Characterization of Bacteriorhodopsin and Halorhodopsin Reconstituted in Lipid Bilayer Membranes

Motivated to produce electricity with photon activated ion pumps, the main purpose of this work was to characterize the photosynthetic membrane proteins bacteriorhodopsin (proton pump) and halorhodopsin (chloride pump). The proteins were re-suspended in lipid bilayers. For this work, an experimental set-up was built which included: chambers for lipid bilayer formation and characterization, lasers for ion pump activation, and an AxoPatch electrophysiology system for small photocurrent measurement. Lipid bilayer membranes were formed using mostly folding method: folding two monolayers together. The membranes were characterized by their resistance, capacitance, and generated photocurrent. Photocurrent was generated upon illumination of lipid-protein membranes with lasers. A green (532 nm) laser was used to illuminate bacteriorhodopsin containing membranes to produce proton-based positive photocurrent, while a purple (405 nm) laser was used to illuminate halorhodopsin containing membranes to generate chloride-based negative photocurrent. The investigation included: the generation of photocurrent using formed lipid-protein membranes and their voltage dependence, the study of the effect of laser intensity and protein concentration on the photocurrent amplitude and efficiency, the TEM-imaging of photocurrent generating lipid-protein solutions, and an equation that can help predict photocurrent amplitude in the defined protein concentration range

Analysis of Bacteriorhodopsin Suspended in a Bilayer Lipid Membrane

The bacteriorhodopsin protein’s unique characteristic of proton pumping can convert light energy to electric energy. The aim of this research was to generate photocurrent using bacteriorhodopsin in a bi-layer lipid membrane. Lipid monolayer and bilayer were formed using painting and folding methods, respectively. Capacitance and resistance of the lipid membranes were measured and used to validate the best methodology. My results show that the folding method is more efficient in incorporating Bacteriorhodopsin. The photocurrent was generated by illuminating a green laser (532 nm) on the bilayer lipid membranes. The patch clamp electrophysiology technique was used to apply voltage across the lipid membrane and to record photocurrent. For the membrane capacitance and resistance, the ranges were (1.70E-01- 7.50E-01 uF/cm2) and (0.30 - 0.49 GΩ), respectively. The photocurrent density produced was between 5.3 pA/cm2 and 7.1 pA/cm2

Photo-induced Proton Transfers of Microbial Rhodopsins

Molecular Photochemistry - Various Aspect

Quantum dot / optical protein bio-nano hybrid system biosensing

The integration of novel nanomaterials with highly-functional biological molecules has advanced multiple fields including electronics, sensing, imaging, and energy harvesting. This work focuses on the creation of a new type of bio-nano hybrid substrate for military biosensing applications. Specifically it is shown that the nano-scale interactions of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots can be utilized as a generic sensing substrate. This work spans from the basic creation of the protein to its application in a novel biosensing system. The functionality of this sensor design originates from the unique interactions between the quantum dot and bacteriorhodopsin molecule when in nanoscale proximity. A direct energy transfer relationship has been established between coreshell quantum dots and the optical protein bacteriorhodopsin that substantially enhances the protein’s native photovoltaic capabilities. This energy transfer phenomena is largely distance dependent, in the sub-10nm realm, and is characterized experimentally at multiple separation distances. Experimental results on the energy transfer efficiency in this hybrid system correlate closely to theoretical predictions. Deposition of the hybrid system with nano-scale control has allowed for the utilization of this energy transfer phenomena as a modulation point for a functional biosensor prototype. This work reveals that quantum dots have the ability to activate the bacteriorhodopsin photocycle through both photonic and non-photonic energy transfer mechanisms. By altering the energy transferred to the bacteriorhodopsin molecule from the quantum dot, the electrical output of the protein can be modulated. A biosensing prototype was created in which the energy transfer relationship is altered upon target binding, demonstrating the applicability of a quantum dot/bacteriorhodopsin hybrid system for sensor applications. The electrical nature of this sensing substrate will allow for its efficient integration into a nanoelectronics array form, potentially leading to a small-low power sensing platform for remote toxin detection applications

Bacteriorhodopsin-based sensors

A sensor comprising a membrane containing bacteriorhodopsin. In one embodiment, the sensor comprises a layer of purple membrane between a first and a second electrode, wherein the electrodes are connected to a circuit such that a signal is produced when a charge is transferred across the membrane. In another embodiment, the sensor comprises a field effect transistor with a layer of purple membrane deposited on the gate. The layer of purple membrane may be further functionalized by adding fluorophores to the layer of purple membrane. The fluorophores may be deposited adjacent to the layer of purple membrane, or the fluorophores may be attached to the layer of purple membrane with linkages. The fluorophores or linkages between the fluorophores and the purple membrane may be functionalized with receptors to produce sensors for targeted chemical or biological species.https://digitalcommons.mtu.edu/patents/1007/thumbnail.jp

Immobilizing bacteriorhodopsin on a single electron transistor

As awareness of potential human and environmental impacts from toxins has increased, so has the development of innovative sensors. Bacteriorhodopsin (bR) is a light activated proton pump contained in the purple membrane (PM) of the bacteria Halobacterium salinarum. Bacteriorhodopsin is a robust protein which can function in both wet and dry states and can withstand extreme environmental conditions. A single electron transistor(SET) is a nano-scale device that exploits the quantum mechanical properties of electrons to switch on and off. SETs have tremendous potential in practical applications due to their size, ultra low power requirements, and electrometer-like sensitivity. The main goal of this research was to create a bionanohybrid device by integrating bR with a SET device. This was achieved by a multidisciplinary approach. The SET devices were created by a combination of sputtering, photolithography, and focused ion beam machining. The bionanomaterial bacteriorhodopsin was created through oxidative fermentation and a series of transmembrane purification processes. The bR was then integrated with the SET by electrophoretic deposition, creating a bionanohybrid device. The bionanohybrid device was then characterized using a semiconductor parametric analyzer. Characterization demonstrated that the bR modulated the operational characteristics of the SET when bR was activated with light within its absorbance spectrum. To effectively integrate bacteriorhodopsin with microelectromechanical systems (MEMS) and nanoelectromechanical systems (NEMS), it is critical to know the electrical properties of the material and to understand how it will affect the functionality of the device. Tests were performed on dried films of bR to determine if there is a relationship between inductance, capacitance, and resistance (LCR) measurements and orientation, light-on/off, frequency, and time. The results indicated that the LCR measurements of the bR depended on the thickness and area of the film, but not on the orientation, as with other biological materials such as muscle. However, there was a transient LCR response for both oriented and unoriented bR which depended on light intensity. From the impedance measurements an empirical model was suggested for the bionanohybrid device. The empirical model is based on the dominant electrical characteristics of the bR which were the parallel capacitance and resistance. The empirical model suggests that it is possible to integrate bR with a SET without influencing its functional characteristics

Photolithography based patterning of bacteriorhodopsin films

The patterning of photoactive purple membrane (PM) films onto electronic substrates to create a biologically based light detection device was investigated. This research is part of a larger collaborative effort to develop a miniaturized toxin detection platform. This platform will utilize PM films containing the photoactive protein bacteriorhodopsin to convert light energy to electrical energy. Following an effort to pattern PM films using focused ion beam machining, the photolithography based bacteriorhodopsin patterning technique (PBBPT) was developed. This technique utilizes conventional photolithography techniques to pattern oriented PM films onto flat substrates. After the basic patterning process was developed, studies were conducted that confirmed the photoelectric functionality of the PM films after patterning. Several process variables were studied and optimized in order to increase the pattern quality of the PM films. Optical microscopy, scanning electron microscopy, and interferometric microscopy were used to evaluate the PM films produced by the patterning technique. Patterned PM films with lateral dimensions of 15 μm have been demonstrated using this technique. Unlike other patterning techniques, the PBBPT uses standard photolithographic processes that make its integration with conventional semiconductor fabrication feasible. The final effort of this research involved integrating PM films patterned using the PBBPT with PMOS transistors. An indirect integration of PM films with PMOS transistors was successfully demonstrated. This indirect integration used the voltage produced by a patterned PM film under light exposure to modulate the gate of a PMOS transistor, activating the transistor. Following this success, a study investigating how this PM based light detection system responded to variations in light intensity supplied to the PM film. This work provides a successful proof of concept for a portion of the toxin detection platform currently under development