Synthetic Silica Nano-Organelles for Regulation of Cascade Reactions in Multi-Compartmentalized Systems

Abstract

In eukaryotic cells, enzymes are compartmentalized into specific organelles so that different reactions and processes can be performed efficiently and with a high degree of control. In this work, we show that these features can be artificially emulated in robust synthetic organelles constructed using an enzyme co-compartmentalization strategy. We describe an in situ encapsulation approach that allows enzymes to be loaded into silica nanoreactors in well-defined compositions. The nanoreactors can be combined into integrated systems to produce a desired reaction outcome. We used the selective enzyme co-compartmentalization and nanoreactor integration to regulate competitive cascade reactions and to modulate the kinetics of sequential reactions involving multiple nanoreactors. Furthermore, we show that the nanoreactors can be efficiently loaded into giant polymer vesicles, resulting in multi-compartmentalized microreactors.

Keywords: cascade reactions; enzymatic reactions; nanoorganelles; nanoreactors; synthetic cells.

Figure 1

Characteristics of enzymatic nanoreactors. a) Schematic illustration of cascade reaction between individual nanoreactors (NRs). b) TEM images of GOx@NRs (top) and HRP@NRs (bottom). Scale bars=1 μm (left column) and 100 nm. c) Encapsulation efficiency (EE, %) of GOx and HRP in the NRs. The EE was determined after centrifuging the dispersions to separate encapsulated from non‐encapsulated enzymes. Disp.: original dispersion, Pellets: NRs collected after centrifugation, SN: supernatant. d–f) Michaelis–Menten kinetics of reactions involving GOx, HRP and GOx+HRP encapsulated and non‐encapsulated into NRs. Tables with 95 % confidence intervals values are shown in the Supporting Information. The NRs concentration in (d–f) was 0.25 mg mL⁻¹.

Figure 2

Compartmentalization in a three‐step sequential cascade reaction (type 1). a) Reaction Scheme for the sequential cascade reaction of β‐G, GOx, and HRP. b) Comparison of reaction kinetics between the co‐compartmentalized GOx and HRP and their separately loaded counterparts. A green color background represents the concentration of H₂O₂ in the system. c) Comparison of reaction kinetics between the co‐compartmentalized β‐G and GOx and their separately loaded counterparts. A red color background represents the concentration of H⁺ and H₂O₂ in the system. d,e) Scheme and reaction kinetics of the selective compartmentalization of enzymes. Pathway A: β‐G was separately loaded; GOx and HRP were co‐loaded. Pathway B: β‐G and GOx were co‐loaded; HRP was separately loaded. The error bars represent s.d. based on three identical measurements.

Figure 3

Compartmentalization in competitive reactions (type 2). a) Reaction Scheme for the competitive reactions of HRP and CAT following the reaction with GOx. b) Fluorescence intensity of reaction product resorufin in pathway 1 in the presence of different ratios of HRP@NR and CAT@NR. Control: experiment without HRP@NR and CAT@NR. c) Scheme of the selective compartmentalization of enzymes. System 1: GOx and HRP were co‐loaded; CAT was loaded separately. System 2: HRP was loaded separately; GOx and CAT were co‐loaded. d) Fluorescence intensity of reaction product resorufin from pathway 1 for system 1 and 2. A model system containing separately loaded GOx and HRP, in the absence of CAT (no competition), was used as reference (considered as 100 % of reaction via pathway 1). e) The selectivity of reaction to pathway 1 or 2 was calculated from the data shown in Figure 3 d. The reaction selectivity towards pathway 1 in system 1 was calculated by dividing the resorufin production rate of system 1 (grey line) by the control system (blue line). While the reaction selectivity towards pathway 2 in system 2 was calculated by 100 % minus the percentage obtained from dividing the resorufin production rate of system 2 (red line) by the control system (blue line). The error bars represent s.d. based on three identical measurements.

Figure 4

a) Scheme of a synthetic cell with nanoreactors encapsulated into a micron‐sized polymer vesicle. b) Fluorescence emission of differently labelled NRs. GOx@NRs were labelled with FITC; HRP@NRs were labelled with Cy5. c) Confocal microscopy images showing the distribution of GOx@NR and HRP@NR in the micron‐sized polymer vesicles. Green signal represents GOx@NR; red signal represents HRP@NR; the overlay image shows the co‐localization of both NRs in the vesicles. Scale bars=100 μm.

Figure 5

Cascade reaction in a synthetic cell. a) Schematic illustration of the cascade reaction promoted by GOx@NR and HRP@NR in a synthetic cell. b) Fluorescence intensity of the product (resorufin) over time obtained by confocal microscopy for 5 individual synthetic cells. Time step size: 6.5 seconds. c) Confocal microscopy images of the reaction end points. Left: resorufin fluorescence. Right: bright field image. Scale bars=50 μm.