Harnessing Liquid Crystal Sensors for High-Throughput Real-Time Detection of Structural Changes in Lysozyme during Refolding Processes.

Despite the rapid advances in process analytical technology, the assessment of protein refolding efficiency has largely relied on off-line protein-specific assays and/or chromatographic procedures such as reversed-phase high-performance liquid chromatography and size exclusion chromatography. Due to the inherent time gap pertaining to traditional methods, exploring optimum refolding conditions for many recombinant proteins, often expressed as insoluble inclusion bodies, has proven challenging. The present study describes a novel protein refolding sensor that utilizes liquid crystals (LCs) to discriminate varying protein structures during unfolding and refolding. An LC layer containing 4-cyano-4'-pentylbiphenyl (5CB) intercalated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used as a sensing platform, and its proof-of-concept performance is demonstrated using lysozyme as a model protein. As proteins unfold or refold, a local charge fluctuation at their surfaces modulates their interaction with zwitterionic phospholipid DOPE. This alters the alignment of DOPE molecules at the aqueous/LC interface, affecting the orientational ordering of bulk LC (i.e., homeotropic to planar for refolding and planar to homeotropic for unfolding). Differential polarized optical microscope images of the LC layer are subsequently generated, whose brightness directly linked to conformational changes of lysozyme molecules is quantified by gray scale analysis. Importantly, our LC-based refolding sensor is compatible with diverse refolding milieus for real-time analysis of lysozyme refolding and thus likely to facilitate the refolding studies of many proteins, especially those lacking a method to determine structure-dependent biological activity.

Bilateral Thygeson's Superficial Punctate Keratitis with Dendritic Corneal Lesion: A Case Report.

Thygeson's superficial punctate keratitis (TSPK) is a recurrent bilateral corneal epithelial disease. Typically, small, multiple discrete epithelial lesions occur in the central cornea. However, dendritic corneal lesions are rare. Herein, we report a rare case of TSPK in both eyes after a unilateral dendritic corneal lesion. A 42-year-old woman presented with decreased vision and foreign body sensation in her right eye that persisted for 1 month. Her uncorrected visual acuity and best-corrected visual acuity (BCVA) were 20/160 in the right eye. Slit-lamp microscopy revealed a dendritic lesion in the central cornea of the right eye. No abnormalities were observed in her left eye. Herpetic keratitis in the right eye was diagnosed and systemic acyclovir was prescribed, along with topical acyclovir ointment and steroids. After one week, most of the corneal lesions had disappeared, and the BCVA in the right eye had improved to 20/25. The corneal epithelium completely recovered after 2 weeks. However, 2 weeks later, the patient visited the hospital with decreased visual acuity in the right eye, and the BCVA decreased to 20/40. Multiple fine corneal lesions were observed under a slit-lamp microscope. The patient was diagnosed with TSPK of the right eye. Topical steroids were started, and after 7 days, the corneal condition improved. However, after 6 weeks, visual acuity decreased in the left eye, and a corneal lesion similar to that in the right eye was observed; therefore, the patient was diagnosed with bilateral TSPK. Short-term topical steroids and long-term topical cyclosporine A 0.1% were used in both eyes, and the disease was maintained without recurrence for 3 months. TSPK can appear as a unilateral dendritic corneal lesion similar to herpetic keratitis. Therefore, in case of unilateral dendritic corneal lesions, it should be considered that TSPK may develop later.

Interfacial Strain-Modulated Nanospherical Ni2 P by Heteronuclei-Mediated Growth on Ti3 C2 Tx MXene for Efficient Hydrogen Evolution.

Interface modulation of nickel phosphide (Ni2 P) to produce an optimal catalytic activation barrier has been considered a promising approach to enhance the hydrogen production activity via water splitting. Herein, heteronuclei-mediated in situ growth of hollow Ni2 P nanospheres on a surface defect-engineered titanium carbide (Ti3 C2 Tx ) MXene showing high electrochemical activity for the hydrogen evolution reaction (HER) is demonstrated. The heteronucleation drives intrinsic strain in hexagonal Ni2 P with an observable distortion at the Ni2 P@Ti3 C2 Tx MXene heterointerface, which leads to charge redistribution and improved charge transfer at the interface between the two components. The strain at the Ni2 P@Ti3 C2 Tx MXene heterointerface significantly boosts the electrochemical catalytic activities and stability toward HER in an acidic medium via a combination between experimental results and theoretical calculations. In a 0.5 m H2 SO4 electrolyte, the Ni2 P@Ti3 C2 Tx MXene hybrid shows excellent HER catalytic performance, requiring an overpotential of 123.6 mV to achieve 10 mA cm-2 with a Tafel slope of 39 mV dec-1 and impressive durability over 24 h operation. This approach presents a significant potential to rationally design advanced catalysts coupled with 2D materials and transition metal-based compounds for state-of-the-art high efficiency energy conversions.

Development of bisphenol A-removing recombinant Escherichia coli by monomeric and dimeric surface display of bisphenol A-binding peptide.

Peptide-displaying Escherichia coli cells were investigated for use in adsorptive removal of bisphenol A (BPA) both in Luria-Bertani medium including BPA or ATM thermal paper eluted wastewater. Two recombinant strains were constructed with monomeric and dimeric repeats of the 7-mer BPA-binding peptide (KSLENSY), respectively. Greater than threefold increased adsorption of BPA [230.4 µmol BPA per g dry cell weight (DCW)] was found in dimeric peptide-displaying cells compared to monomeric strains (63.4 µmol per g DCW) in 15 ppm BPA solution. The selective removal of BPA from a mixture of BPA analogs (bisphenol F and bisphenol S) was verified in both monomeric and dimeric peptide-displaying cells. The binding chemistry of BPA with the peptide was assumed, based on molecular docking analysis, to be the interaction of BPA with serine and asparagine residues within the 7-mer peptide sequence. The peptide-displaying cells also functioned efficiently in thermal paper eluted wastewater containing 14.5 ppm BPA.

Harnessing aptamers for electrochemical detection of endotoxin.

Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (K(d)) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay.

Affinity analysis of DNA aptamer-peptide interactions using gold nanoparticles.

Gold nanoparticles (AuNPs) were used as colorimetric probe and fluorescence quencher for affinity analysis of DNA aptamers toward their target mucin 1 (MUC1) peptide. Single-stranded DNA (ssDNA) aptamer-coated AuNPs showed increased stability (i.e., more resistant to aggregation induced by NaCl) in the presence of their target peptide due to the increase in steric protection conferred by the ssDNA-peptide complexes formed on the AuNPs. Based on changes in the UV-vis extinction spectrum of AuNPs (a measure of AuNPs aggregation) and fluorescence restoration of CY5-ssDNA upon ssDNA-peptide complex formation, the formation of the complexes and ssDNA sequence-dependent dissociation constant (K(d)) were determined. Besides the UV-vis and fluorescence measurements, the hydrodynamic diameters, zeta potential measurements, and transmission electron microscopy (TEM) images of AuNPs after various coatings supported the assay principle. The methodology presented herein provides a rapid and sensitive alternative solution for the identification of high affinity binders from systematic evolution of ligands by exponential enrichment (SELEX).

Spontaneous resolution of a unilateral cataract in an adult: A case report.

RATIONALE: Cataracts are a disease that is usually caused by aging and involve the irreversible degeneration of the lens material. On the other hand, transient cataracts have also been reported, mainly due to systemic hyperglycemia, which often occurs bilaterally. However, reports of the spontaneous regression of unilaterally occurring cataracts in patients with normal blood glucose levels are rare. Here, we report a rare case of spontaneous regression of unilateral posterior subcapsular cataracts in an adult with normal blood glucose levels. PATIENT CONCERNS: A 42-year-old woman presented with distorted vision in her right eye. The patient was taking medication for diabetes, and her blood sugar level was well-controlled. DIAGNOSIS: Upon examination, her uncorrected visual acuity and best-corrected visual acuity were 20/70 in her right eye. Slit lamp microscopy revealed fine, feathery, and streak-like posterior subcapsular opacities. Color fundus photography revealed a star-shaped shadow due to the cataract, and no diabetic retinopathy was observed. Her two hour postprandial glucose level was 115 mg/dL. The patient was diagnosed with posterior subcapsular cataracts, and cataract surgery was planned. The patient was scheduled to visit the clinic again after seven days. INTERVENTIONS: Close observation for one week without any intervention. OUTCOMES: After one week, most of the posterior subcapsular opacities disappeared, and the uncorrected visual acuity and best-corrected visual acuity in the right eye improved to 20/40 and 20/30, respectively. LESSONS: This case report demonstrates that unilateral posterior subcapsular cataracts may spontaneously regress in patients with normal blood glucose levels. Therefore, it is important to check whether cataracts improve spontaneously through short-term close follow-up before planning cataract surgery to avoid unnecessary surgery.

A highly activated iron phosphate over-layer for enhancing photoelectrochemical ammonia decomposition.

Environmentally friendly ammonia (NH3) decomposition has attracted a lot of interests in recent years to resolve the issue of water eutrophication from a wastewater and achieve a clean H2 storage. Here, we report a novel strategy for solar-driven ammonia decomposition by introducing a highly-activated iron phosphate (FePi) over-layer on the surface of α-Fe2O3 nanorods photoanode (FePi/Fe2O3), and innovatively propose a photoelectrochemical (PEC) ammonia degradation system with enhanced performance. After a facile electrochemical (EC) activation, the FePi over-layer is converted into FeOOH. The EC-activated over-layer provides the efficient active sites for the ammonia adsorption process, which promotes the high catalytic kinetics for ammonia oxidation reaction (AOR). Due to the synergistic effect of the electrocatalytic and the photocatalytic process, the FePi/Fe2O3 exhibits the enhanced PEC AOR performance, which competes with water oxidation reaction (WOR). Comparing to the initial concentration of ammonia, the FePi/Fe2O3 achieves a 54.4% ammonia degradation rate within 3 h at 1.23 V vs. reversible hydrogen electrode (RHE) under 1 sun illumination, which demonstrates the reliable ammonia decomposition performance. This study confirms that it is feasible to achieve PEC ammonia decomposition in an aqueous solution without chloride mediators and provides a promising strategy for the harmless treatment of ammonia wastewater.

Mechanism Study of Thermally Induced Anti-Tumor Drug Loading to Engineered Human Heavy-Chain Ferritin Nanocages Aided by Computational Analysis.

Diverse drug loading approaches for human heavy-chain ferritin (HFn), a promising drug nanocarrier, have been established. However, anti-tumor drug loading ratio and protein carrier recovery yield are bottlenecks for future clinical application. Mechanisms behind drug loading have not been elaborated. In this work, a thermally induced drug loading approach was introduced to load anti-tumor drug doxorubicin hydrochloride (DOX) into HFn, and 2 functionalized HFns, HFn-PAS-RGDK, and HFn-PAS. Optimal conditions were obtained through orthogonal tests. All 3 HFn-based proteins achieved high protein recovery yield and drug loading ratio. Size exclusion chromatography (SEC) and transmission electron microscopy (TEM) results showed the majority of DOX loaded protein (protein/DOX) remained its nanocage conformation. Computational analysis, molecular docking followed by molecular dynamic (MD) simulation, revealed mechanisms of DOX loading and formation of by-product by investigating non-covalent interactions between DOX with HFn subunit and possible binding modes of DOX and HFn after drug loading. In in vitro tests, DOX in protein/DOX entered tumor cell nucleus and inhibited tumor cell growth.

Impact of feedstocks and downstream processing technologies on the economics of caproic acid production in fermentation by Megasphaera elsdenii T81.

Caproic acid (CA) was produced by Megasphaera elsdenii T81 with Jerusalem artichoke tubers (JA) as a feedstock. More CA was produced under the medium with the acid hydrolysate of JA than the comparative medium with a carbon composition similar to that of JA. CA was produced up to 13.0 g/L and 0.52 g/L/h with extractive fermentation using a mixed solvent of alamine 336 in oleyl alcohol at 37 °C. The JA cost to produce 1 ton of CA is only 505 USD, which is much lower than that required for purchasing sucrose (860 USD) in CA production. As a result of the analysis performed using SuperPro Designer, including the cost of distillation to obtain pure CA, the estimated production cost for CA from dry JA is 1869 USD/ton CA at the production scale of 2000 ton/year, which is lower than the current market price for petroleum-derived CA (~2500 USD/ton).

Metal-Organic Decomposition-Mediated Nanoparticulate Vanadium Oxide Hole Transporting Buffer Layer for Polymer Bulk-Heterojunction Solar Cells.

In this study, a solution-processable compact vanadium oxide (V2O5) film with a globular nanoparticulate structure is introduced to the hole transport layer (HTL) of polymer bulk-heterojunction based solar cells comprised of PTB7:PC70BM by using a facile metal-organic decomposition method to replace the conventionally utilized poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS). For this, a biocompatible structure-determining agent, polyethylene glycol (PEG, Mn 300), is used as an additive in the precursor to form the nanoparticulate compact V2O5 (hereafter referred to as NP-V2O5) film, which possesses an outstandingly smooth surface morphology. The introduction of NP-V2O5 HTL via the solution process with a neutral pH condition successfully improved the stability by preventing the decomposition of indium tin oxide (ITO) glass and the penetration of heavy-metal components and moisture, which are considered as the crucial drawbacks of using PEDOT:PSS. Over 1440 h (60 days) of the stability test, an organic solar cell (OSC) with NP-V2O5 showed a significant durability, maintaining 82% of its initial power conversion efficiency (PCE), whereas an OSC with PEDOT:PSS maintained 51% of its initial PCE. Furthermore, due to the positive effects of the modified surface properties of NP-V2O5, the PCE was slightly enhanced from 7.47% to 7.89% with a significant improvement in the short-circuit current density and fill factor.

Spin-assembled layer-by-layer films of weakly charged polyelectrolyte multilayer.

We investigate the effect of film deposition methods on the film properties of layer-by-layer assembled polyelectrolyte multilayers. Multilayered structures of linear polyethylenimine (LPEI) and poly(acrylic acid) (PAA) are prepared by using conventional dipping-based assembly as well as spin-assisted assembly. While the polyelectrolyte interpenetration by the diffusion motion of LPEI species is allowed in dipping-based assembly, an instantaneously and kinetically frozen phase of the film deposition is obtainable from spin-assisted assembly. Being kept from the interdiffusion of LPEI, a stratified internal structure is expected in the spin-assisted assembly, which is completely contrasted to the intermixed phase in the dipping-based assembly. The ability to control the inner structure of the multilayered film enables us to manipulate the physical properties or chemical activity of the functionalized thin films. We also demonstrate that the control over the strength of polyelectrolyte interdiffusion on a very top surface can be utilized for a quantitative manipulation on the degree of macromolecular self-assembly of nanomaterials.

Chaperone-assisted column refolding of gloshedobin with the use of refolding cocktail.

Gloshedobin, a recently isolated thrombin-like enzyme from the snake venom of Gloydius shedaoensis, is expressed mainly in the form of inclusion bodies (IBs) in Escherichia coli due to its cysteine-rich nature. Following extraction and solubilization of the IBs, one-step immobilized metal affinity chromatography purification produces highly purified (>99%) denatured-solubilized gloshedobin ready to enter the subsequent refolding process. However, the traditional dilution or column refolding strategy, based on gradual denaturant removal, is found to be inefficient for the recovery of protein activity. In this study, a new refolding strategy harnessing the ClpB and DnaK/DnaJ/GrpE bichaperone system is demonstrated to be superior to the conventional refolding methods in either batch dilution or column refolding mode. It is noted that the efficacy of bichaperone-mediated column refolding strategy is further highlighted especially when refolding reaction is attempted at a higher protein concentration with the recirculation of the refolding cocktail containing the bichaperone system. This is evidenced by an uncompromised refolding efficiency (ca. 21.4%) achieved at 2000 microg/mL of initial protein concentration, which is comparable to the refolding efficiency (ca. 22.5%) obtained at 20 times lower protein concentration (i.e. 100 microg/mL) in the conventional batch dilution refolding technique. The demonstrated chaperone-assisted column refolding strategy thus provides an effective tool for refolding-recalcitrant proteins whose reactivation is otherwise difficult to achieve.

Polyethyleneimine-mediated chemical extraction of cytoplasmic His-tagged inclusion body proteins from Escherichia coli.

The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His-tagged model proteins: gloshedobin, a thrombin-like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine-induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost-prohibitive spermine and EDTA. The residual PEI in the post-extraction mixture was efficiently counteracted by addition of Mg2+, allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI-mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.

Optimization of key process variables for enhanced hydrogen production by Enterobacter aerogenes using statistical methods.

The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box-Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H(2)(g dry cell h)(-1) was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 degrees C and pH 6.13. The experimental results showed that the RSM with the Box-Behnken design was a useful tool for achieving high rate of hydrogen production by E. aerogenes.

Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions.

Separation of negatively charged molecules, such as plasmid DNA (pDNA), RNA and endotoxin forms a bottleneck for the development of pDNA vaccine production process. The use of affinity interactions of transition metal ions with these molecules may provide an ideal separation methodology. In this study, the binding behaviour of pDNA, RNA and endotoxin to transition metal ions, either in immobilised or free form, was investigated. Transition metal ions: Cu2+, Ni2+, Zn2+, Co2+ and Fe3+, typically employed in the immobilised metal affinity chromatography (IMAC), showed very different binding behaviour depending on the type of metal ions and their existing state, i.e. immobilised or free. In the alkaline cell lysate, pDNA showed no binding to any of the IMAC chemistries tested whereas RNA interacted significantly with Cu2+-iminodiacetic acid (IDA) and Ni2+-IDA but showed no substantial binding to the rest of the IMAC chemistries. pDNA and RNA, however, interacted to varying degrees with free metal ions in the solution. The greatest selectivity in terms of pDNA and RNA separation was achieved with Zn2+ which enabled almost full precipitation of RNA while keeping pDNA soluble. For both immobilised and free metal ions, ionic strength of solution affected the metal ion-nucleic acid interaction significantly. Endotoxin, being more flexible, was able to interact better with the immobilised metal ions than the nucleic acids and showed binding to all the IMAC chemistries. The specific interactions of immobilised and/or free metal ions with pDNA, RNA and endotoxin showed a good potential, by selectively removing RNA and endotoxin at high efficiency, to develop a simplified pDNA purification process with improved process economics.

Toxicity of imidazolium salt with anion bromide to a phytoplankton Selenastrum capricornutum: effect of alkyl-chain length.

Room-temperature ionic liquids are regarded as environmentally benign alternatives to volatile organic solvents. However, the product designs for this promising group of compounds should take account, not only the technological needs, but also the eco-toxicological hazards. Therefore, this study aimed to evaluate the toxicity of some important ionic liquids on the growth of the freshwater alga, Selenastrum capricornutum. The ionic liquids examined in this study included 1-propyl-3-methylimidazolium [PMIM], 1-butyl-3-methylimidazolium [BMIM], 1-hexyl-3-methylimidazolium [HMIM] and 1-octyl-3-methylimidazolium [OMIM] with a bromide anion. The susceptibility of alga to ionic liquids was strongly dependent on the alkyl-chain length. According to our results, a longer alkyl-chain resulted in stronger inhibition of algal growth. In general, the toxicity could be summarized as decreasing in the following order: [OMIM]>[HMIM]>[BMIM]>or=[PMIM]. Among the ionic liquids examined, [OMIM] [Br] was found to be most toxic to S. capricornutum, with EC(50) values ranging from 26.3 microM to 54.9 microM after an incubating time of 96 h. Although [BMIM] [Br] and [PMIM] [Br] was relatively less toxic than [OMIM] and [HMIM], their toxicity increased as increasing the incubation time from 48 h to 96 h. This fact indicates that these kinds of ionic liquids may become more toxic after being released and contacted to freshwater ecosystem.

Harnessing denatured protein for controllable bipolar doping of a monolayer graphene.

In this work, we demonstrated tunable p- and/or n-type doping of chemical vapor deposition-grown graphene with the use of protein bovine serum albumin (BSA) as a dopant. BSA undergoes protonation or deprotonation reaction subject to solution pH, thereby acting as either an electron donor or an electron acceptor on the graphene surface layered with denatured BSA through π-stacking interaction. This direct annealing of graphene with denatured BSA of amphoteric nature rendered facilitated fabrication of a p- and/or n-type graphene transistor by modulating pH-dependent net charges of the single dopant. Following AFM confirmation of the BSA/graphene interface assembly, the carrier transport properties of BSA-doped graphene transistors were assessed by I-V measurement and Raman spectra to show effective charge modulation of the graphene enabled by BSA doping at various pH conditions. The protein-mediated bipolar doping of graphene demonstrated in our work is simple, scalable, and straightforward; the proposed scheme is therefore expected to provide a useful alternative for fabricating graphene transistors of novel properties and promote their implementation in practice.

Electrochemical detection of Bisphenol A with high sensitivity and selectivity using recombinant protein-immobilized graphene electrodes.

A novel Bisphenol A (4,4'-isopropylidenediphenol, BPA) sensor was developed harnessing an electrochemical platform comprising a layer-by-layer assembled reduced graphene oxide (rGO) electrode and a designer probe specifically recognizing BPA. The BPA detection probe, a recombinant protein (LacI-BPA), was constructed by fusing a disulfide-constrained high affinity BPA binding peptide (CKSLENSYC) to the C-terminus of Lac repressor (LacI). Following expression and purification, the LacI-BPA was heat-denatured on-purpose to facilitate its direct adhesion on the rGO electrode surface via pi-stacking interaction. When the performance of the fabricated BPA sensor (LacI-BPA/rGO) was assessed by electrochemical impedance spectroscopy (EIS), it showed a wide linear dynamic range of BPA detection spanning from 100 fM to 10nM. Moreover, our BPA sensor exhibited negligible cross reactivity to BPA analogs such as Bisphenol S (BPS) and Bisphenol F (BPF) and almost complete spike recovery of BPA from plastic extracts containing various potential interferents. With these merits, the BPA sensor developed in the present study is expected to find practical application in selective and sensitive detection of BPA from diverse sample solutions.

Incorporation of a Metal Oxide Interlayer using a Virus-Templated Assembly for Synthesis of Graphene-Electrode-Based Organic Photovoltaics.

Transition metal oxide (TMO) thin films have been exploited as interlayers for charge extraction between electrodes and active layers in organic photovoltaic (OPV) devices. Additionally, graphene-electrode-based OPVs have received considerable attention as a means to enhance device stability. However, the film deposition process of a TMO thin-film layer onto the graphene electrode is highly restricted owing to the hydrophobic nature of the graphene surface; thus, the preparation of the device should rely on a vacuum process that is incompatible with solution processing. In this study, we present a novel means for creating a thin tungsten oxide (WO3 ) interlayer on a graphene electrode by employing an engineered biotemplate of M13 viruses, whereby nondestructive functionalization of the graphene and uniform synthesis of a WO3 thin interlayer are concurrently achieved. As a result, the incorporated virus-templated WO3 interlayer exhibited solar-conversion efficiency that was 20 % higher than that of conventional OPVs based on the use of a (3,4-ethylenedioxythiophene):poly(styrenesulfonate) ( PEDOT: PSS) interlayer. Notably, bilayer-structured OPVs with synergistically integrated WO3 /PEDOT:PSS achieved >60 % enhancement in device performance.