Electrochemical Scanning Tunneling Microscopic Study of the Potential Dependence of Germanene Growth on Au(111) at pH 9.0.

Germanene is a 2D material whose structure and properties are of great interest for integration with Si technology. Preparation of germanene experimentally remains a challenge because, unlike graphene, bulk germanene does not exist. Thus, germanene cannot be directly exfoliated and is mostly grown in ultrahigh vacuum. The present report uses electrodeposition in an aqueous HGeO3- solution at pH 9. Germanene deposition has been limited to 2-3 monolayers, thus greatly restricting many applicable characterization methods. The in situ technique of electrochemical scanning tunneling microscopy was used to follow Ge deposition on Au(111) as a function of potential. Previous work by this group at pH 4.5 suggested germanene growth, but no buffer was used, resulting in change in surface pH. The addition of borate buffer to create pH 9.0 solution has reduced hydrogen formation and stabilized the surface pH, allowing systematic characterization of germanene growth versus potential. Initial germanene nucleated at defects in the Au(111) herringbone (HB) reconstruction. Subsequent growth proceeded down the face-centered cubic troughs, slowly relaxing the HB. The resulting honeycomb (HC) structure displayed an average lattice constant of 0.41 ± 0.06 nm. Continued growth resulted in the addition of a second layer on top, formed initially by nucleating around small islands and subsequent lateral 2D growth. Near atomic resolution of the germanene layers displayed small coherent domains, 2-3 nm, of the HC structure composed of six-membered rings. Domain walls were based on defective, five- and seven-membered rings, which resulted in small rotations between adjacent HC domains.

Enhanced Kinetics of Electrochemical Hydrogen Uptake and Release by Palladium Powders Modified by Electrochemical Atomic Layer Deposition.

Electrochemical atomic layer deposition (E-ALD) is a method for the formation of nanofilms of materials, one atomic layer at a time. It uses the galvanic exchange of a less noble metal, deposited using underpotential deposition (UPD), to produce an atomic layer of a more noble element by reduction of its ions. This process is referred to as surface limited redox replacement and can be repeated in a cycle to grow thicker deposits. It was previously performed on nanoparticles and planar substrates. In the present report, E-ALD is applied for coating a submicron-sized powder substrate, making use of a new flow cell design. E-ALD is used to coat a Pd powder substrate with different thicknesses of Rh by exchanging it for Cu UPD. Cyclic voltammetry and X-ray photoelectron spectroscopy indicate an increasing Rh coverage with increasing numbers of deposition cycles performed, in a manner consistent with the atomic layer deposition (ALD) mechanism. Cyclic voltammetry also indicated increased kinetics of H sorption and desorption in and out of the Pd powder with Rh present, relative to unmodified Pd.

Gd-encapsulated carbonaceous dots with efficient renal clearance for magnetic resonance imaging.

Nanoprobes for MRI and optical imaging are demonstrated. Gd@C-dots possess strong fluorescence and can effectively enhance signals on T1 -weighted MR images. The nanoprobes have low toxicity, and, despite a relatively large size, can be efficiently excreted by renal clearance from the host after systemic injection.

Atomic-layer electroless deposition: a scalable approach to surface-modified metal powders.

Palladium has a number of important applications in energy and catalysis in which there is evidence that surface modification leads to enhanced properties. A strategy for preparing such materials is needed that combines the properties of (i) scalability (especially on high-surface-area substrates, e.g. powders); (ii) uniform deposition, even on substrates with complex, three-dimensional features; and (iii) low-temperature processing conditions that preserve nanopores and other nanostructures. Presented herein is a method that exhibits these properties and makes use of benign reagents without the use of specialized equipment. By exposing Pd powder to dilute hydrogen in nitrogen gas, sacrificial surface PdH is formed along with a controlled amount of dilute interstitial hydride. The lattice expansion that occurs in Pd under higher H2 partial pressures is avoided. Once the flow of reagent gas is terminated, addition of metal salts facilitates controlled, electroless deposition of an overlayer of subnanometer thickness. This process can be cycled to create thicker layers. The approach is carried out under ambient processing conditions, which is an advantage over some forms of atomic layer deposition. The hydride-mediated reaction is electroless in that it has no need for connection to an external source of electrical current and is thus amenable to deposition on high-surface-area substrates having rich, nanoscale topography as well as on insulator-supported catalyst particles. STEM-EDS measurements show that conformal Rh and Pt surface layers can be formed on Pd powder with this method. A growth model based on energy-resolved XPS depth profiling of Rh-modified Pd powder is in general agreement. After two cycles, deposits are consistent with 70-80% coverage and a surface layer with a thickness from 4 to 8 Å.

PtRu nanofilm formation by electrochemical atomic layer deposition (E-ALD).

The high CO tolerance of PtRu electrocatalysis, compared with pure Pt and other Pt-based alloys, makes it interesting as an anode material in proton exchange membrane fuel cells (PEMFC) and direct methanol fuel cells (DMFC). This report describes the formation of bimetallic PtRu nanofilms using the electrochemical form of atomic layer deposition (E-ALD). Metal nanofilm formation using E-ALD is facilitated by use of surface-limited redox replacement (SLRR), where an atomic layer (AL) of a sacrificial metal is first formed by UPD. The AL is then spontaneously exchanged for a more noble metal at the open-circuit potential (OCP). In the present study, PtRu nanofilms were formed using SLRR for Pt and Ru, and Pb UPD was used to form the sacrificial layers. The PtRu E-ALD cycle consisted of Pb UPD at -0.19 V, followed by replacement using Pt(IV) ions at OCP, rinsing with blank, then Pb UPD at -0.19 V, followed by replacement using Ru(III) ions at OCP. PtRu nanofilm thickness was controlled by the number of times the cycle was repeated. PtRu nanofilms with atomic proportions of 70/30, 82/18, and 50/50 Pt/Ru were formed on Au on glass slides using related E-ALD cycles. The charge for Pb UPD and changes in the OCP during replacement were monitored during the deposition process. The PtRu films were then characterized by CO adsorption and electrooxidation to determine their overpotentials. The 50/50 PtRu nanofilms displayed the lowest CO electrooxidation overpotentials as well as the highest currents, compared with the other alloy compositions, pure Pt, and pure Ru. In addition, CO electrooxidation studies of the terminating AL on the 50/50 PtRu nanostructured alloy were investigated by deposition of one or two SLRR of Pt, Ru, or PtRu on top.

Formation of palladium nanofilms using electrochemical atomic layer deposition (E-ALD) with chloride complexation.

Pd thin films were formed by electrochemical atomic layer deposition (E-ALD) using surface-limited redox replacement (SLRR) of Cu underpotential deposits (UPD) on polycrystalline Au substrates. An automated electrochemical flow deposition system was used to deposit Pd atomic layers using a sequence of steps referred to as a cycle. The initial step was Cu UPD, followed by its exchange for Pd ions at open circuit, and finishing with a blank rinse to complete the cycle. Deposits were formed with up to 75 cycles and displayed proportional deposit thicknesses. Previous reports by this group indicated excess Pd deposition at the flow cell ingress, from electron probe microanalysis (EPMA). Those results suggested that the SLRR mechanism did not involve direct transfer between a Cu(UPD) atom and a Pd(2+) ion that would take its position. Instead, it was proposed that electrons are transferred through the metallic surface to reduce Pd(2+) ions near the surface where their activity is highest. It was proposed that if the cell was filled completely before a significant fraction of the Cu(UPD) atoms had been oxidized then the deposit would be homogeneous. Previous work with EDTA indicated that the hypothesis had merit, but it proved to be very sensitive to the EDTA concentration. In the present study, chloride was used to complex Pd(2+) ions, forming PdCl(4)(2-), to slow the exchange rate. Both complexing agents led to a decrease in the rate of replacement, producing more homogeneous films. Although the use of EDTA improved the homogeneity, it also decreased the deposit thickness by a factor of 3 compared to the thickness obtained via the use of chloride.

Electrodeposition of CuInSe2 (CIS) via electrochemical atomic layer deposition (E-ALD).

The growth of stoichiometric CuInSe(2) (CIS) on Au substrates using electrochemical atomic layer deposition (E-ALD) is reported here. Parameters for a ternary E-ALD cycle were investigated and included potentials, step sequence, solution compositions and timing. CIS was also grown by combining cycles for two binary compounds, InSe and Cu(2)Se, using a superlattice sequence. The formation, composition, and crystal structure of each are discussed. Stoichiometric CIS samples were formed using the superlattice sequence by performing 25 periods, each consisting of 3 cycles of InSe and 1 cycle of Cu(2)Se. The deposits were grown using 0.14, -0.7, and -0.65 V for Cu, In, and Se precursor solutions, respectively. XRD patterns displayed peaks consistent with the chalcopyrite phase of CIS, for the as-deposited samples, with the (112) reflection as the most prominent. AFM images of deposits suggested conformal deposition, when compared with corresponding image of the Au on glass substrate.

Growth of Ge nanofilms using electrochemical atomic layer deposition, with a "bait and switch" surface-limited reaction.

Ge nanofilms were deposited from aqueous solutions using the electrochemical analog of atomic layer deposition (ALD). Direct electrodeposition of Ge from an aqueous solution is self-limited to a few monolayers, depending on the pH. This report describes an E-ALD process for the growth of Ge films from aqueous solutions. The E-ALD cycle involved inducing a Ge atomic layer to deposit on a Te atomic layer formed on Ge, via underpotential deposition (UPD). The Te atomic layer was then reductively stripped from the deposit, leaving the Ge and completing the cycle. The Te atomic layer was bait for Ge deposition, after which the Te was switched out, reduced to a soluble telluride, leaving the Ge (one "bait and switch" cycle). Deposit thickness was a linear function of the number of cycles. Raman spectra indicated formation of an amorphous Ge film, consistent with the absence of a XRD pattern. Films were more stable and homogeneous when formed on Cu substrates, than on Au, due to a larger hydrogen overpotential, and the corresponding lower tendency to form bubbles.

Aqueous electrodeposition of Ge monolayers.

The electrodeposition of germanium on Au(111) in aqueous solutions has been investigated by means of cyclic voltammetry, Auger electron spectroscopy, and in situ scanning tunneling microscopy (STM). The data yield a picture of germanium deposition, which starts with the formation of two well-ordered hydroxide phases, with 1/3 ML and 4/9 ML coverages upon initial reduction of the Ge(IV) species (probably H(2)GeO(3) at pH 4.7). Those structures appear to result from a three-electron reduction to form surface-limited structures with (square root(3) x square root(3))R30 degrees or (3 x 3) unit cells, respectively. Further reduction, probably in a two-electron process from the hydroxide structures, resulted in a germanium hydride structure, again surface-limited, with a coverage of close to 0.8 ML. The hydride structure is very flat, though with the periodic modulation characteristic of a Moiré pattern. Longer deposition times and lower potentials resulted in increased coverage of Ge in some cases, but with apparently limited coverage as a function of pH. The maximum Ge coverage, about 4 ML, was observed using a pH 9.32 deposition solution. At potentials negative of the Moiré pattern, about -850 mV versus Ag/AgCl, a "corruption" of the smooth Moiré pattern occurred. This roughening appears to mark the initial formation of a Au-Ge alloy, accounting for the observation of coverage in excess of that needed to form the Moiré pattern at some pH values.

Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2(-/-) mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

Pb deposition on I-coated Au(111). UHV-EC and EC-STM studies.

This article concerns the growth of an atomic layer of Pb on the Au(111)( radical3 x radical3)R30 degrees -I structure. The importance of this study lies in the use of Pb underpotential deposition (UPD) as a sacrificial layer in surface-limited redox replacement (SLRR). SLRR reactions are being applied in the formation of metal nanofilms via electrochemical atomic layer deposition (ALD). Pb UPD is a surface-limited reaction, and if it is placed in a solution of ions of a more noble metal, redox replacement can occur, but limited by the amount of Pb present. Pb UPD is a candidate for use as a sacrificial layer for replacement by any more noble element. It has been used by this group for both Cu and Pt nanofilm formation using electrochemical ALD. The I atom layer was intended to facilitate electrochemical annealing during nanofilm growth. Two distinctly different Pb atomic layer structures are reported, studied using in situ scanning tunneling microscopy (STM) with an electrochemical flow cell and ultrahigh vacuum surface analysis combined directly with electrochemical reactions (UHV-EC). Starting with the initial Au(111)( radical3 x radical3)R30 degrees -I, 1/3 monolayer of I on the Au(111) surface, Pb deposition began at approximately 0.1 V. The first Pb UPD structure was observed just below -0.2 V and displayed a (2 x radical3)-rect unit cell, for a structure composed of 1/4 monolayer each of Pb and I. The I atoms fit in Pb 4-fold sites, on the Au(111) surface. The structure was present in domains rotated by 120 degrees. Deposition to -0.4 V resulted in complete loss of the I atoms and formation of a Pb monolayer on the Au(111), which produced a Moiré pattern, due to the Pb and Au lattice mismatch. These structures represent two well-defined starting points for the growth of nanofilms of other more noble elements. It is apparent from these studies that the adsorption of I- on Pb is weak, and it will rinse away. If Pb is used as a sacrificial metal in an electrochemical ALD cycle and adsorbed I atoms are employed for electrochemical annealing, I atoms will need to be applied each cycle.

Preliminary studies in the electrodeposition of PbSe/PbTe superlattice thin films via electrochemical atomic layer deposition (ALD).

This paper concerns the electrochemical growth of compound semiconductor thin film superlattice structures using electrochemical atomic layer deposition (ALD). Electrochemical ALD is the electrochemical analogue of atomic layer epitaxy (ALE) and ALD, methods based on nanofilm formation an atomic layer at a time, using surface-limited reactions. Underpotential deposition (UPD) is a type of electrochemical surfaced-limited reaction used in the present studies for the formation of PbSe/PbTe superlattices via electrochemical ALD. PbSe/PbTe thin-film superlattices with modulation wavelengths (periods) of 4.2 and 7.0 nm are reported here. These films were characterized using electron probe microanalysis, X- ray diffraction, atomic force microscopy (AFM), and infrared reflection absorption measurements. The 4.2 nm period superlattice was grown after deposition of 10 PbSe cycles, as a prelayer, resulting in an overall composition of PbSe0.52Te0.48. The 7.0 nm period superlattice was grown after deposition of 100 PbTe cycle prelayer, resulting for an overall composition of PbSe0.44Te0.56. The primary Bragg diffraction peak position, 2theta, for the 4.2 superlattice was consistent with the average (111) angles for PbSe and PbTe. First-order satellite peaks, as well as a second, were observed, indicating a high-quality superlattice film. For the 7.0 nm superlattice, Bragg peaks for both the (200) and (111) planes of the PbSe/PbTe superlattice were observed, with satellite peaks shifted 1 degrees closer to the (111), consistent with the larger period of the superlattice. AFM suggested conformal superlattice growth on the Au on glass substrate. Band gaps for the 4.2 and 7.0 nm period superlattices were measured as 0.48 and 0.38 eV, respectively.

Platinum nanofilm formation by EC-ALE via redox replacement of UPD copper: studies using in-situ scanning tunneling microscopy.

The growth of Pt nanofilms on well-defined Au(111) electrode surfaces, using electrochemical atomic layer epitaxy (EC-ALE), is described here. EC-ALE is a deposition method based on surface-limited reactions. This report describes the first use of surface-limited redox replacement reactions (SLR(3)) in an EC-ALE cycle to form atomically ordered metal nanofilms. The SLR(3) consisted of the underpotential deposition (UPD) of a copper atomic layer, subsequently replaced by Pt at open circuit, in a Pt cation solution. This SLR(3) was then used a cycle, repeated to grow thicker Pt films. Deposits were studied using a combination of electrochemistry (EC), in-situ scanning tunneling microscopy (STM) using an electrochemical flow cell, and ultrahigh vacuum (UHV) surface studies combined with electrochemistry (UHV-EC). A single redox replacement of upd Cu from a PtCl(4)(2-) solution yielded an incomplete monolayer, though no preferential deposition was observed at step edges. Use of an iodine adlayer, as a surfactant, facilitated the growth of uniformed films. In-situ STM images revealed ordered Au(111)-(square root 3 x square root 3)R30 degrees-iodine structure, with areas partially distorted by Pt nanoislands. After the second application, an ordered Moiré pattern was observed with a spacing consistent with the lattice mismatch between a Pt monolayer and the Au(111) substrate. After application of three or more cycles, a new adlattice, a (3 x 3)-iodine structure, was observed, previously observed for I atoms adsorbed on Pt(111). In addition, five atom adsorbed Pt-I complexes randomly decorated the surface and showed some mobility. These pinwheels, planar PtI(4) complexes, and the ordered (3 x 3)-iodine layer all appeared stable during rinsing with blank solution, free of I(-) and the Pt complex (PtCl(4)(2-)).

CdTe electrodeposition on InP(100) via electrochemical atomic layer epitaxy (EC-ALE): studies using UHV-EC.

The II-VI compound semiconductor CdTe was electrodeposited on InP(100) surfaces using electrochemical atomic layer epitaxy (EC-ALE). CdTe was deposited on a Te-modified InP(100) surface using this atomic layer by atomic layer methodology. The deposit started with formation of an atomic layer of Te on the InP(100) surface, as Cd was observed not to form an underpotential deposition (UPD) layer on InP(100), although it was found to UPD on Te atomic layers. On the In-terminated 'clean' InP(100) surface, Te was deposited at -0.80 V from a 0.1 mM solution of TeO2, resulting in formation of a Te atomic layer and some small amount of bulk Te. The excess bulk Te was then removed by reduction in blank solution at -0.90 V, leaving a Te atomic layer. Given the presences of the Te atomic layer, it was then possible to form an atomic layer of Cd by UPD at -0.58 V to complete the formation of a CdTe monolayer by EC-ALE. That cycle was then repeated to demonstrate the applicability of the cycle to the formation of CdTe nanofilms. Auger spectra recorded after the first three cycles of CdTe deposition on InP(100) were consistent with the layer-by-layer CdTe growth. It is interesting to note that Cd did not form a UPD deposit on the In-terminated InP(100) surface and only formed Cd clusters at an overpotential. This issue is probably related to the inability of the Cd and In to form a stable surface compound.

Prenylation-defective human connexin32 mutants are normally localized and function equivalently to wild-type connexin32 in myelinating Schwann cells.

Mutations in GJB1, the gene encoding the gap junction protein connexin32 (Cx32), cause the X-linked form of Charcot-Marie-Tooth disease, an inherited demyelinating neuropathy. The C terminus of human Cx32 contains a putative prenylation motif that is conserved in Cx32 orthologs. Using [3H]mevalonolactone ([3H]MVA) incorporation, we demonstrated that wild-type human connexin32 can be prenylated in COS7 cells, in contrast to disease-associated mutations that are predicted to disrupt the prenylation motif. We generated transgenic mice that express these mutants in myelinating Schwann cells. Male mice expressing a transgene were crossed with female Gjb1-null mice; the male offspring were all Gjb1-null, and one-half were transgene positive; in these mice, all Cx32 was derived from expression of the transgene. The mutant human protein was properly localized in myelinating Schwann cells in multiple transgenic lines and did not alter the localization of other components of paranodes and incisures. Finally, both the C280G and the S281x mutants appeared to "rescue" the phenotype of Gjb1-null mice, because transgene-positive male mice had significantly fewer abnormally myelinated axons than did their transgene-negative male littermates. These results indicate that Cx32 is prenylated, but that prenylation is not required for proper trafficking of Cx32 and perhaps not even for certain aspects of its function, in myelinating Schwann cells.

Methods to study protein-protein interactions.

Protein-protein interactions are the underpinnings of a vast number of cellular processes. In recent years, the convergence of biochemistry, cellular, and molecular biology has made available a number of powerful techniques for studying such interactions. These techniques vary in their sensitivity, efficiency, and rapidity, but judicial deployment of a combination of them has proved to be effective and reliable. Here, we highlight a version of the yeast two-hybrid assay originally pioneered by Fields and Song (1989) and subsequently enhancements by other investigators. We also briefly describe a number of new fluorescent imaging-based biophysical techniques for studying protein-protein interactions FRET, FCS, and BiFC. Together, these constitute an impressive collection of tools for studying interactions among proteins.

Activation of the tumor suppressor merlin modulates its interaction with lipid rafts.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by bilateral schwannomas of the eighth cranial nerve. The NF2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane/F-actin linkers. Merlin resists solubilization by the detergent Triton X-100 (TX-100), a property commonly attributed to association with the cytoskeleton. Accordingly, NF2 patient mutations that encode merlins with enhanced TX-100 solubility have been explained previously in terms of loss of cytoskeletal attachment. However, here we present data to suggest that the detergent resistance of merlin is a result of its constitutive residence in lipid rafts. Furthermore, when cells are grown to high density, merlin shifts to a more buoyant lipid raft fraction in a density gradient. This shift is mimicked in subconfluent cells treated with cytochalasin D, suggesting that the shift results from merlin dissociation from the actin cytoskeleton, but not from lipid rafts. Intramolecular NH(2)- and COOH-terminal binding, which occurs when merlin transitions to the growth-suppressive form, also brings about a similar change in buoyant density. Our results suggest that constitutive residence of merlin in lipid rafts is crucial for its function and that as merlin becomes growth suppressive in vivo, one significant molecular event may be the loss of interaction with the actin cytoskeleton. To our knowledge, merlin is the first tumor suppressor known to reside within lipid rafts, and the significance of this finding is underscored by known loss-of-function NF2 patient mutations that encode merlins with enhanced TX-100 solubility.

Electrodeposition of Au-Cd alloy nanostructures on Au(111).

This report concerns an in-situ scanning tunneling microscopy study of the initial stages in the formation of a Au-Cd alloy on the Au(111) herringbone reconstruction. Although Au-Cd nanoclusters of alloy have been observed in sulfate electrolyte by this group, alloy "nanowires" were observed to form preferentially in the hcp regions between the sets of "soliton" walls of the reconstruction only in the presence of chloride. The nanowires were formed at -0.55 V versus 3 M Ag/AgCl, corresponding to Cd underpotential deposition (upd). Upd is electrodeposition at a potential prior to that needed to deposit the bulk element.

High-Resolution Electrochemical Scanning Tunneling Microscopy (EC-STM) Flow-Cell Studies.

Atomic-level studies involving an electrochemical scanning tunneling microscope (EC-STM) flow-cell are presented. Multiple electrochemical atomic layer epitaxy (EC-ALE) cycles of CdTe formation were observed. For a binary compound (i.e., CdTe), an EC-ALE cycle involves exposure of the substrate to a solution of the first precursor (CdSO4), followed by exposure to the second precursor (TeO2), while maintaining potential control. Interleaving blank rinses may also be used, but were omitted in the present studies. To allow the exchange of solutions, the EC-STM cell was modified to allow solution exchange via a single peristaltic pump. A selection valve was used to choose the solution to be introduced into the cell. There is evidence that the growth of the initial layer of CdTe on Au(111), the (√7 × âˆš7)-CdTe monolayer, can be improved in homogeneity and morphology by repeatedly depositing and stripping the Cd atomic layer. Therefore, a new starting cycle, which should improve the quality of deposits formed via EC-ALE, has been developed.