Single-molecular protein-based bioelectronics via electronic transport: fundamentals, devices and applications.

Biomolecular electronics is a rapidly growing multidisciplinary field that combines biology, nanoscience, and engineering to bridge the two important fields of life sciences and molecular electronics. Proteins are remarkable for their ability to recognize molecules and transport electrons, making the integration of proteins into electronic devices a long sought-after goal and leading to the emergence of the field of protein-based bioelectronics, also known as proteotronics. This field seeks to design and create new biomolecular electronic platforms that allow for the understanding and manipulation of protein-mediated electronic charge transport and related functional applications. In recent decades, there have been numerous reports on protein-based bioelectronics using a variety of nano-gapped electrical devices and techniques at the single molecular level, which are not achievable with conventional ensemble approaches. This review focuses on recent advances in physical electron transport mechanisms, device fabrication methodologies, and various applications in protein-based bioelectronics. We discuss the most recent progress of the single or few protein-bridged electrical junction fabrication strategies, summarise the work on fundamental and functional applications of protein bioelectronics that enable high and dynamic electron transport, and highlight future perspectives and challenges that still need to be addressed. We believe that this specific review will stimulate the interdisciplinary research of topics related to protein-related bioelectronics, and open up new possibilities for single-molecule biophysics and biomedicine.

Bacterial mineralization of chromium-copper spinel with highly performance in electroplating effluent.

Cr (VI) contamination has posed severe challenges to water quality, food safety, and land resources. Microbial reduction of Cr(VI) to Cr(III) has drawn considerable attention due to its low cost and environmental friendliness. However, recent reports have shown that Cr(VI) generates highly migratable organo-Cr(III) rather than stable inorganic chromium minerals during the biological reduction process. In this work, it was reported for the first time that spinel structure CuCr2O4 was formed by Bacillus cereus in Cr biomineralization process. Different from known biomineralization models (biologically controlled mineralization and biologically induced mineralization), the chromium-copper minerals here appeared as specialized minerals with extracellular distribution. In view of this, a possible mechanism of biologically secretory mineralization was proposed. In addition, Bacillus cereus demonstrated a high conversion ability in the treatment of electroplating wastewater. The Cr(VI) removal percentage reached 99.7%, which satisfied the Chinese emission standard of pollutants for electroplating (GB 21,900-2008), indicating its application potential. Altogether, our work elucidated a bacterial chromium spinel mineralization pathway and evaluated the potential of this system for application in actual wastewater, opening a new avenue in the field of chromium pollution treatment and control.

Mesoporous Silica Nanoparticles Induce Intracellular Peroxidation Damage of Phytophthora infestans: A New Type of Green Fungicide for Late Blight Control.

Nanopesticides are considered to be a promising alternative strategy for enhancing bioactivity and delaying the development of pathogen resistance to pesticides. Here, a new type of nanosilica fungicide was proposed and demonstrated to control late blight by inducing intracellular peroxidation damage to Phytophthora infestans, the pathogen associated with potato late blight. Results indicated that the structural features of different silica nanoparticles were largely responsible for their antimicrobial activities. Mesoporous silica nanoparticles (MSNs) exhibited the highest antimicrobial activity with a 98.02% inhibition rate of P. infestans, causing oxidative stress responses and cell structure damage in P. infestans. For the first time, MSNs were found to selectively induce spontaneous excess production of intracellular reactive oxygen species in pathogenic cells, including hydroxyl radicals (•OH), superoxide radicals (•O2-), and singlet oxygen (1O2), leading to peroxidation damage in P. infestans. The effectiveness of MSNs was further tested in the pot experiments as well as leaf and tuber infection, and successful control of potato late blight was achieved with high plant compatibility and safety. This work provides new insights into the antimicrobial mechanism of nanosilica and highlights the use of nanoparticles for controlling late blight with green and highly efficient nanofungicides.

Measuring conductance switching in single proteins using quantum tunneling.

Interpreting the electrical signatures of single proteins in electronic junctions has facilitated a better understanding of the intrinsic properties of proteins that are fundamental to chemical and biological processes. Often, this information is not accessible using ensemble and even single-molecule approaches. In addition, the fabrication of nanoscale single-protein junctions remains challenging as they often require sophisticated methods. We report on the fabrication of tunneling probes, direct measurement, and active control (switching) of single-protein conductance with an external field in solution. The probes allowed us to bridge a single streptavidin molecule to two independently addressable, biotin-terminated electrodes and measure single-protein tunneling response over long periods. We show that charge transport through the protein has multiple conductive pathways that depend on the magnitude of the applied bias. These findings open the door for the reliable fabrication of protein-based junctions and can enable their use in future protein-embedded bioelectronics applications.

Water evaporation-induced electricity with Geobacter sulfurreducens biofilms.

Water evaporation-induced electricity generators (WEGs) have recently attracted extensive research attention as an emerging renewable energy-harvesting technology that harvests electricity directly from water evaporation. However, the low power output, limited available material, complicated fabrication process, and extremely high cost have restricted wide applications of this technology. Here, a facile and efficient WEG prototype based on Geobacter sulfurreducens biofilm was demonstrated. The device can generate continuous electric power with a maximum output power density of ~685.12 µW/cm2, which is two orders of magnitude higher than that of previously reported analogous devices. The superior performance of the device is attributed to the intrinsic properties of the G. sulfurreducens biofilm, including its hydrophilicity, porous structure, conductivity, etc. This study not only presents the unprecedented evaporating potential effect of G. sulfurreducens biofilms but also paves the way for developing hydrovoltaic technology with biomaterials.

Electronic Transport in Molecular Wires of Precisely Controlled Length Built from Modular Proteins.

DNA molecular wires have been studied extensively because of the ease with which molecules of controlled length and composition can be synthesized. The same has not been true for proteins. Here, we have synthesized and studied a series of consensus tetratricopeptide repeat (CTPR) proteins, spanning 4 to 20 nm in length, in increments of 4 nm. For lengths in excess of 6 nm, their conductance exceeds that of the canonical molecular wire, oligo(phenylene-ethylenene), because of the more gradual decay of conductance with length in the protein. We show that, while the conductance decay fits an exponential (characteristic of quantum tunneling) and not a linear increase of resistance with length (characteristic of hopping transport), it is also accounted for by a square-law dependence on length (characteristic of weakly driven hopping). Measurements of the energy dependence of the decay length rule out the quantum tunneling case. A resonance in the carrier injection energy shows that allowed states in the protein align with the Fermi energy of the electrodes. Both the energy of these states and the long-range of hopping suggest that the reorganization induced by hole formation is greatly reduced inside the protein. We outline a model for calculating the molecular-electronic properties of proteins.

Probing Bioelectronic Connections Using Streptavidin Molecules with Modified Valency.

As molecular electronic components, proteins are distinguished by a remarkably long electronic decay length (∼10 nm) together with high contact resistance and extreme sensitivity to the chemical details of the contact. As a consequence, the conductance of even a large bioelectronic assembly is largely controlled by the conductance of the contacts. Streptavidin is a versatile linker protein that can tether together biotinylated electrodes and biotinylated proteins but with an ambiguity about the contact geometry that arises from its four possible binding sites for biotin. Here, we use engineered streptavidin tetramers, selected to contain a defined ratio of active monomers to "dead" monomers so as to define the biotin binding sites. We find a strong dependence of conductance on the separation of the biotin molecules, consistent with a short-range tunneling interaction within the streptavidin and in contrast to the long-range transport observed inside larger proteins. Hexaglutamate tails label the active monomers, and the additional negative charge enhances conductance significantly. This effect is quantitatively accounted for by an electronic resonance in the protein conductance.

Electronic Conductance Resonance in Non-Redox-Active Proteins.

Bioelectronics research has mainly focused on redox-active proteins because of their role in biological charge transport. In these proteins, electronic conductance is a maximum when electrons are injected at the known redox potential of the protein. It has been shown recently that many non-redox-active proteins are good electronic conductors, though the mechanism of conduction is not yet understood. Here, we report single-molecule measurements of the conductance of three non-redox-active proteins, maintained under potential control in solution, as a function of electron injection energy. All three proteins show a conductance resonance at a potential ∼0.7 V removed from the nearest oxidation potential of their constituent amino acids. If this shift reflects a reduction of reorganization energy in the interior of the protein, it would account for the long-range conductance observed when carriers are injected into the interior of a protein.

Engineering an Enzyme for Direct Electrical Monitoring of Activity.

Proteins have been shown to be electrically conductive if tethered to an electrode by means of a specific binding agent, allowing single molecules to be wired into an electrical sensing circuit. Such circuits allow enzymes to be used as sensors, detectors, and sequencing devices. We have engineered contact points into a Φ29 polymerase by introducing biotinylatable peptide sequences. The modified enzyme was bound to electrodes functionalized with streptavidin. Φ29 connected by one biotinylated contact, and a second nonspecific contact showed rapid small fluctuations in current when activated. Signals were greatly enhanced with two specific contacts. Features in the distributions of DC conductance increased by a factor 2 or more over the open to closed conformational transition of the polymerase. Polymerase activity is manifested by a rapid (millisecond) large (25% of background) current fluctuations imposed on the DC conductance.

Electronic Decay Length in a Protein Molecule.

Antibodies have two identical binding domains and can therefore form a well-defined conducting bridge by binding a pair of electrodes functionalized with an epitope. The conductance measured between these two fixed points on the antibody does not change with the size of the electrode gap. A second conduction path is via one specific attachment to an epitope and a second nonspecific attachment to the surface of the antibody. In this case, the conductance does change with gap size, yielding an estimated electronic decay length >6 nm, long enough that it is not possible to distinguish between an exponential or a hyperbolic distance dependence. This decay length is substantially greater than that measured for hopping transport in an organic molecular wire.

Role of contacts in long-range protein conductance.

Proteins are widely regarded as insulators, despite reports of electrical conductivity. Here we use measurements of single proteins between electrodes, in their natural aqueous environment to show that the factor controlling measured conductance is the nature of the electrical contact to the protein, and that specific ligands make highly selective electrical contacts. Using six proteins that lack known electrochemical activity, and measuring in a potential region where no ion current flows, we find characteristic peaks in the distributions of measured single-molecule conductances. These peaks depend on the contact chemistry, and hence, on the current path through the protein. In consequence, the measured conductance distribution is sensitive to changes in this path caused by ligand binding, as shown with streptavidin-biotin complexes. Measured conductances are on the order of nanosiemens over distances of many nanometers, orders of magnitude more than could be accounted for by electron tunneling. The current is dominated by contact resistance, so the conductance for a given path is independent of the distance between electrodes, as long as the contact points on the protein can span the gap between electrodes. While there is no currently known biological role for high electronic conductance, its dependence on specific contacts has important technological implications, because no current is observed at all without at least one strongly bonded contact, so direct electrical detection is a highly selective and label-free single-molecule detection method. We demonstrate single-molecule, highly specific, label- and background free-electronic detection of IgG antibodies to HIV and Ebola viruses.

Cucurbituril mediated single molecule detection and identification via recognition tunneling.

Recognition tunneling (RT) is an emerging technique for investigating single molecules in a tunnel junction. We have previously demonstrated its capability of single molecule detection and identification, as well as probing the dynamics of intermolecular bonding at the single molecule level. Here by introducing cucurbituril as a new class of recognition molecule, we demonstrate a powerful platform for electronically investigating the host-guest chemistry at single molecule level. In this report, we first investigated the single molecule electrical properties of cucurbituril in a tunnel junction. Then we studied two model guest molecules, aminoferrocene and amantadine, which were encapsulated by cucurbituril. Small differences in conductance and lifetime can be recognized between the host-guest complexes with the inclusion of different guest molecules. By using a machine learning algorithm to classify the RT signals in a hyper dimensional space, the accuracy of guest molecule recognition can be significantly improved, suggesting the possibility of using cucurbituril molecule for single molecule identification. This work enables a new class of recognition molecule for RT technique and opens the door for detecting a vast variety of small molecules by electrical measurements.

Retraction: Synthesis and facet-dependent photocatalytic activity of BiOBr single-crystalline nanosheets.

Retraction of 'Synthesis and facet-dependent photocatalytic activity of BiOBr single-crystalline nanosheets' by Wenwen Lin et al., Chem. Commun., 2014, DOI: 10.1039/c3cc41498a

Observation of Giant Conductance Fluctuations in a Protein.

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically-inactive protein, the ~200 kD αVß3 extracelluar domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly-binding protein (α4ß1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (~ nS) state were observed, the protein being "on" for times from ms to tenths of a second. The high-conductance states only occur above ~ 100mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states.

[Ru(bpy)3]²âº-mediated photoelectrochemical detection of bisphenol A on a molecularly imprinted polypyrrole modified SnO2 electrode.

A ruthenium-mediated photoelectrochemical sensor was developed for the detection of BPA, using molecularly imprinted polymers (MIPs) as the recognition element, a tin oxide (SnO2) nanoparticle-modified ITO as the electrode, and a blue 473-nm LED as the excitation light source. Photoelectrochemical oxidation of BPA on SnO2 electrode was achieved by [Ru(bpy)3](2+) under the irradiation of light. It was found that BPA was oxidized by Ru(3+) species produced in the photoelectrochemical reaction, resulting in the regeneration of Ru(2+) and the concomitant photocurrent enhancement. MIPs film was prepared by electropolymerization of pyrrole on SnO2 electrode using BPA as the template. Surface morphology and properties of the as-prepared electrode were characterized by SEM, electrochemical impedance spectroscopy, and photocurrent measurement. In the presence of BPA, an enhanced photocurrent was observed, which was dependent on the amount of BPA captured on the electrode. A detection limit of 1.2 nM was obtained under the optimized conditions, with a linear range of 2-500 nM. Selectivity of the sensor was demonstrated by measuring five BPA analogs. To verify its practicality, this sensor was applied to analyze BPA spiked tap water and river water. With advantages of high sensitivity and selectivity, low-cost instrument, and facile sensor preparation procedure, this sensor is potentially suitable for the rapid monitoring of BPA in real environmental samples. Moreover, the configuration of this sensor is universal and can be extended to organic molecules that can be photoelectrochemically oxidized by Ru(3+).

An electrochemiluminescence biosensor for 8-oxo-7,8-dihydro-2'-deoxyguanosine quantification and DNA repair enzyme activity analysis using a novel bifunctional probe.

A new electrochemiluminescence (ECL) sensor was developed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) quantification and Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) activity assay. The sensor employed a novel spermine conjugated ruthenium tris-(bipyridine) derivative (spermine-Ru) which binds specifically with 8-oxodGuo through a one-step reaction and also acts as an ECL signal reporter. In the sensor, an 8-oxodGuo-containing ds-DNA film was first immobilized on a gold electrode by self-assembly. The DNA film was then incubated with spermine-Ru under oxidative condition for 8-oxodGuo labeling. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Addition of FPG resulted in some loss of the signal due to the excision of 8-oxodGuo by the enzyme. An inverse relationship between ECL intensity and FPG concentration was observed in a range from 0 to 4.0U/µL, demonstrating that this sensor could be used for FPG activity assay. A number of metal ions were screened by the sensor for their inhibition effect on FPG activity. Among them, Hg(2+) and methyl Hg(II) shown very potent inhibition, with IC50 values of 4.04µM and 4.34nM respectively. The result may suggest that interference on the DNA repair system could be another mechanism for the high toxicity of MeHg.

ZnO nanoflower-based photoelectrochemical DNAzyme sensor for the detection of Pb2+.

Lead contamination is now widespread, and exposure to lead may cause adverse effects on human beings. In this study, a photoelectrochemical sensor based on flower-like ZnO nanostructures was developed for Pb(2+) detection, using a Pb(2+)-dependent DNAzyme as the recognition unit and a double-strand DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c] phenazine) as the photoelectrochemical signal reporter. The ZnO nanoflower was fabricated on an indium tin oxide (ITO) electrode by the convenient hydrothermal decomposition method. The morphology and photoelectrochemical property of the ZnO nanoflowers were characterized by SEM, XRD and photocurrent measurements. DNAzyme-substrate duplex was assembled on an ITO/ZnO electrode through electrostatic adsorption. In the presence of Pb(2+), RNA-cleavage activity of the DNAzyme was activated and its substrate strand was cleaved, resulting in the release of Ru(bpy)2(dppz)(2+) from the DNA film and the concomitant photocurrent decrease. The detection principle was verified by fluorescence measurements. Under the optimized conditions, a linear relationship between photocurrent and Pb(2+) concentration was obtained over the range of 0.5-20 nM, with a detection limit of 0.1 nM. Interference from other common metal ions was found negligible. Applicability of the sensor was demonstrated by analyzing lead level in human serum and Pb(2+) spiked water samples. This facile and economical sensor system showed high sensitivity and selectivity, thus can be potentially applied for on-site monitoring of lead contaminant.

Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNA repair enzymes.

Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 10(5) normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes.

Hydroxylated polybrominated diphenyl ethers exhibit different activities on thyroid hormone receptors depending on their degree of bromination.

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions in experimental animals, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to TH receptors (TRs). However, previous data on TH receptor binding and TH activity of OH-PBDEs were very limited and sometimes inconsistent. In the present paper, we examined the binding potency of ten OH-PBDEs with different degrees of bromination to TR using a fluorescence competitive binding assay. The results showed that the ten OH-PBDEs bound to TR with potency that correlated to their bromination level. We further examined their effect on TR using a coactivator binding assay and GH3 cell proliferation assay. Different TR activities of OH-PBDEs were observed depending on their degree of bromination. Four low-brominated OH-PBDEs (2'-OH-BDE-28, 3'-OH-BDE-28, 5-OH-BDE-47, 6-OH-BDE-47) were found to be TR agonists, which recruited the coactivator peptide and enhanced GH3 cell proliferation. However, three high-brominated OH-PBDEs (3-OH-BDE-100, 3'-OH-BDE-154, 4-OH-BDE-188) were tested to be antagonists. Molecular docking was employed to simulate the interactions of OH-PBDEs with TR and identify the structural determinants for TR binding and activity. According to the docking results, low-brominated OH-PBDEs, which are weak binders but TR agonists, bind with TR at the inner side of its binding pocket, whereas high-brominated compounds, which are potent binders but TR antagonists, reside at the outer region. These results indicate that OH-PBDEs have different activities on TR (agonistic or antagonistic), possibly due to their different binding geometries with the receptor.

Quantification of 8-oxodGuo lesions in double-stranded DNA using a photoelectrochemical DNA sensor.

Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO(2) nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ∼1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine-DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 µM Fe(2+)/200 µM H(2)O(2). Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage.