Abstract
AbstractLocal oxidation lithography has the potential for patterning proteins on conductive substrates such as silicon with nanometer accuracy, guided by and extending the nanoscale architectures found in native bioenergetic membranes. Such membranes foster energy and electron transfers between two or more types of protein complex, so the potential of this lithographic technique is investigated for copatterning multiple types of protein complex. Composite patterns consisting of light‐harvesting 2 (LH2) and reaction center‐light‐harvesting 1‐PufX (RCLH1) complexes purified from Rhodobacter (Rba.) sphaeroides, and light‐harvesting complex II (LHCII) purified from spinach, are fabricated. Atomic force microscopy (AFM) images demonstrate the successful sequential deposition of single‐molecule layers of RCLH1 and LH2 molecules. In the case of LHCII, a mixture of single‐layer and multilayer patterns is found on the silicon substrate. Experimental conditions are established for the most efficient substrate surface modification and for protein immobilization. Spectral imaging and fluorescence lifetime imaging microscopy (FLIM) show that the immobilized photosynthetic complexes retain their native light‐harvesting and energy transfer functions, and provide evidence for excitation energy transfer from LH2 to RCLH1. Local oxidation lithography has the capacity to pattern proteins singly, or in small domains, for fabricating bioinspired nanoscale architectures for biosensors and solar cells.
Highlights
Introduction proteinsThe surface properties of these substrates are generally modified by physical or chemical means to control theIn bacterial and plant photosynthesis, sunlight harvested by attachment of the target molecule
Most of the efforts in nanopatterning of photosynthetic complexes have been focused on attaching a single type of complex such as light-harvesting 2 (LH2), reaction center-light-harvesting 1-PufX (RCLH1), or light-harvesting complex II (LHCII) to a surface, and it was only very recently that we demonstrated excitation energy transfer between intersecting lines of LH2 and RCLH1 patterned sequentially onto glass substrate with a spatial distribution controlled on a micrometer rather than a nanometer scale.[38]
The most efficient experimental conditions have been found for mPEG molecule’s oxidation and protein immobilization, and at the points of intersection of the copatterns we found some evidence for shortened lifetimes of LH2 energy donor complexes, where they are in proximity to RCLH1 acceptor complexes
Summary
To fabricate nanopatterns with a high protein occupancy, we investigated a range of experimental conditions for more effective mPEG oxidation and better protein immobilization. The presence of RCLH1 aggregates on top of the LH2 square pattern (green ovals in Figure 7A) has clearly quenched the LH2 emission shortening the LH2 lifetime down to around 0.3 ns We interpret this as a possible indication of energy transfer between the two types of complexes when they sit on top of each other, providing larger interface area. The AFM topographic images of the copatterned RCLH1 and LH2 nanolines unequivocally show that the two proteins co-exist within the same area on the substrate (Figures 6 and 7A), while the acquired fluorescence emission spectra clearly demonstrate the retained structural and optical properties of the protein complexes. Previous studies have already demonstrated the usefulness of LH2 or RCLH1 proteins randomly immobilized onto conductive substrates (metals) to study their electrical properties and their potential applications in biohybrid devices and biosensors.[15,18,20,21,71,72] our method for a precise immobilization of multiple light-harvesting proteins on the nanoscale onto a semiconductive substrate that is widely used in microelectronics and nanofabrication (silicon wafers) is highly relevant when investigating future biohybrid and biophotovoltaic applications of the light-harvesting proteins
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