ordered NMR spectroscopy (DOSY) spectra were recorded on a Bruker DRX 400 MHz and a Bruker Ultrashield Plus 500 MHz spectrometer.

Synthesis of the Acrylamide Copolymer Chains
As the acrydite modification is only available on the 5' end of DNA, H 1 (1) was modified directly while H 2 (3) was hybridized to a short tether strand (2) that itself was modified with acrydite. The acrydite-modified oligonucleotides were used to synthesize the copolymer chains. To solution consisting of 0.75 mM acrydite-modified oligonucleotide and 0.18 mM acrylamide was added 0.4% of the initiator (prepared as 17 mg of 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in 100 µL of DMSO).
After 10 minutes of nitrogen bubbling, the sample was exposed to UV light (λ = 365 nm) for 10 minutes and then incubated at 4°C for a time interval of 12 hours to form the copolymer chains. Finally, the samples were filtered through the Amicon (Millipore) spin filter unit to remove all the unreacted compounds. Polymers P A and P C were filtered through a MWCO 30 kDa column, while polymers P B and P D (before hybridization of (3)) were filtered through a MWCO 10 kDa column. Next, the purified polymers were subsequently subjected to freeze-drying, followed by dissolution in buffer to achieve a S5 concentration of 1 mg/20 µL. After determination of (2) concentration, (3) was added in a 1:1 molar ratio in the hybridization chain reaction (HCR) buffer (25 mM Tris, 25 mM MgCl 2 , pH 7.4). Polymers were then incubated at 95°C for 5 minutes, followed immediately by a 30-minute ice incubation to ensure efficient closing of hairpins. Synthesis of polymers P A and P B containing the i-motif and the complementary strand was performed according to the procedure above, with the addition of sequences (4) and (5), while synthesis of polymers P C and P D containing the G-quadruplex was performed with the addition of sequences (7) and (8).

Determination of Ratio of Acrylamide/Acrydite-Nucleic Acids
To a solution containing the respective acrydite-modified nucleic acids, variable concentrations of pure acrylamide were added, and the absorption spectra of the different solutions were recorded. The increase in the absorbance at λ = 200 nm corresponded to the nonsubstituted polyacrylamide chains, while the absorbance at λ = 260 nm corresponded to the acrydite-modified nucleic acid units. An appropriate calibration curve corresponding to the molar ratio of the nucleic acids in the copolymer and the acrylamide monomer units was derived. Based on this calibration curve, the ratio of acrylamide/acrydite-nucleic acid in the different copolymers was evaluated spectroscopically ( Figure S4).

Modification of Gold Surface
A solution of 15 µM of (6) was mixed with a 150-fold excess of TCEP and incubated for 10 minutes in coating buffer (Tris, 25 mM, pH 7.4, MgCl 2 , 5 mM). Gold-coated glass S8 slides were boiled in pure ethanol for 30 minutes, followed by immersion of slides in solution of (6). Slides were incubated for 12 hours at room temperature and then washed with ultrapure water before use.

Gelation
Surface-integrated hydrogels were prepared by placing the polymer P B or P D (7.5 µL of 1 mg/20 µL) onto the gold surface coated by (6) and the polymer P A or P C (7.5 µL of 1 mg/20 µL) was added and mixed, correspondingly. Samples were then incubated for 12 hours at 4 ºC in a closed chamber with buffer reservoir to reduce evaporation of solution on surface.

The insulin/GOx-fluorophore conjugates
The insulin and GOx -fluorophore conjugates were prepared as it was described previously 3 . GOx was modified with fluorescein isothiocyanate isomer I (FITC) (λ ex = 490 nm; λ em = 525 nm) and insulin was modified with 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester (Coumarin) (λ ex =360 nm; λ em = 410 nm).  2) The switchable bioelectrocatalytic oxidation of glucose and release of drugs from the i-motif-modified hydrogel matrix were performed under anaerobic conditions in HEPES buffer (10 mM. pH=7.4, NaCl 50 mM, MgCl 2 5 mM) by applying S10 potential of E=0.35 V vs. SCE for diffusional system, or potential of E=0.4 V vs. SCE for integrated system and addition of different glucose concentrations.
3) The switchable bioelectrocatalytic oxidation of glucose by the G-quadruplex-  To monitor these changes, the pH-sensitive dye bromocresol purple (5′,5″-dibromo-o-cresolsulfophthalein) was used, which has a pK A = 6.3 and exhibits a yellow color at pH = 5.5 and violet color above pH = 7. The glucose concentration was 250 µM.