Gold-binding Peptide Research Articles

Engineered M13-Derived Bacteriophages Capable of Gold Nanoparticle Synthesis and Nanogold Manipulations

For years, gold nanoparticles (AuNPs) have been widely used in medicine and industry. Although various experimental procedures have been reported for their preparation and manipulation, none of them is optimal for all purposes. In this work, we engineered the N-terminus of the pIII minor coat protein of bacteriophage (phage) M13 to expose a novel HLYLNTASTHLG peptide that effectively and specifically binds gold. In addition to binding gold, this engineered phage could synthesize spherical AuNPs of 20 nm and other sizes depending on the reaction conditions, aggregate them, and precipitate gold from a colloid, as revealed by transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), as well as ultraviolet–visible (UV–vis) and Fourier-transform infrared (FTIR) spectroscopic methods. We demonstrated that the engineered phage exposing a foreign peptide selected from a phage-displayed library may serve as a sustainable molecular factory for both the synthesis of the peptide and the subsequent overnight preparation of AuNPs from gold ions at room temperature and neutral pH in the absence of strong reducing agents, such as commonly used NaBH4. Taken together, the results suggest the potential applicability of the engineered phage and the new, in vitro-identified gold-binding peptide in diverse biomimetic manipulations.

Generation and Evaluation of Gold-binding Peptide Fused Protein A

Generation and Evaluation of Gold-binding Peptide Fused Protein A

Construction of Solid Binding Peptide (SBP)-Fused Non-Native Chimeric Enzyme to Promote Bienzymatic Cascade Induced Bioelectrocatalysis

In nature, most enzymes form a multienzyme complex called “metabolon” in the cellular system. The natural metabolon has been evolved to increase the efficiency of multi-step biocatalysis involved in such conditions. Such principle of enzymatic cascade reaction could be applied to the system which mimics pertinent reaction in vitro. Especially, its pertaining to bioelectrocatalytic system is quite promising to enhance its performance in terms of electrical generation or biochemical production, depending on systematic applications. In the previous studies , it was abundantly reported that multi-enzymatic reaction has been utilized to generate higher electrical current via sequential enzymatic oxidation of substrates (e.g., methanol, glucose, etc.) in bioelectrochemical systems, and to operate biochemical production system utilizing multi-enzymic conversion or reduction reactions. However, it is still challenging to implement multi-step enzymatic reaction in the electrode platforms due to unmanageable regulations of inter-enzyme distance or electrical connection between enzymatic cofactor and electrode, during multi-enzyme co-immobilization on electrode surface.Herein, we intentionally design and construct variants of chimeric enzyme in which two enzymes, invertase (INV) and glucose dehydrogenase (GDH), are conjugated with peptide linkers. We expected that INV hydrolyzes sucrose into fructose and glucose, then glucose is oxidized at the catalytic subunit of GDH, releasing electrons toward the electrode. It also employed solid binding peptides (SBPs) fused to GDH in chimeric enzyme for the electrical connection of GDH-SBP and electrode. In order to accomplish the desired reaction with chimeric enzyme, we have conducted the following tests; 1) The recombinant plasmid harboring the genetic sequence of two enzymes with an in-between linker was constructed. 2) By production of chimeric proteins, the chain reaction efficiency of fusion constructs was compared depending on the length of the linker. 3) The gold binding peptide (GBP) was fused to sites (N- and C-terminus, or other sites) of the catalytic subunit of GDH. And finally, 4) the electron transfer rates were monitored and compared depending on the GBP tethering sites. Throughout the whole study, we found the factors affecting the occurrence of multienzyme cascade-based bioelectrochemical reactions and how to control these factors to improve the reaction efficiency Figure 1

In Vitro and Ex Planta Gold-Bonded and Gold-Mineralized Tobacco Mosaic Virus.

Biotemplated mineralization is a promising and ecofriendly approach to manufacture metal nanoparticles and composites with precise size control. Plant viruses are suitable templates for biomineralization because they are chemically robust and highly scalable through molecular farming. Here, we report a gold-nanoparticle-coated tobacco mosaic virus (TMV) synthesized in a test tube or in plant extracts making use of a TMV displaying a gold-binding peptide (GBP). The methods developed are a step toward engineered living materials, where gold nanowires could be formed in plant tissues for sensing or energy harvest applications.

Modular Design for Proteins Assembling into Antifouling Coatings: Case of Gold Surfaces.

We analyze modularity for a B-M-E triblock protein designed to self-assemble into antifouling coatings. Previously, we have shown that the design performs well on silica surfaces when B is taken to be a silica-binding peptide, M is a thermostable trimer domain, and E is the uncharged elastin-like polypeptide (ELP), E = (GSGVP)40. Here, we demonstrate that we can modulate the nature of the substrate on which the coatings form by choosing different solid-binding peptides as binding domain B and that we can modulate antifouling properties by choosing a different hydrophilic block E. Specifically, to arrive at antifouling coatings for gold surfaces, as binding block B we use the gold-binding peptide GBP1 (with the sequence MHGKTQATSGTIQS), while we replace the antifouling blocks E by zwitterionic ELPs of different lengths, EZn = (GDGVP-GKGVP)n/2, with n = 20, 40, or 80. We find that even the B-M-E proteins with the shortest E blocks make coatings on gold surfaces with excellent antifouling against 1% human serum (HS) and reasonable antifouling against 10% HS. This suggests that the B-M-E triblock protein can be easily adapted to form antifouling coatings on any substrate for which solid-binding peptide sequences are available.

Highly sensitive and label-free electrochemical detection of C-reactive protein on a peptide receptor−gold nanoparticle−black phosphorous nanocomposite modified electrode

C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein found in blood serum, and an enhanced CRP level is indicative of inflammatory conditions such as infection and cancer, among others. In this work, we developed a novel high CRP-affinity peptide-functionalized label-free electrochemical biosensor for the highly sensitive and selective detection of CRP. Throughout biopanning with random peptide libraries, high affinity peptides for CRP was successfully identified, and then a series of synthetic peptide receptor, of which C-terminus was incorporated to gold binding peptide (GBP) as an anchoring motif was covalently immobilized onto gold nanoparticle (AuNPs) tethered polydopamine (PDA)‒black phosphorus (BP) (AuNPs@BP@PDA) nanocomposite electrode. Interaction between the CRP-binding peptide and CRP was confirmed via enzyme-linked immunosorbent assay along with various physicochemical and electrochemical analyses. Under the optimized experimental conditions, the proposed peptide-based biosensor detects CRP in the range of 0–0.036 μg/mL with a detection limit (LOD) of 0.7 ng/mL. The developed sensor effectively detects CRP in the real samples of serum and plasma of Crohn's disease patients. Thus, the fabricated peptide-based biosensor has potential applications in clinical diagnosis and medical applications.

Gold-binding peptide as a selective layer for electrochemical detection of SARS-CoV-2 antibodies

Electrochemical immunosensors are excellent alternatives to prepare portable platforms used for rapid and inexpensive diagnostic of infectious diseases such as the recently emerged COVID-19. Incorporating synthetic peptides as selective recognition layers combined with nanomaterials such as gold nanoparticles (AuNPs) can significantly enhance the analytical performance of immunosensors. In the present study, an electrochemical immunosensor based on solid-binding peptide was built and evaluated towards SARS-CoV-2 Anti-S antibodies detection. The peptide used as recognition site has two important portions: one based on the viral receptor binding domain (RBD), capable of recognizing antibodies of the spike protein (Anti-S), and another suitable for interacting with gold nanoparticles. Gold-binding peptide (Pept/AuNP) dispersion was used directly to modify a screen-printed carbon electrode (SPE). The voltammetric behavior of the [Fe(CN)6]3-/4- probe after every construction and detection step was recorded using cyclic voltammetry by assessing the stability of the Pept/AuNP as a recognition layer onto the electrode surface. Differential pulse voltammetry was used as a detection technique, and a linear working range from 75 ng mL−1 to 15 μg mL−1 was established, with 1.059 μA dec−1 of sensitivity and R2 = 0.984. The response selectivity against SARS-CoV-2 Anti-S antibodies was investigated in presence of concomitant species. The immunosensor was used to detect SARS-CoV-2 Anti-spike protein (Anti-S) antibodies in human serum samples, successfully differentiating between negative and positive responses of samples at a 95% confidence level. Therefore, the gold-binding peptide is a promising tool to be applied as a selective layer for antibody detection.

Versatile Biosynthetic Approach to Regioselective Enzyme Patterning for Direct Bioelectrocatalysis Application

AbstractDue to the outstanding attributes of oxidoreductases, they have been utilized as biomaterials for bioelectrocatalytic systems. Herein, a simple and versatile biosynthetic approach that can designate binding position of enzymes on electrode with their surface‐orientation is suggested. In this regard, material‐selective properties of gold‐binding peptide (GBP) are exploited and genetically fused GBP to enzyme. To optimize the design of synthetic enzyme, a variable repeat number of GBP are fused to flavin adenine dinucleotide‐dependent glucose dehydrogenase gamma‐alpha complex (GDHγα) and their catalytic and gold‐binding activities are determined. The substrate catalysis and direct electrocatalytic capability of selected construct, GDHγα with three GBP repeats (GDHγα‐3GBP), are investigated on electrode. In an inorganic‐binding characterization, GDHγα‐3GBP exhibits fourfold higher affinity on gold (Au) surface and 215‐fold lower binding affinity for silicon dioxide (SiO2) than wild‐type GDHγα. Utilizing those regioselective features, fusion GDHγα is incorporated into nanotemplates comprising Au nanopatterns and SiO2 background. Thereby, nanoscale patterned GDHγα‐3GBP molecules are successfully obtained with their binding locations controlled specifically by Au nanopatterns, not SiO2. The results reveal that genetic SBP fusions enable highly selective template‐based surface assembly of biomolecules with electrically intimate cofactor‐surface interfaces. The proposed technology has remarkable potential to fabricate small‐scale biochips applied for enzyme‐based bioelectronics.

Sequence rules for gold-binding peptides.

Metal-binding peptides play a central role in bionanotechnology, wherein they are responsible for directing growth and influencing the resulting properties of inorganic nanomaterials. One of the key advantages of using peptides to create nanomaterials is their versatility, wherein subtle changes in the sequence can have a dramatic effect on the structure and properties of the nanomaterial. However, precisely knowing which position and which amino acid should be modified within a given sequence to enhance a specific property can be a daunting challenge owing to combinatorial complexity. In this study, classification based on association rules was performed using 860 gold-binding peptides. Using a minimum support threshold of 0.035 and confidence of 0.9, 30 rules with confidence and lift values greater than 0.9 and 1, respectively, were extracted that can differentiate high-binding from low-binding peptides. The test performance of these rules for categorizing the peptides was found to be satisfactory, as characterized by accuracy = 0.942, F1 = 0.941, MCC = 0.884. What stands out from the extracted rules are the importance of tryptophan and arginine residues in differentiating peptides with high binding affinity from those with low affinity. In addition, the association rules revealed that positions 2 and 4 within a decapeptide are frequently involved in the rules, thus suggesting their importance in influencing peptide binding affinity to AuNPs. Collectively, this study identified sequence rules that may be used to design peptides with high binding affinity.

Gold nanoparticles-decorated M13 phage SPR probe for dual detection of antigen biomarkers in serum

Label-free SPR is rarely used for biomarker detection in real samples due to its low sensitivity. Herein, we report a phage-based SPR (P-SPR) strategy for the detection of carcinoembryonic antigen (CEA) in undiluted serums. M13 phage is genetically engineered to display an anti-CEA single-chain variable region fragment on pIII protein for antigen targeting and to display the gold binding peptide (GBP) at the pVIII protein for gold nanoparticles (GNPs) binding to form the GNPs decorated M13 phage probe that can amplify the plasmon resonance signal. The proposed P-SPR method demonstrated ultrasensitive detection of CEA within 10 min. Under ideal conditions, P-SPR showed exceptional sensitivity with a limit of detection (LOD) of 0.83 fM, which is approximately 2000 times that of the conventional SPR and four orders of magnitude more sensitive than enzyme-linked immunosorbent assay. In particular, the proposed strategy can cross-validate the SPR results through naked-eye counting of the plaques generated by the P-SPR probes, enabling a reliable dual detection system. In addition, P-SPR scheme has excellent sensing performance for the detection of real samples at concentrations lower than LOD of ELISA. Therefore, the proposed P-SPR strategy holds potential for early detection of tumour biomarkers in liquid biopsy.

Chimeric Peptide-Based Biomolecular Constructs for Versatile Nucleic Acid Biosensing.

Nucleic acid biomarkers hold great potential as key indicators for the diagnosis and monitoring of diseases. Herein we design and implement bifunctional chimeric biomolecules composed of a solid-binding peptide (SBP) domain that specifically adsorbs onto solid sensor surfaces and a peptide nucleic acid (PNA) moiety that facilitates anchoring of antisense oligonucleotide (ASO) probes for the detection of nucleic acid targets. A gold-binding peptide, AuBP1, previously selected by directed evolution to specifically bind to gold, served as the basis for immobilizing nucleic acid probes onto gold substrates. Using surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) analyses, we demonstrate the sequential biomolecular assembly of the heterofunctional solid-binding peptide-antisense oligomer (SBP-ASO) construct onto a sensor surface and the subsequent detection of DNA in an aqueous environment. The effect of steric hindrance on optimal probe assembly is observed, establishing that less packing density results in greater target capture efficacy. In addition, an adsorbed layer of chimeric solid-binding peptide-peptide nucleic acid (SBP-PNA) undergoes viscoelastic changes at the solid-liquid interface upon probe immobilization and DNA target capture, whereby the rigid biofunctional layer becomes more flexible. The dual nature of the chimeric construct is highly amenable to a variety of platforms allowing for both specific recognition and probe immobilization on the sensor surface, while the modular design of the solid-binding peptide-antisense oligonucleotide provides facile functionalization of a wide diversity of solid substrates.

Direct electrical contact of NAD+/NADH-dependent dehydrogenase on electrode surface enabled by non-native solid-binding peptide as a molecular binder

NAD+/NADH-dependent redox enzymes constitute most known oxidoreductases, but their inherent complexity due to their reliance on the diffusional nature of soluble cofactors limits their application in bioelectrocatalytic systems. Herein, a gold-binding peptide (gbp) is genetically introduced at the NAD+/NADH-dependent formate dehydrogenase (FDH) to add a non-native gold-binding activity to directly wiring the enzyme to the electrode surface. Our gold-binding kinetics studies on the native and synthetic FDHs revealed that the gold-binding properties of the fused enzymes are highly dependent on the fusion site and that the tertiary structure of the fusion enzyme controls the efficiency of gold-binding domain display fusion. As such, the highest gold-binding activity was observed with the fusion of gbp at the C-terminus (FDHgbpC), whereas binding at both termini FDHgbpNC and FDHgbpN appeared to be less active. Moreover, the presence of gbp increased the stability of an integrated enzyme electrode in the bioelectrocatalytic reactions occurring at the enzyme-electrode interface. Direct electrochemical NADH oxidation produced by the enzymatic reaction in the presence of formate and NAD+ was observed at a low overpotential range of −0.45 to −0.15 V vs Ag/Ag+ for all types of enzyme-electrodes, which indicates direct-electrical contact between the cofactor binding site and the electrode surface. The enhanced electron transfer kinetics could be explained by the shorter distance between the cofactor-binding site and electrode surface. Furthermore, the addition of NADH and CO2 increased the reductive catalytic current, which suggested the enzymatic CO2 reduction to formate using a hydride of NADH and a subsequent reduction of the generated NAD+. We demonstrated that biotechnology can directly contact enzymes onto the electrode surface, which has proven to be a reliable method for bio-electronic applications, especially involving NAD+/NADH-dependent enzymes.

Control of carbon monoxide dehydrogenase orientation by site-specific immobilization enables direct electrical contact between enzyme cofactor and solid surface

Controlling the orientation of redox enzymes on electrode surfaces is essential in the development of direct electron transfer (DET)-based bioelectrocatalytic systems. The electron transfer (ET) distance varies according to the enzyme orientation when immobilized on an electrode surface, which influences the interfacial ET rate. We report control of the orientation of carbon monoxide dehydrogenase (CODH) as a model enzyme through the fusion of gold-binding peptide (gbp) at either the N- or the C-terminus, and at both termini to strengthen the binding interactions between the fusion enzyme and the gold surface. Key factors influenced by the gbp fusion site are described. Collectively, our data show that control of the CODH orientation on an electrode surface is achieved through the presence of dual tethering sites, which maintains the enzyme cofactor within a DET-available distance (<14 Å), thereby promoting DET at the enzyme–electrode interface.

Peptide modification on the interior surface of red blood cell ghosts for construction of catalytic reactors.

We have succeeded in in situ synthesis of gold nanoparticles (Au NPs) on the interior surfaces of red blood cell ghosts (RBCGs) with a cytoskeleton chemically conjugated to a gold-binding peptide. The Au NP-deposited RBCG with the peptide exhibits enhanced catalytic reactivity for the reduction of 4-nitrophenol due to the uniform distribution of the Au NPs on the inner surface and the coverage of the Au NPs surface with the peptide.

Construction of an enzyme-based all-fiber SPR biosensor for detection of enantiomers

Chiral analysis of amino acids (AAs) is of great importance in medical science due to the distinctive effect of AA isomers on human health. Although various chiral recognition techniques have been developed, the quantitative chiral recognition of low-level AA isomers remains challenging. Here, we combined the fiber optic SPR with an enzyme-substrate recognition mechanism to construct a direct-assay-type chiral AA biosensor. As a proof-of-concept attempt, a recently discovered Rasamsonia emersonii D-amino acid oxidase (ReDAAO) with a wide substrate spectrum and high stability was immobilized on the graphene oxide and gold nanorods composites (GO-AuNRs), using both EDC/NHS coupling and the gold-binding peptide (GBP) method. Such a biosensor can distinguish two AA isomers at the same concentration. It achieved specific detection of D-amino acids (D-AAs) with a linear range from 5x10-4 mM to 30 mM. Furthermore, it showed good resistance to enantiomeric interference. When the percentage of D-AA increases in the isomer mixture, a good linear relationship between the D/(D + L)-AA ratio and SPR spectral shift was obtained. This unique combination of the enzyme, nanocomposite, and SPR taps into the rich reservoir of proteins for chiral receptors. It lays the foundation for protein-based chiral recognition of other clinically important small molecules in future biosensor designs.

Peptide sequence-driven direct electron transfer properties and binding behaviors of gold-binding peptide-fused glucose dehydrogenase on electrode

SummaryOriented enzyme immobilization on electrodes is crucial for interfacial electrical coupling of direct electron transfer (DET)-based enzyme-electrode systems. As inorganic-binding peptides are introduced as molecular binders and enzyme-orienting agents, inorganic-binding peptide-fused enzymes should be designed and constructed to achieve efficient DET. In this study, it is aimed to compare the effects of various gold-binding peptides (GBPs) fused to enzymes on electrocatalytic activity, bioactivity, and material-binding behaviors. Here, GBPs with identical gold-binding properties but different amino acid sequences were fused to the FAD-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) to generate four GDHγα variants. The structural, biochemical, mechanical, and bioelectrochemical properties of these GDHγα variants immobilized on electrode were determined by their fused GBPs. Our results confirmed that the GBP type is vital in the design, construction, and optimization of GBP-fused enzyme-modified electrodes for facile interfacial DET and practical DET-based enzyme-electrode systems.

Probing Selective Self-Assembly of Putrescine Oxidase with Controlled Orientation Using a Genetically Engineered Peptide Tag.

Controlling enzyme orientation and location on surfaces is a critical step for their successful deployment in diverse applications from biosensors to lab-on-a-chip devices. Functional activity of the enzymes on the surface will largely depend on the spatial arrangement and orientation. Solid binding peptides have been proven to offer versatility for immobilization of biomolecules on inorganic materials including metals, oxides, and minerals. Previously, we demonstrated the utility of a gold binding peptide genetically incorporated into the enzyme putrescine oxidase (PutOx-AuBP), enabling self-enzyme assembly on gold substrates. PutOx is an attractive biocatalyst among flavin oxidases, using molecular oxygen as an electron acceptor without requiring a dissociable coenzyme. Here, we explore the selective self-assembly of this enzyme on a range of surfaces using atomic force microscopy (AFM) along with the assessment of functional activity. This work probes the differences in surface coverage, distribution, size, shape, and activity of PutOx-AuBP in comparison to those of native putrescine oxidase (PutOx) on multiple surfaces to provide insight for material-selective enzymatic assembly. Surfaces investigated include metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers (SAMs) with a range of hydrophobicity and charge. Supported by both the coverage and the dimensions of immobilized enzymes, our results indicate that of the surfaces investigated, material-selective binding takes place with orientation control only for PutOx-AuBP onto the TSG substrate. These differences are consistent with the measurements of surface-bound enzymatic activities. Substrate-dependent differences observed indicate significant variations in enzyme-surface interactions ranging from peptide-directed self-assembly to enzyme aggregation. The implications of this study provide insight for the fabrication of enzymatic patterns directed by self-assembling peptide tags onto localized surface regions. Enabling functional enzyme-based nanoscale materials offers a fascinating path for utilization of sustainable biocatalysts integrated into multiscale devices.

Designing Gold Binding Peptides: Modelling and Experiment at the Bio-Abiotic Interface

The interface of biological and abiological material is an area of intense interest, touching on a widespread application space of sensors, bioelectronics, designer coatings, self-healing materials. Building upon our work in reconfigurable electronic devices, part of our efforts were focused on engineering E. coli to display metal binding peptides using a bacterial display system, eCPX. Here, we present our efforts in understanding cell binding to Au(111) surfaces as well as gold nanoparticles (AuNPs) through a series of known and designed gold binding peptides (GBPs) that have been engineered into eCPX. First, we demonstrate that integration of specific GBPs into the E. coli outer membrane protein increases binding to both Au(111) surfaces and AuNPs, that the binding to gold is selective, and is influenced by interactions with specific amino acids. Second, from molecular dynamics (MD) simulations of select GBPs on Au(111), we show that interaction energies between the peptides and surface correlate with experimental data, and that both thermodynamic and entropic effects play a role in binding. And finally, we demonstrate a novel fragment-based computer-aided biomaterial design approach using Au(111) as a proof of concept. These findings will be discussed in the greater context of our work in transformational synthetic biology for military environments.

Electrochemical Detection of Insulin with a Novel Protein-Based Artificial Receptor

In this paper, we have developed a new electrochemical method to detect insulin with a protein-based artificial receptor. Diabetes is one of the most serious diseases all over the world. For the treatment of diabetes and maintenance of the patient's quality of life, the control of blood glucose level is important. Usually the blood glucose level in human body is regulated by insulin. Therefore, it is very important to know the status of both glucose and insulin in blood for the treatment of diabetes. Currently, it has become possible to measure the blood glucose level by patients themselves with bioelectrochemical sensors, however, there is no convenient method to measure insulin by patients themselves. In recent years, antibody-based and aptamer-based electrochemical insulin sensors have been developed. Although these sensors are highly sensitive, they may also detect physiologically inactive insulin such as partially digested or mutation insulin because the insulin recognition molecules of these sensors specifically recognize a part of insulin. However, it is desired to detect only active insulin for accurate evaluation of insulin which is functional in blood. Therefore, it is necessary to develop a novel sensor based on a new target recognition mechanism for the accurate evaluation of insulin. In our strategy, we utilized an insulin receptor, because the recognition by the insulin receptor is one of the essential processes for insulin to exhibit its functional role in blood. It has been reported that two domains in the insulin receptor, αCT segment and L1CR domain, recognize an insulin molecule by sandwiching format. However, the insulin receptor is typically buried in a cellular membrane and thus it is difficult to use the receptor with a native format for electrochemical detection. Hence, we have developed a novel artificial insulin receptor by connecting these two domains with a linker. We considered that this new artificial insulin receptor alters its structure in response to insulin recognition, and thus electrochemical properties of an electrode modified with the artificial receptor should reflect the structural change. In order to modify the electrode, we utilized three-time-repeats of a gold-binding peptide (GBP) that is known to assemble on a gold surface spontaneously. These domains were genetically fused and resulting artificial insulin receptors were termed as 3GαL and αL3G depending on their sequence of the domains, respectively. The novel artificial receptors were expressed by introducing plasmids encoding each recombinant gene to animal cells, then purified utilizing histidine-tag affinity. The expression and purification of each receptor was confirmed by SDS-PAGE. The ability of self-assembling on a gold surface was evaluated by QCM-D. The artificial receptor modified electrode was prepared by placing each protein solution on the gold surface of electrodes. The electrochemical response of the modified electrode to insulin was evaluated using electrochemical impedance spectroscopy (EIS). EIS measurement was performed in buffer solution in the presence of 5 mM [Fe(CN)6]3-/4- (the frequency range: 100 mHz to 100 kHz, potential: REST, amplitude: 10 mV). The comparisons of Nyquist plots revealed that the diameters of semicircle increased in the case of electrodes modified with the artificial receptors, when compared with an unmodified electrode. This is probably because the artificial receptors are proteins which inhibit interfacial electron transfer. When insulin was added to the solution, the diameters of semicircle further increased, while no drastic increase was observed when C-peptide was added. Comparison of the responses between electrodes modified with 3GαL and αL3G revealed that the electrode modified with 3GαL exhibited a better S/N ratio in response to insulin. In case of 3GαL, the side of αCT segment was immobilized on the gold surface and L1CR domain whose size is much larger than αCT segment kept rather free. In contrast, in case of αL3G, the side of larger domain of L1CR was already fixed and the smaller part of αCT segment kept rather free. Therefore, it was considered that the effect of structural change induced by the recognition of insulin on the electrochemical property of electrode should be more significant in the case of 3GαL than the case of αL3G. From these results, it has been concluded that insulin can be measured electrochemically with the electrode modified with the protein-based artificial receptors.

Self-Immobilized Putrescine Oxidase Biocatalyst System Engineered with a Metal Binding Peptide.

Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.