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Quantum dot enhancement of bacteriorhodopsin-based electrodes
Biosensors and Bioelectronics 25 (2010) 1493–1497
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Quantum dot enhancement of bacteriorhodopsin-based electrodes Mark H. Griep a , Karl A. Walczak a , Eric M. Winder b , Donald R. Lueking b , Craig R. Friedrich a,∗ a b
Dept. of Mechanical Engineering-Engineering Mechanics, Multi-Scale Technologies Institute, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA Dept. of Biological Sciences, Multi-Scale Technologies Institute, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA
a r t i c l e
i n f o
Article history: Received 11 May 2009 Received in revised form 4 November 2009 Accepted 7 November 2009 Available online 11 November 2009 Keywords: Bacteriorhodopsin Quantum dots Bionanosensing Hybrid electrode
a b s t r a c t Nanoscale sensing arrays utilizing the unique properties of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots are being developed for toxin detection applications. This paper describes an innovative method to activate bacteriorhodopsin-based electrodes with the optical output of quantum dots, producing an enhanced electrical response from the protein. Results show that the photonic emission of CdSe/ZnS quantum dots is absorbed by the bacteriorhodopsin retinal and initiates the proton pumping sequence, resulting in an electrical output from a bacteriorhodopsin-based electrode. It is also shown that activated quantum dots in sub-10 nm proximity to bacteriorhodopsin further amplify the photovoltaic response of the protein by approximately 23%, compared to without attached quantum dots, suggesting direct energy transfer mechanisms beyond photonic emission alone. The ability of quantum dots to activate nanoscale regions on bacteriorhodopsin-based electrodes could allow sub-micron sensing arrays to be created due to the ability to activate site-specific regions on the array. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Throughout history the use of biological materials in engineered applications has led to dramatic innovations. The field of nanotechnology is no different, as there are arrays of natural nanoscale materials with capabilities beyond that of current technology. The efficient integration of select biological materials with engineered nanomaterials will be a critical step for expanding nanotechnological applications. One such biological material with broad nanoengineering potential is the integral membrane protein bacteriorhodopsin (bR). Bacteriorhodopsin is a retinal containing protein found in the cell membrane of the extremophile Halobacterium salinarum and is utilized to generate a proton motive force that energizes adenine triphosphate (ATP) synthase to drive the conversion of adenine diphosphate (ADP) and Pi to ATP and H2 O, thereby providing the energy to drive the cell’s internal machinery. The proton motive force is achieved when bR’s attached retinal chromophore absorbs visible light in the 570 nm region, resulting in a trans–cis isomerization of the prosthetic group. This structural alteration initiates proton transport from the cytoplasmic side to the extracellular side of the cell membrane, creating a proton gradient across the membrane system (Lanyi, 2004). For engineered applications, coupling the bR protein with an electrically conductive substrate rather than an ATP synthase allows the efficient quantum conversion of light energy into an
∗ Corresponding author. Tel.: +1 906 487 1922. E-mail address: [email protected] (C.R. Friedrich). 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.11.005
electric potential by charge translocation. Bacteriorhodopsin can be purified as membrane patches, known as purple membrane (PM). Purple membrane, intrinsic to the cellular membrane of H. salinarum, has an average diameter of 500 nm and is comprised of multiple bR molecules and associated phospholipids. In the dried and wet states, PM retains its light absorption properties and photochemical activity for years (Vsevolodov, 1998). The potential applications for PM in engineered devices are extensive; including information storage, information processing, charge transport membranes, and photoelectric systems (Birge et al., 1994; Min et al., 2001; Choi et al., 2005). In the current work, the photoelectric property of bR was used as the transducer substrate for a bionanosensing device. For this application PM needs to be patterned and activated on a microelectronics array to create a multitude of sensing sites in a small chip area. To photonically activate individual PM sensing sites without contributing signal to neighboring sites, a 570 nm light-source needs to be incorporated that is on the same size scale as the bR molecules. To facilitate the activation of a bR array at the nanoscale, the photonic output of quantum dots (QD) was explored. As will be shown, the usefulness of quantum dots is two-fold; contributing both to bR activation, and to the proposed sensing mechanism. The QDs used in this study are colloidal semiconductor QDs composed of a cadmium selenide (CdSe) core and an optional zinc sulfide (ZnS) protective shell. They are semiconductor particles that have dimensions on the nanometer scale, typically 2–10 nm. Due to their small dimensions, below the exciton Bohr radius of the semiconducting material, QDs have discrete energy levels with bandgaps that can be fine tuned based on the size of the dot. Since
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Fig. 1. Normalized bR absorption and 575 nm QD emission, displaying spectral overlap.
the size of the bandgap determines the energy of the electromagnetic radiation emitted by the electron when it falls back to the valence band, the wavelength emitted by the QD can be selected by its size. In this study, the QD emission peak was selected to correspond to the absorption maximum of bacteriorhodopsin, as shown in Fig. 1. Along with providing a narrow emission spectrum, QDs have a broad absorption range with a local maximum in the ultra-violet (UV) region. When compared to other fluorescing materials, such as fluorescent dyes, QDs have vastly improved characteristics including increased quantum yield, chemical stability, lifetime, ease of emission tunability, broad absorption range, and a greater resistance to photo-bleaching (Jovin, 2003). Apart from the intrinsic properties, the surface chemistry of QDs has also been shown to be amenable to the addition of functionalized binding materials, allowing the use of QDs in a wide array of sensor applications. 2. Materials and methods 2.1. Protein preparation and electrode construction The PM used in these studies was obtained from H. salinarium strain S9P (courtesy J. Spudich) and prepared on-site according to a modified version of previously reported methods (Oesterhelt and Stoeckenius, 1974). The S9P strain is an over-producer of bR so the PM patches are highly dense with protein. Details of the preparation methodology are provided in the supplementary materials. Due to the uni-directional proton pumping direction of bR, the PM films used to make the photosensitive electrodes must be highly oriented (Gergely et al., 1993). This allows a maximum number of protons to be vectorially shifted in the same direction, yielding a higher electrical voltage upon illumination. The high degree of protein orientation for these studies was obtained by electrophoretic sedimentation (EPS) (Keszthelyi, 1980; Varo and Keszthelyi, 1983). Two different approaches were undertaken for stimulating the PM by quantum dots; first by physical separation of the PM and QDs by glass, and second by chemically linking the PM and QDs utilizing a biotin and streptavidin binding scheme. These two separate setups isolate QD optical activation in the first, and potential direct energy transfer mechanisms in the second. For both investigations, the PM electrodes were fabricated in the same basic manner. The electrode (anode) was a glass slide with a thin sputtered layer of indium tin oxide (ITO). The dimensions of the electrode were 5 mm × 10 mm and the ITO had a sheet resistance of 5–10 /sq. A 40 l suspension of 15 mg ml−1 PM in pH 6.5 ddI water was pipetted on top of the ITO and a brass electrode (cathode) was placed on
top of the suspension with 1 mm spacing between the electrodes. An electric field of 40 VDC cm−1 was applied across the electrodes for 1 min. Due to the elevated negative dipole charge of bR’s cytoplasmic region, the PM will preferentially orient and attach to the ITO cathode when the electric field potential is applied. Following the 1 min exposure to the electric field, the top brass electrode was removed and the ITO electrode with attached PM was agitated for 10 s in a beaker of ddI water to remove any weakly attached PM. The ITO electrode was dried in a humidity chamber, maintained at 52% relative humidity with a magnesium nitrate salt solution, for 24 h. The dimensions of the dried bR film were approximately 5 mm × 5 mm with a thickness of approximately 20 m. After drying, another ITO-coated glass electrode of similar dimensions was placed on top of the oriented PM film to create the measurement circuit. The PM electrode was wired directly into a custom-built, T input impedance, non-inverting operational amplifier, similar to those previously reported (Wang et al., 2006), and configured to 10× amplification. The ITO electrode with the cytoplasmic side of the attached PM was set to ground (−) and the opposing ITO electrode was attached to the op-amp (+). Both the op-amp and PM electrode were placed in a Faraday cage. The non-inverting opamp was powered by an HP 6237A triple output voltage supply. The signal from the op-amp was passed through a highpass filter (Krohn-Hite Model 3364) with a cutoff frequency set at 30 Hz. From the filter, the conditioned signal was input to a 60 MHz, 200 MSa/s oscilloscope (Agilent 54621D) for digital display and recording. 2.2. Quantum dot quenching effects of bR To help clarify the amount of energy and the energy transfer mechanism, the fluorescent alteration of QDs in response to close proximity bR molecules was analyzed. The emission of thin film QDs on glass, dried bR, and bleached bR films was measured. Bleaching of bR yield the apo-protein, bacterio-opsin (bO), in which the associated prosthetic retinal group has been released. The retinal is responsible for the light absorption around 570 nm and the proton pumping cycle, therefore without the retinal all functional properties of bR are lost. One method to remove the bR retinal is through continuous illumination with high intensity light (Dancshazy et al., 1999). Depending on the light intensity, the bleaching process can take several days. To increase the speed of this process, the pH of the PM solution can be increased and hydroxylamine can be added. For these studies a 10 mg/ml PM solution was used. Hydroxylamine was added to the solution at 0.3 M and the pH was increased to 7.5. The solution was then subjected to continuous high intensity light illumination at 500 mW/cm2 . The degree of retinal release was analyzed by monitoring the bR570 absorbance peak. It was revealed that a high degree of bleaching, >80%, was achieved in less than 24 h. Upon reaching near complete bleaching, the bO membrane solution was pelleted out of solution at 40,000 times gravity centrifugation and washed multiple times with ddI water to remove any residual hydroxylamine. The experimental setup compared the dried film fluorescence of 566 nm CdSe and 575 nm CdSe/ZnS QDs (Evident Technologies) when layered on a control (blank glass slide), dried bR film (5 l of 5 mg/ml PM), and dried bO film (5 l of 5 mg/ml). Each of the QD samples was suspended in toluene at a concentration of 0.5 nmol/ml. A total of 1 l of the 0.5 nmol/ml QD solution was applied to the desired substrate for each particular test. The estimated 3 mm diameter dimensions of deposited QDs would create a film no more than 1 QD thick, on average, thus putting a majority of QDs in direct contact with the selected substrate.
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2.3. Quantum dot integration with bR electrodes In the first investigation of the QD activation of the bR photoresponse, the quantum dots used for remote photonic activation of bR were 575 nm emission CdSe/ZnS QDs (Evident Technologies). The QDs were suspended in toluene at a concentration of 63 nmol ml−1 . A total of 40 l of the CdSe/ZnS suspension was added to a well constructed on top of the bR-based electrode, providing a glass barrier between the bR and QDs to eliminate any unwanted physical reactions between the two materials and to isolate the interactions solely to QD photonic emission and bR photonic absorption. Electrical measurements were made as described. In the second QD/bR electrode investigation, electrodes were created as described above but with biotinylated bR, which would provide a binding point for streptavidin-coated QDs. The small biotin molecule has an extremely high binding affinity for the streptavidin protein, which ensures specific and tight binding at all locations. Streptavidin, a 53 kDa protein, and biotin, also known as vitamin H or B7 , are together widely regarded as the strongest non-covalent bond in nature, with a dissociation constant (Kd ) of 4 × 10−14 M (Weber et al., 1989). With its robust nature and virtually unbreakable association, the biotin–streptavidin binding scheme has been well studied in applications of nanoparticle linkers (Cui et al., 2001; Oh et al., 2005). The initial step was to biotinylate the single Lysine-129 amino acid residue on the bR molecule, which has been reported previously (Chen et al., 2003; Sharma et al., 2004) and is further elaborated upon in the supplementary materials. This procedure resulted in a liquid suspension of PM fragments containing covalently bound biotin groups on the exposed bR molecules. Using a HABA/avidin assay the molar ratio of biotins per bR molecule was determined to be 0.4:1, respectively, showing that nearly 1 in every 2 bR molecules was effectively biotinylated. The biotinylated bR was then put into electrode form and the output was measured. For this work, streptavidin-coated 595 nm emission CdSe/ZnS QDs (Invitrogen) were used. Fresh biotinylated bR electrodes described above were submerged in a 0.5 nmol ml−1 QD solution for 5 min to facilitate full biotin–streptavidin linkage formation. The functionalized slide was vigorously rinsed for 2 min in ddI water to remove any unbound QD particles and the electrode output was measured. 3. Results and discussion The initial studies monitoring the effects of bR films on QD emission properties focused upon determining if the bR retinal possessed the ability to absorb the emitted QD energy. In Fig. 2(a) it can be seen that the deposition of a thin 566 nm CdSe QD film on top of a bR film resulted in a 54.5% reduction in the measured QD fluorescence output as compared to the same amount of QDs on a cleaned glass substrate. Due to the large spectral overlap of the QD emission and bR absorption peaks, as shown in Fig. 1, a large portion of the quenching effect is likely due to absorption of the emitted QD photons by the bR retinal. To confirm that the bR retinal was in fact responsible for the QD energy reduction, an identical substrate was created using bleached bR, where the retinal was removed from the bR molecule. Without the retinal these bO films were white in color, as compared to the distinct purple color of bR, due to its inability to absorb photons in the visible light spectrum. As shown in Fig. 2(b), the bleached bR substrate induced no quenching effects on the QDs, similar to that of the glass substrate control. This confirms that the bR retinal is the source of QD quenching in the experimental dried films. With results confirming that the QDs emitted energy can be absorbed by the bR retinal in dried films, work was performed to analyze the effect of separation distance between the bR film and deposited QDs. The alteration in bR–QD separation distance is
Fig. 2. Influence of substrate on QD fluorescence properties for (a) 566 nm CdSe and 575 nm CdSe/ZnS QDs on glass (control) and bR electrode substrates, and (b) 566 nm CdSe QDs on glass, bR electrode, and bleached bR electrode substrates.
accomplished through the use of CdSe (core) and CdSe/ZnS (coreshell) QDs. The ZnS polymeric shell on the CdSe will provide an approximate additional 1–1.5 nm spacer between the CdSe semiconducting material and the bR retinal in the dried films. When layering CdSe and CdSe/ZnS QDs directly on top of dried bR films, results show that the bR substrate induces a strong quenching effect on the emission of both QD types, as shown in Fig. 2(a). The magnitude of the induced QD quenching is 54.5% for the CdSe QDs and 43.8% for the core-shell CdSe/ZnS QDs, thus the slight change in separation distance produces a noticeable alteration in the QD quenching effects. There is a distinct difference between the degree of quenching for the core and core-shell QDs, as the addition of a ZnS shell on the CdSe QD reduces the energy loss to the bR molecules by 19.5% compared to the plain CdSe QDs. An explanation for the difference in quenching effects is the potential existence of a direct energy transfer relationship, or forster resonance energy transfer (FRET), between the CdSe QD core and the bR retinal. The FRET process is sensitive to the donor/acceptor (QD/bR) separation distance, particularly in the sub-10 nm region. Thus by increasing the QD/bR separation distance by adding polymeric shells to the QDs less energy was transferred non-radiatively from the QD to the bR retinal. This results in the increased QD emission over closer proximity non-shell QDs. With results suggesting that both photonic and non-photonic energy transfer mechanisms potentially exist in a close proximity bR–QD system, different bR–QD activation setups were created to isolate each possible mechanism. The first setup implemented a spacer to take the QDs out of direct energy coupling range of the bR, thus isolating QD photonic activation of the bR photovoltaic response. To establish a control response for comparison, a 310 nm UV-light emitting diode (LED) light source was used. Previous work has shown that the energy of normal incidence UV light, absorbed by the aromatic amino acids in bR, can be transferred to the retinal through resonance and initiate the photocycle (Kalisky et al., 1981). Therefore the angle of the UV source for QD activation was adjusted to avoid bR illumination and activation of the bR photocycle. In this control experiment, and prior to introducing QDs in the three
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Fig. 3. bR photoelectric response to (a) ∼4 Hz UV flashing – no QD’s (1 mV/vertical division, 500 ms/horizontal division) and (b) 4 Hz remote QD illumination (2 mv/vertical division, 500 ms/horizontal division).
methods described, the UV-LED was adjusted to an approximate 60◦ incidence angle. With no QDs in the proximity of the bR electrode, no apparent bR photovoltaic response was elicited from the angled UV illumination. The electrical response of this system during ∼4 Hz UV flashing is shown in Fig. 3(a). To investigate whether QDs can remotely activate bR, 40 l CdSe/ZnS of QD suspension (63 nmol/ml) was placed into the well separated from the top of the bR electrode. Upon subsequent ∼4 Hz UV excitation a detectable photovoltaic response is seen from the PM as shown in Fig. 3(b). With the addition of 575 nm emission CdSe/ZnS QDs on top of the bR electrode and subsequent UV illumination, a measurable electrical response was generated from the bR. The flashing of the UV-LED visibly activated the QD suspension and the electrical output of the bR electrode was monitored, as shown in Fig. 3(b). The cyclic signal from the bR-remote-QD electrode directly correlates to the flashing of the UV-LED. In the test system, it was calculated that there was approximately 1 QD for every 10 bR molecules. Assuming that every bR molecule was photonically activated, each quantum dot was potentially activating several bR molecules. This shows the potential capability of utilizing quantum dots in a nanoscale system since a single top monolayer of quantum dots might activate several monolayers of PM. To analyze the ability for QDs to activate bR through both photonic and non-photonic means, dried films were created to place a single QD monolayer in direct contact with the bR electrode. Achieving this bR–QD thin film structure was accomplished through the use of biotinylated bR as the attachment mechanism to bind streptavidin-coated quantum dots to the PM-based electrode. To determine the presence and functionality of the biotin groups for the biotinylated bR-based electrodes, the fluorescence emission of biotinylated and non-biotinylated PM electrodes exposed to QDs was analyzed on a Hitachi-F7000 fluorospectrometer to determine the presence of bound QDs. The resulting fluorescence spectrum, displayed in Fig. 4(a), shows that the streptavidin-coated QDs only bind to the biotinylated PM electrodes. Thus the biotinylated bR electrodes created by EPS retain the capacity to effectively bind to a functional streptavidin group. This linkage scheme allows for the
Fig. 4. (a) Emission spectra confirming attachment of single QD monolayer to the biotinylated PM electrode, while non-biotinylated PM resulted in no QD attachment. (b) bR electrode activation with and without integrated QD monolayer. Inset displays spectrum of incident white-light source.
binding of a single QD monolayer on top of the bR electrode, holding the QD–bR separation distance at approximately 6–8 nm. With the binding of a QD monolayer to the bR electrode achieved, the effects of the added nanoscale power source to the overall bR photovoltaic output was tested and the resulting voltage characteristics are shown in Fig. 4(b). It can be seen in Fig. 4(b) that the binding of the QDs to the surface of the PM electrode resulted in an increased bR photovoltaic response to white-light illumination. Without any QDs present, the bR electrode yielded a 0.249 V peak transient response upon illumination followed by a −0.158 V peak upon termination of the light. When a QD layer was then bound to the same sample and re-activated, the initial peak transient yielded a bR photovoltage of 0.306 V followed by a −0.2 V peak upon termination of the light. The addition of the QDs to the PM electrode increased the bR photovoltaic output by approximately 57 mV, or 23%. The photovoltaic on/off peak ratios of 1.42 and 1.55 for the PM and PM–QD electrodes are similar to the ratio of 1.5 that has been previously reported for PM electrodes. In dried PM films it has been shown that the decreased water content of the bR molecule significantly alters the normal photocycle process (Váró and Keszthelyi, 1985; Ganea et al., 1997). In particular the N and O photocycle intermediates, which are required for the proton uptake and release mechanism, cease to function. Therefore the photoelectric responses produced in dried PM films are due to charge translocation via retinal isomerization (Wang et al., 2006). Since the innate functionality of the bR retinal group is retained in dried films, it has the ability to both accept photons and potential resonance energy directly from the close proximity QD energy source. The photoelectric responses of the PM and PM–QD electrodes are similar in structure, only varying in magnitude, thus it is assumed that the dried film photocycle is not significantly altered with the accepted QD energy. These studies demonstrate that QDs can enhance the bR photovoltaic response, even with white-light illumination that overlaps
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the visible spectra of the QD absorption band. Since the QD are within a sub-10 nm proximity to the bR molecules and there is maximal bR–QD spectral overlap, the potential for a direct energy transfer relationship also exists in addition to photonic activation. The large magnitude photovoltaic increase of 23%, potentially consisting of both direct energy transfer and photonic activation of the bR, is of sufficient magnitude that alterations to the QD emission could detectably affect the bR output. This establishes a novel bionanosensing platform in which the disruption of the QD energy transmission to the bR molecule, upon target molecule binding, would result in a detectably altered electrical output. 4. Conclusions With the ability to initiate and amplify the bR photovoltage output with the energy released from chemically linked CdSe quantum dots, the development of a nanosensing array utilizing these nanomaterials is feasible. Results demonstrate the ability to activate the bR photovoltaic response from the photonic emission of a size-selected CdSe QD suspension with UV illumination. Creating a solid-state bR–QD nano-bio-hybrid material, with a dried QD monolayer in sub-10 nm proximity to the bR molecules, allows for a large amplification in the protein photovoltaic response even with a traditional white-light source. Due to the close proximity and optimal spectral overlap, it is feasible that the bR activation energy results in part from a direct energy transfer mechanism from the QDs, in addition to normal photonic absorptive processes. The utility of a direct energy transfer mechanism in a bR–QD hybrid system would not only allow for isolated pixels in an array platform, but may also provide a potential signal modulation point to achieve a bR–QD bio-nanosensing system. Acknowledgements We thank Chris Anton and Dawdon Cheam of the Michigan Technological University for their aid and input on various aspects of this
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project. The research reported in this document was performed in connection with contract DAAD17-03-C-0115 with the U.S. Army Research Laboratory. The research was also partially supported under a National Science Foundation Graduate Research Fellowship for Mark Griep. Appendix A. Supplementary data Further details pertaining to the bR production/purification techniques and the bR biotinylation procedure are given in the supplementary information section. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2009.11.005. References Birge, R.R., Govender, D.S.K., et al., 1994. Bioelectronics, Three-dimensional Memories and Hybrid Computers. Electron Devices Meeting, 1994. IEDM’94. Technical Digest. International. Chen, D.-l., Lu, Y.-j., et al., 2003. J. Phys. Chem. 107, 3598–3605. Choi, H., Lee, H., et al., 2005. Nanotechnology 16, 1589–1597. Cui, Y., Wei, Q., et al., 2001. Science 293 (5533), 1289–1292. Dancshazy, Z., Tokaji, Z., et al., 1999. FEBS Lett. 450, 154–157. Ganea, C., Gergely, C., et al., 1997. Biophys. J. 73, 2718–2725. Gergely, C., Ganea, C., et al., 1993. Biophys. J. 65, 2478–2483. Jovin, T.M., 2003. Nat. Biotechnol. 21, 32–33. Kalisky, O., Feitelson, J., et al., 1981. Biochemistry 20, 205–209. Keszthelyi, L., 1980. Biochem. Biophys. Acta 598, 429–436. Lanyi, J.K., 2004. Annu. Rev. Physiol. 66 (1), 665–688. Min, J., Choi, H.-G., et al., 2001. Biosens. Bioelectron. 16, 917–923. Oesterhelt, D., Stoeckenius, W., 1974. Methods Enzymol. 31, 667–678. Oh, E., Hong, M.Y., et al., 2005. J. Am. Chem. Soc. 127 (10), 3270–3271. Sharma, M.K., Jatteni, H., et al., 2004. Bioconjugate Chem. 15, 942–947. Varo, G., Keszthelyi, L., 1983. Biophys. J. 43, 47–51. Váró, G., Keszthelyi, L., 1985. Biophys. J 47 (2), 243–246. Vsevolodov, N., 1998. Biomolecular Electronics an Introduction via Photosensitive Proteins. Birkhauser, Boston. Wang, W., Knopf, G., et al., 2006. Biosens. Bioelectron. 21, 1309–1319. Weber, P.C., Ohlendorf, D.H., et al., 1989. Science 243 (4887), 85–88.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Quantum dot enhancement of bacteriorhodopsin-based electrodes Mark H. Griep a , Karl A. Walczak a , Eric M. Winder b , Donald R. Lueking b , Craig R. Friedrich a,∗ a b
Dept. of Mechanical Engineering-Engineering Mechanics, Multi-Scale Technologies Institute, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA Dept. of Biological Sciences, Multi-Scale Technologies Institute, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA
a r t i c l e
i n f o
Article history: Received 11 May 2009 Received in revised form 4 November 2009 Accepted 7 November 2009 Available online 11 November 2009 Keywords: Bacteriorhodopsin Quantum dots Bionanosensing Hybrid electrode
a b s t r a c t Nanoscale sensing arrays utilizing the unique properties of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots are being developed for toxin detection applications. This paper describes an innovative method to activate bacteriorhodopsin-based electrodes with the optical output of quantum dots, producing an enhanced electrical response from the protein. Results show that the photonic emission of CdSe/ZnS quantum dots is absorbed by the bacteriorhodopsin retinal and initiates the proton pumping sequence, resulting in an electrical output from a bacteriorhodopsin-based electrode. It is also shown that activated quantum dots in sub-10 nm proximity to bacteriorhodopsin further amplify the photovoltaic response of the protein by approximately 23%, compared to without attached quantum dots, suggesting direct energy transfer mechanisms beyond photonic emission alone. The ability of quantum dots to activate nanoscale regions on bacteriorhodopsin-based electrodes could allow sub-micron sensing arrays to be created due to the ability to activate site-specific regions on the array. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Throughout history the use of biological materials in engineered applications has led to dramatic innovations. The field of nanotechnology is no different, as there are arrays of natural nanoscale materials with capabilities beyond that of current technology. The efficient integration of select biological materials with engineered nanomaterials will be a critical step for expanding nanotechnological applications. One such biological material with broad nanoengineering potential is the integral membrane protein bacteriorhodopsin (bR). Bacteriorhodopsin is a retinal containing protein found in the cell membrane of the extremophile Halobacterium salinarum and is utilized to generate a proton motive force that energizes adenine triphosphate (ATP) synthase to drive the conversion of adenine diphosphate (ADP) and Pi to ATP and H2 O, thereby providing the energy to drive the cell’s internal machinery. The proton motive force is achieved when bR’s attached retinal chromophore absorbs visible light in the 570 nm region, resulting in a trans–cis isomerization of the prosthetic group. This structural alteration initiates proton transport from the cytoplasmic side to the extracellular side of the cell membrane, creating a proton gradient across the membrane system (Lanyi, 2004). For engineered applications, coupling the bR protein with an electrically conductive substrate rather than an ATP synthase allows the efficient quantum conversion of light energy into an
∗ Corresponding author. Tel.: +1 906 487 1922. E-mail address: [email protected] (C.R. Friedrich). 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.11.005
electric potential by charge translocation. Bacteriorhodopsin can be purified as membrane patches, known as purple membrane (PM). Purple membrane, intrinsic to the cellular membrane of H. salinarum, has an average diameter of 500 nm and is comprised of multiple bR molecules and associated phospholipids. In the dried and wet states, PM retains its light absorption properties and photochemical activity for years (Vsevolodov, 1998). The potential applications for PM in engineered devices are extensive; including information storage, information processing, charge transport membranes, and photoelectric systems (Birge et al., 1994; Min et al., 2001; Choi et al., 2005). In the current work, the photoelectric property of bR was used as the transducer substrate for a bionanosensing device. For this application PM needs to be patterned and activated on a microelectronics array to create a multitude of sensing sites in a small chip area. To photonically activate individual PM sensing sites without contributing signal to neighboring sites, a 570 nm light-source needs to be incorporated that is on the same size scale as the bR molecules. To facilitate the activation of a bR array at the nanoscale, the photonic output of quantum dots (QD) was explored. As will be shown, the usefulness of quantum dots is two-fold; contributing both to bR activation, and to the proposed sensing mechanism. The QDs used in this study are colloidal semiconductor QDs composed of a cadmium selenide (CdSe) core and an optional zinc sulfide (ZnS) protective shell. They are semiconductor particles that have dimensions on the nanometer scale, typically 2–10 nm. Due to their small dimensions, below the exciton Bohr radius of the semiconducting material, QDs have discrete energy levels with bandgaps that can be fine tuned based on the size of the dot. Since
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Fig. 1. Normalized bR absorption and 575 nm QD emission, displaying spectral overlap.
the size of the bandgap determines the energy of the electromagnetic radiation emitted by the electron when it falls back to the valence band, the wavelength emitted by the QD can be selected by its size. In this study, the QD emission peak was selected to correspond to the absorption maximum of bacteriorhodopsin, as shown in Fig. 1. Along with providing a narrow emission spectrum, QDs have a broad absorption range with a local maximum in the ultra-violet (UV) region. When compared to other fluorescing materials, such as fluorescent dyes, QDs have vastly improved characteristics including increased quantum yield, chemical stability, lifetime, ease of emission tunability, broad absorption range, and a greater resistance to photo-bleaching (Jovin, 2003). Apart from the intrinsic properties, the surface chemistry of QDs has also been shown to be amenable to the addition of functionalized binding materials, allowing the use of QDs in a wide array of sensor applications. 2. Materials and methods 2.1. Protein preparation and electrode construction The PM used in these studies was obtained from H. salinarium strain S9P (courtesy J. Spudich) and prepared on-site according to a modified version of previously reported methods (Oesterhelt and Stoeckenius, 1974). The S9P strain is an over-producer of bR so the PM patches are highly dense with protein. Details of the preparation methodology are provided in the supplementary materials. Due to the uni-directional proton pumping direction of bR, the PM films used to make the photosensitive electrodes must be highly oriented (Gergely et al., 1993). This allows a maximum number of protons to be vectorially shifted in the same direction, yielding a higher electrical voltage upon illumination. The high degree of protein orientation for these studies was obtained by electrophoretic sedimentation (EPS) (Keszthelyi, 1980; Varo and Keszthelyi, 1983). Two different approaches were undertaken for stimulating the PM by quantum dots; first by physical separation of the PM and QDs by glass, and second by chemically linking the PM and QDs utilizing a biotin and streptavidin binding scheme. These two separate setups isolate QD optical activation in the first, and potential direct energy transfer mechanisms in the second. For both investigations, the PM electrodes were fabricated in the same basic manner. The electrode (anode) was a glass slide with a thin sputtered layer of indium tin oxide (ITO). The dimensions of the electrode were 5 mm × 10 mm and the ITO had a sheet resistance of 5–10 /sq. A 40 l suspension of 15 mg ml−1 PM in pH 6.5 ddI water was pipetted on top of the ITO and a brass electrode (cathode) was placed on
top of the suspension with 1 mm spacing between the electrodes. An electric field of 40 VDC cm−1 was applied across the electrodes for 1 min. Due to the elevated negative dipole charge of bR’s cytoplasmic region, the PM will preferentially orient and attach to the ITO cathode when the electric field potential is applied. Following the 1 min exposure to the electric field, the top brass electrode was removed and the ITO electrode with attached PM was agitated for 10 s in a beaker of ddI water to remove any weakly attached PM. The ITO electrode was dried in a humidity chamber, maintained at 52% relative humidity with a magnesium nitrate salt solution, for 24 h. The dimensions of the dried bR film were approximately 5 mm × 5 mm with a thickness of approximately 20 m. After drying, another ITO-coated glass electrode of similar dimensions was placed on top of the oriented PM film to create the measurement circuit. The PM electrode was wired directly into a custom-built, T input impedance, non-inverting operational amplifier, similar to those previously reported (Wang et al., 2006), and configured to 10× amplification. The ITO electrode with the cytoplasmic side of the attached PM was set to ground (−) and the opposing ITO electrode was attached to the op-amp (+). Both the op-amp and PM electrode were placed in a Faraday cage. The non-inverting opamp was powered by an HP 6237A triple output voltage supply. The signal from the op-amp was passed through a highpass filter (Krohn-Hite Model 3364) with a cutoff frequency set at 30 Hz. From the filter, the conditioned signal was input to a 60 MHz, 200 MSa/s oscilloscope (Agilent 54621D) for digital display and recording. 2.2. Quantum dot quenching effects of bR To help clarify the amount of energy and the energy transfer mechanism, the fluorescent alteration of QDs in response to close proximity bR molecules was analyzed. The emission of thin film QDs on glass, dried bR, and bleached bR films was measured. Bleaching of bR yield the apo-protein, bacterio-opsin (bO), in which the associated prosthetic retinal group has been released. The retinal is responsible for the light absorption around 570 nm and the proton pumping cycle, therefore without the retinal all functional properties of bR are lost. One method to remove the bR retinal is through continuous illumination with high intensity light (Dancshazy et al., 1999). Depending on the light intensity, the bleaching process can take several days. To increase the speed of this process, the pH of the PM solution can be increased and hydroxylamine can be added. For these studies a 10 mg/ml PM solution was used. Hydroxylamine was added to the solution at 0.3 M and the pH was increased to 7.5. The solution was then subjected to continuous high intensity light illumination at 500 mW/cm2 . The degree of retinal release was analyzed by monitoring the bR570 absorbance peak. It was revealed that a high degree of bleaching, >80%, was achieved in less than 24 h. Upon reaching near complete bleaching, the bO membrane solution was pelleted out of solution at 40,000 times gravity centrifugation and washed multiple times with ddI water to remove any residual hydroxylamine. The experimental setup compared the dried film fluorescence of 566 nm CdSe and 575 nm CdSe/ZnS QDs (Evident Technologies) when layered on a control (blank glass slide), dried bR film (5 l of 5 mg/ml PM), and dried bO film (5 l of 5 mg/ml). Each of the QD samples was suspended in toluene at a concentration of 0.5 nmol/ml. A total of 1 l of the 0.5 nmol/ml QD solution was applied to the desired substrate for each particular test. The estimated 3 mm diameter dimensions of deposited QDs would create a film no more than 1 QD thick, on average, thus putting a majority of QDs in direct contact with the selected substrate.
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2.3. Quantum dot integration with bR electrodes In the first investigation of the QD activation of the bR photoresponse, the quantum dots used for remote photonic activation of bR were 575 nm emission CdSe/ZnS QDs (Evident Technologies). The QDs were suspended in toluene at a concentration of 63 nmol ml−1 . A total of 40 l of the CdSe/ZnS suspension was added to a well constructed on top of the bR-based electrode, providing a glass barrier between the bR and QDs to eliminate any unwanted physical reactions between the two materials and to isolate the interactions solely to QD photonic emission and bR photonic absorption. Electrical measurements were made as described. In the second QD/bR electrode investigation, electrodes were created as described above but with biotinylated bR, which would provide a binding point for streptavidin-coated QDs. The small biotin molecule has an extremely high binding affinity for the streptavidin protein, which ensures specific and tight binding at all locations. Streptavidin, a 53 kDa protein, and biotin, also known as vitamin H or B7 , are together widely regarded as the strongest non-covalent bond in nature, with a dissociation constant (Kd ) of 4 × 10−14 M (Weber et al., 1989). With its robust nature and virtually unbreakable association, the biotin–streptavidin binding scheme has been well studied in applications of nanoparticle linkers (Cui et al., 2001; Oh et al., 2005). The initial step was to biotinylate the single Lysine-129 amino acid residue on the bR molecule, which has been reported previously (Chen et al., 2003; Sharma et al., 2004) and is further elaborated upon in the supplementary materials. This procedure resulted in a liquid suspension of PM fragments containing covalently bound biotin groups on the exposed bR molecules. Using a HABA/avidin assay the molar ratio of biotins per bR molecule was determined to be 0.4:1, respectively, showing that nearly 1 in every 2 bR molecules was effectively biotinylated. The biotinylated bR was then put into electrode form and the output was measured. For this work, streptavidin-coated 595 nm emission CdSe/ZnS QDs (Invitrogen) were used. Fresh biotinylated bR electrodes described above were submerged in a 0.5 nmol ml−1 QD solution for 5 min to facilitate full biotin–streptavidin linkage formation. The functionalized slide was vigorously rinsed for 2 min in ddI water to remove any unbound QD particles and the electrode output was measured. 3. Results and discussion The initial studies monitoring the effects of bR films on QD emission properties focused upon determining if the bR retinal possessed the ability to absorb the emitted QD energy. In Fig. 2(a) it can be seen that the deposition of a thin 566 nm CdSe QD film on top of a bR film resulted in a 54.5% reduction in the measured QD fluorescence output as compared to the same amount of QDs on a cleaned glass substrate. Due to the large spectral overlap of the QD emission and bR absorption peaks, as shown in Fig. 1, a large portion of the quenching effect is likely due to absorption of the emitted QD photons by the bR retinal. To confirm that the bR retinal was in fact responsible for the QD energy reduction, an identical substrate was created using bleached bR, where the retinal was removed from the bR molecule. Without the retinal these bO films were white in color, as compared to the distinct purple color of bR, due to its inability to absorb photons in the visible light spectrum. As shown in Fig. 2(b), the bleached bR substrate induced no quenching effects on the QDs, similar to that of the glass substrate control. This confirms that the bR retinal is the source of QD quenching in the experimental dried films. With results confirming that the QDs emitted energy can be absorbed by the bR retinal in dried films, work was performed to analyze the effect of separation distance between the bR film and deposited QDs. The alteration in bR–QD separation distance is
Fig. 2. Influence of substrate on QD fluorescence properties for (a) 566 nm CdSe and 575 nm CdSe/ZnS QDs on glass (control) and bR electrode substrates, and (b) 566 nm CdSe QDs on glass, bR electrode, and bleached bR electrode substrates.
accomplished through the use of CdSe (core) and CdSe/ZnS (coreshell) QDs. The ZnS polymeric shell on the CdSe will provide an approximate additional 1–1.5 nm spacer between the CdSe semiconducting material and the bR retinal in the dried films. When layering CdSe and CdSe/ZnS QDs directly on top of dried bR films, results show that the bR substrate induces a strong quenching effect on the emission of both QD types, as shown in Fig. 2(a). The magnitude of the induced QD quenching is 54.5% for the CdSe QDs and 43.8% for the core-shell CdSe/ZnS QDs, thus the slight change in separation distance produces a noticeable alteration in the QD quenching effects. There is a distinct difference between the degree of quenching for the core and core-shell QDs, as the addition of a ZnS shell on the CdSe QD reduces the energy loss to the bR molecules by 19.5% compared to the plain CdSe QDs. An explanation for the difference in quenching effects is the potential existence of a direct energy transfer relationship, or forster resonance energy transfer (FRET), between the CdSe QD core and the bR retinal. The FRET process is sensitive to the donor/acceptor (QD/bR) separation distance, particularly in the sub-10 nm region. Thus by increasing the QD/bR separation distance by adding polymeric shells to the QDs less energy was transferred non-radiatively from the QD to the bR retinal. This results in the increased QD emission over closer proximity non-shell QDs. With results suggesting that both photonic and non-photonic energy transfer mechanisms potentially exist in a close proximity bR–QD system, different bR–QD activation setups were created to isolate each possible mechanism. The first setup implemented a spacer to take the QDs out of direct energy coupling range of the bR, thus isolating QD photonic activation of the bR photovoltaic response. To establish a control response for comparison, a 310 nm UV-light emitting diode (LED) light source was used. Previous work has shown that the energy of normal incidence UV light, absorbed by the aromatic amino acids in bR, can be transferred to the retinal through resonance and initiate the photocycle (Kalisky et al., 1981). Therefore the angle of the UV source for QD activation was adjusted to avoid bR illumination and activation of the bR photocycle. In this control experiment, and prior to introducing QDs in the three
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Fig. 3. bR photoelectric response to (a) ∼4 Hz UV flashing – no QD’s (1 mV/vertical division, 500 ms/horizontal division) and (b) 4 Hz remote QD illumination (2 mv/vertical division, 500 ms/horizontal division).
methods described, the UV-LED was adjusted to an approximate 60◦ incidence angle. With no QDs in the proximity of the bR electrode, no apparent bR photovoltaic response was elicited from the angled UV illumination. The electrical response of this system during ∼4 Hz UV flashing is shown in Fig. 3(a). To investigate whether QDs can remotely activate bR, 40 l CdSe/ZnS of QD suspension (63 nmol/ml) was placed into the well separated from the top of the bR electrode. Upon subsequent ∼4 Hz UV excitation a detectable photovoltaic response is seen from the PM as shown in Fig. 3(b). With the addition of 575 nm emission CdSe/ZnS QDs on top of the bR electrode and subsequent UV illumination, a measurable electrical response was generated from the bR. The flashing of the UV-LED visibly activated the QD suspension and the electrical output of the bR electrode was monitored, as shown in Fig. 3(b). The cyclic signal from the bR-remote-QD electrode directly correlates to the flashing of the UV-LED. In the test system, it was calculated that there was approximately 1 QD for every 10 bR molecules. Assuming that every bR molecule was photonically activated, each quantum dot was potentially activating several bR molecules. This shows the potential capability of utilizing quantum dots in a nanoscale system since a single top monolayer of quantum dots might activate several monolayers of PM. To analyze the ability for QDs to activate bR through both photonic and non-photonic means, dried films were created to place a single QD monolayer in direct contact with the bR electrode. Achieving this bR–QD thin film structure was accomplished through the use of biotinylated bR as the attachment mechanism to bind streptavidin-coated quantum dots to the PM-based electrode. To determine the presence and functionality of the biotin groups for the biotinylated bR-based electrodes, the fluorescence emission of biotinylated and non-biotinylated PM electrodes exposed to QDs was analyzed on a Hitachi-F7000 fluorospectrometer to determine the presence of bound QDs. The resulting fluorescence spectrum, displayed in Fig. 4(a), shows that the streptavidin-coated QDs only bind to the biotinylated PM electrodes. Thus the biotinylated bR electrodes created by EPS retain the capacity to effectively bind to a functional streptavidin group. This linkage scheme allows for the
Fig. 4. (a) Emission spectra confirming attachment of single QD monolayer to the biotinylated PM electrode, while non-biotinylated PM resulted in no QD attachment. (b) bR electrode activation with and without integrated QD monolayer. Inset displays spectrum of incident white-light source.
binding of a single QD monolayer on top of the bR electrode, holding the QD–bR separation distance at approximately 6–8 nm. With the binding of a QD monolayer to the bR electrode achieved, the effects of the added nanoscale power source to the overall bR photovoltaic output was tested and the resulting voltage characteristics are shown in Fig. 4(b). It can be seen in Fig. 4(b) that the binding of the QDs to the surface of the PM electrode resulted in an increased bR photovoltaic response to white-light illumination. Without any QDs present, the bR electrode yielded a 0.249 V peak transient response upon illumination followed by a −0.158 V peak upon termination of the light. When a QD layer was then bound to the same sample and re-activated, the initial peak transient yielded a bR photovoltage of 0.306 V followed by a −0.2 V peak upon termination of the light. The addition of the QDs to the PM electrode increased the bR photovoltaic output by approximately 57 mV, or 23%. The photovoltaic on/off peak ratios of 1.42 and 1.55 for the PM and PM–QD electrodes are similar to the ratio of 1.5 that has been previously reported for PM electrodes. In dried PM films it has been shown that the decreased water content of the bR molecule significantly alters the normal photocycle process (Váró and Keszthelyi, 1985; Ganea et al., 1997). In particular the N and O photocycle intermediates, which are required for the proton uptake and release mechanism, cease to function. Therefore the photoelectric responses produced in dried PM films are due to charge translocation via retinal isomerization (Wang et al., 2006). Since the innate functionality of the bR retinal group is retained in dried films, it has the ability to both accept photons and potential resonance energy directly from the close proximity QD energy source. The photoelectric responses of the PM and PM–QD electrodes are similar in structure, only varying in magnitude, thus it is assumed that the dried film photocycle is not significantly altered with the accepted QD energy. These studies demonstrate that QDs can enhance the bR photovoltaic response, even with white-light illumination that overlaps
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the visible spectra of the QD absorption band. Since the QD are within a sub-10 nm proximity to the bR molecules and there is maximal bR–QD spectral overlap, the potential for a direct energy transfer relationship also exists in addition to photonic activation. The large magnitude photovoltaic increase of 23%, potentially consisting of both direct energy transfer and photonic activation of the bR, is of sufficient magnitude that alterations to the QD emission could detectably affect the bR output. This establishes a novel bionanosensing platform in which the disruption of the QD energy transmission to the bR molecule, upon target molecule binding, would result in a detectably altered electrical output. 4. Conclusions With the ability to initiate and amplify the bR photovoltage output with the energy released from chemically linked CdSe quantum dots, the development of a nanosensing array utilizing these nanomaterials is feasible. Results demonstrate the ability to activate the bR photovoltaic response from the photonic emission of a size-selected CdSe QD suspension with UV illumination. Creating a solid-state bR–QD nano-bio-hybrid material, with a dried QD monolayer in sub-10 nm proximity to the bR molecules, allows for a large amplification in the protein photovoltaic response even with a traditional white-light source. Due to the close proximity and optimal spectral overlap, it is feasible that the bR activation energy results in part from a direct energy transfer mechanism from the QDs, in addition to normal photonic absorptive processes. The utility of a direct energy transfer mechanism in a bR–QD hybrid system would not only allow for isolated pixels in an array platform, but may also provide a potential signal modulation point to achieve a bR–QD bio-nanosensing system. Acknowledgements We thank Chris Anton and Dawdon Cheam of the Michigan Technological University for their aid and input on various aspects of this
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project. The research reported in this document was performed in connection with contract DAAD17-03-C-0115 with the U.S. Army Research Laboratory. The research was also partially supported under a National Science Foundation Graduate Research Fellowship for Mark Griep. Appendix A. Supplementary data Further details pertaining to the bR production/purification techniques and the bR biotinylation procedure are given in the supplementary information section. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2009.11.005. References Birge, R.R., Govender, D.S.K., et al., 1994. Bioelectronics, Three-dimensional Memories and Hybrid Computers. Electron Devices Meeting, 1994. IEDM’94. Technical Digest. International. Chen, D.-l., Lu, Y.-j., et al., 2003. J. Phys. Chem. 107, 3598–3605. Choi, H., Lee, H., et al., 2005. Nanotechnology 16, 1589–1597. Cui, Y., Wei, Q., et al., 2001. Science 293 (5533), 1289–1292. Dancshazy, Z., Tokaji, Z., et al., 1999. FEBS Lett. 450, 154–157. Ganea, C., Gergely, C., et al., 1997. Biophys. J. 73, 2718–2725. Gergely, C., Ganea, C., et al., 1993. Biophys. J. 65, 2478–2483. Jovin, T.M., 2003. Nat. Biotechnol. 21, 32–33. Kalisky, O., Feitelson, J., et al., 1981. Biochemistry 20, 205–209. Keszthelyi, L., 1980. Biochem. Biophys. Acta 598, 429–436. Lanyi, J.K., 2004. Annu. Rev. Physiol. 66 (1), 665–688. Min, J., Choi, H.-G., et al., 2001. Biosens. Bioelectron. 16, 917–923. Oesterhelt, D., Stoeckenius, W., 1974. Methods Enzymol. 31, 667–678. Oh, E., Hong, M.Y., et al., 2005. J. Am. Chem. Soc. 127 (10), 3270–3271. Sharma, M.K., Jatteni, H., et al., 2004. Bioconjugate Chem. 15, 942–947. Varo, G., Keszthelyi, L., 1983. Biophys. J. 43, 47–51. Váró, G., Keszthelyi, L., 1985. Biophys. J 47 (2), 243–246. Vsevolodov, N., 1998. Biomolecular Electronics an Introduction via Photosensitive Proteins. Birkhauser, Boston. Wang, W., Knopf, G., et al., 2006. Biosens. Bioelectron. 21, 1309–1319. Weber, P.C., Ohlendorf, D.H., et al., 1989. Science 243 (4887), 85–88.