Multimodal Enzyme-Carrying Suprastructures for Rapid and Sensitive Biocatalytic Cascade Reactions

Abstract

Colloidal assemblies of mesoporous suprastructures provide effective catalysis in an advantageous volume-confined environment. However, typical fabrication methods of colloidal suprastructures are carried out under toxic or harmful conditions for unstable biomolecules, such as, biocatalytic enzymes. For this reason, biocatalytic enzymes have rarely been used with suprastructures, even though biocatalytic cascade reactions in confined environments are more efficient than in open conditions. Here, multimodal enzyme- and photocatalyst-carrying superstructures with efficient cascade reactions for colorimetric glucose detection are demonstrated. The suprastructures consisting of various functional nanoparticles, including enzyme-carrying nanoparticles, are fabricated by surface-templated evaporation driven suprastructure synthesis on polydimethylsiloxane-grafted surfaces at ambient conditions. For the fabrication of suprastructures, no additional chemicals and reactions are required, which allows maintaining the enzyme activities. The multimodal enzymes (glucose oxidase and peroxidase)-carrying suprastructures exhibit rapid and highly sensitive glucose detection via two enzyme cascade reactions in confined geometry. Moreover, the combination of enzymatic and photocatalytic cascade reactions of glucose oxidase to titanium dioxide nanoparticles is successfully realized for the same assay. These results show promising abilities of multiple colloidal mixtures carrying suprastructures for effective enzymatic reactions and open a new door for advanced biological reactions and enzyme-related works.

Keywords: biocatalytic cascade reactions; colloidal suprastructures; glucose assays; liquid repellent surfaces; surface-templated suprastructure synthesis.

Figure 1

Enzyme‐carrying suprastructures of nanoparticle fabricated by a S‐TED method. a) Schematics for the fabrication of GOX‐ and HRP‐carrying suprastructures on the PDMS‐grafted surface by evaporation. TEM images of silica‐NPs carrying b) GOX and c) HRP to prepare the suprastructure. Scale bars represent 200 nm. d) Evaporation progress for the preparation of the suprastructures on the PDMS‐grafted surface (scale bar: 500 µm). e) SEM images of the suprastructure. Scale bars represent 200 nm for a high magnification image and 500 µm for a low magnification.

Figure 2

GOX/HRP cascade reactions of the suprastructure at millimolar glucose levels. a) Enzymatic reactions and signal generation principle of glucose assay using KI as a chromogen. b) Schematics for the suprastructure‐based glucose assay. c) Pictures of glucose assay for 100, 10, and 1 mm concentration on suprastructures (smaller drops on left side) and non‐dried NP dispersions (larger drops on right side) after different reaction times. The yellow‐brown color indicates the progression of the cascade reactions. d–f) Reaction progression respective to (c) through nanodrop spectroscopy (absorbance at 400 nm). All measurements were conducted three times to get average values.

Figure 3

Reaction kinetics of GOX/HRP cascade reactions. a) Enzymatic reactions and signal generation principle of glucose assay using Amplex red dye. b) GOX/HRP cascade reactions at micromolar glucose levels. Pictures of glucose assay for 50, 10, and 1 µM solution drops on suprastructures (left drops) and non‐dried NP dispersions (right drops) in time relapse. The red color indicates the progression of the cascade reactions. c) Schematics of GOX/HRP cascade reactions in dispersion (left) and Michaelis‐Menten kinetics in terms of GOX (right). d) Schematics of GOX/HRP cascade reactions in suprastructure (left) and Michaelis‐Menten kinetics in terms of GOX (right).

Figure 4

GOX/TiO₂ cascade reactions on suprastructure. a) Schematics of GOX‐ and TiO₂‐carrying suprastructure‐based glucose assay, and SEM image of the cross‐sectioned GOX‐TiO₂ suprastructure (scale bar: 1 µm). b) Photographic pictures of glucose assay on suprastructures for 100, 10, and 1 mm solution drops. The yellow‐brown color indicates the progression of the reactions. The GOX reactions were allowed for 9 min, then, UV‐A was irradiated for 1 min to allow TiO₂ reactions. c–e) Color intensities of solutions after finishing the cascade reactions, characterized by image analysis of pictures (the left plain bars: Before reactions; the right diagonal‐checked bars: After reactions). Each result was averaged from 3 samples.

Figure 5

Glucose assay in human urine and serum through a GOX/HRP cascade reactions. a) Pictures of glucose assay for human urine samples. The generation of yellow‐brown color after 20 min indicates the progression of the reactions. b) A standard curve of GOX/HRP cascade reactions for respective glucose concentrations in human urine. KI was used as the indicator. The absorbance was measured at 400 nm. c) Comparison of urine glucose quantification by assay of suprastructures and a commercial glucometer. d) A standard curve of GOX/HRP cascade reactions for respective glucose concentrations in human serum. Amplex red was used as the indicator. Fluorescence was obtained at excitation 555 nm and emission 595 nm. e) Comparison of urine glucose quantification by assay of suprastructures and a commercial glucometer. (Note: Unit conversion for glucose concentration; 37.3 mg dL⁻¹ (2.1 mm), 484 mg dL⁻¹ (27 mm), 343 mg dL⁻¹ (19 mm), 268 mg dL⁻¹ (15 mm), 120 mg dL⁻¹ (6.7 mm), 225 mg dL⁻¹ (12.5 mm), 179 mg dL⁻¹ (9.9 mm), 145 mg dL⁻¹ (8.1 mm)).