Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Printed Antifouling Electrodes for Biosensing Applications.

Biosensors based on miniaturized, functional electrodes are of high potential for various biosensing applications, especially at the point-of-care setting among others. However, the sensor performance of such electrochemical devices is still strongly limited, especially due to surface fouling in complex sample fluids, such as blood serum. Electrode coatings based on conductive nanomaterials embedded in antifouling matrices offer a promising strategy to overcome this limitation. However, known composite coatings require long (typically >24 h) and complex fabrication processes, which pose a strong barrier for cost-effective mass manufacturing and successful commercialization. Here, we describe a novel polymer/carbon nanotube (CNT) composite coating that can be produced from an ink containing a photoreactive and antifouling copolymer as well as conductive CNTs using fast and highly scalable printing processes. Coatings were prepared on screen-printed electrodes and characterized using cyclic voltammetry (CV) and protein fouling experiments. The coatings offered an electroactive surface area (EASA) comparable to uncoated screen-printed electrodes and retained >90% of initial EASA after 1 h of exposure to concentrated bovine serum albumin solution, while uncoated electrodes decreased to <20% of initial EASA after the same treatment. Utilizing the universal crosslinking reaction of the polymer coating, antibodies against the inflammatory biomarker C-reactive protein (CRP) were photochemically immobilized on the electrodes. Functionalized electrodes were fabricated in <2 h and were successfully used to quantify nanogram-range concentrations of CRP spiked in undiluted human blood serum using a sandwich-immunoassay with electrochemical read-out, demonstrating the high potential of the platform for biosensing applications.