Gold-binding Peptide Research Articles

Peptide-functionalized iron oxide magnetic nanoparticle for gold mining

Here, we present our work on preparing a novel nanomaterial composed of inorganic binding peptides and magnetic nanoparticles for inorganic mining. Two previously selected and well-characterized gold-binding peptides from cell surface display, AuBP1 and AuBP2, were exploited. This nanomaterial (AuBP-MNP) was designed to fulfill the following two significant functions: the surface conjugated gold-binding peptide will recognize and selectively bind to gold, while the magnetic nano-sized core will respond and migrate according to the applied external magnetic field. This will allow the smart nanomaterial to mine an individual material (gold) from a pool of mixture, without excessive solvent extraction, filtration, and concentration steps. The working efficiency of AuBP-MNP was determined by showing a dramatic reduction of gold nanoparticle colloid concentration, monitored by spectroscopy. The binding kinetics of AuBP-MNP onto the gold surface was determined using surface plasmon resonance (SPR) spectroscopy, which exhibits around 100 times higher binding kinetics than peptides alone. The binding capacity of AuBP-MNP was demonstrated by a bench-top mining test with gold microparticles.Graphical abstract

Metal enhanced fluorescence on super-hydrophobic clusters of gold nanoparticles

We used optical lithography, electroless deposition and deep reactive ion etching techniques to realize arrays of super-hydrophobic gold nanoparticles arranged in a hierarchical structure. At the micro-scale, silicon-micro pillars in the chip permit to manipulate and concentrate biological solutions, at the nano-scale, gold nanoparticles enable metal enhanced fluorescence (MEF) effects, whereby fluorescence signal of fluorophores in close proximity to a rough metal surface is amplified by orders of magnitude. Here, we demonstrated the device in the analysis of fluorescein derived gold-binding peptides (GBP-FITC). While super-hydrophobic schemes and MEF effects have been heretofore used in isolation, their integration in a platform may advance the current state of fluorescence-based sensing technology in medical diagnostics and biotechnology. This scheme may be employed in protein microarrays where the increased sensitivity of the device may enable the early detection of cancer biomarkers or other proteins of biomedical interest.

Synthesis of gold structures by gold-binding peptide governed by concentration of gold ion and peptide.

Although biological synthesis methods for the production of gold structures by microorganisms, plant extracts, proteins, and peptide have recently been introduced, there have been few reports pertaining to controlling their size and morphology. The gold ion and peptide concentrations affected on the size and uniformity of gold plates by a gold-binding peptide Midas-11. The higher concentration of gold ions produced a larger size of gold structures reached 125.5μm, but an increased amount of Midas-11 produced a smaller size of gold platelets and increased the yield percentage of polygonal gold particles rather than platelets. The mechanisms governing factors controlling the production of gold structures were primarily related to nucleation and growth. These results indicate that the synthesis of gold architectures can be controlled by newly isolated and substituted peptides under different reaction conditions.

Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing

Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a “renewable” capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings.

Viral-templated gold/polypyrrole nanopeapods for an ammonia gas sensor

One-dimensional gold/polypyrrole (Au/PPy) nanopeapods were fabricated using a viral template: M13 bacteriophage. The genetically modified filamentous virus displayed gold-binding peptides along its length, allowing selective attachment of gold nanoparticles (Au NPs) under ambient conditions. A PPy shell was electropolymerized on the viral-templated Au NP chains forming nanopeapod structures. The PPy shell morphology and thickness were controlled through electrodeposition potential and time, resulting in an ultra-thin conductive polymer shell of 17.4 ± 3.3 nm. A post-electrodeposition acid treatment was used to modify the electrical properties of these hybrid materials. The electrical resistance of the nanopeapods was monitored at each assembly step. Chemiresistive ammonia (NH3) gas sensors were developed from networks of the hybrid Au/PPy nanostructures. Room temperature sensing performance was evaluated from 5 to 50 ppmv and a mixture of reversible and irreversible chemiresistive behavior was observed. A sensitivity of 0.30%/ppmv was found for NH3 concentrations of 10 ppmv or less, and a lowest detection limit (LDL) of 0.007 ppmv was calculated. Furthermore, acid-treated devices exhibited an enhanced sensitivity of 1.26%/ppmv within the same concentration range and a calculated LDL of 0.005 ppmv.

Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant.

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants.

Phage-templated gold nano-aggregates for cancer treatment

Event Abstract Back to Event Phage-templated gold nano-aggregates for cancer treatment Esen Sokullu1, Aziz Berchtikou1, Amy Blum2, Tsuneyuki Ozaki1 and Marc A. Gauthier1 1 Institut national de la recherche scientifique (INRS), Énergie, Matériaux et Télécommunications (EMT), Canada 2 McGill University, Chemistry Department, Canada Theranostic is an approach combining diagnosis and therapy to increase the precision and efficacy of cancer treatment. Different nanoparticle (NP) platforms have been engineered to obtain both functions by uniting them in one agent; however none of these can overcome the limitation in specificity of NP targeting, which is still a major challenge in cancer treatment. Plasmonic nanobubbles (PNB) are a new class of nano-agent that was developed to overcome this limitation. A PNB is a vapor nanobubble transiently generated around superheated gold NPs by a short laser pulse. The rapidly expanding bubble mechanically disrupts the surrounding tissue and achieves micrometer precision in tissue ablation. In general, PNB therapy relies on receptor-mediated endocytosis of gold NPs and formation of gold clusters within the cells [1],[2]. However, variability in the cellular uptake and subsequent aggregation of gold NPs may be detrimental to reproducibility of PNB. In this study, M13 and T4 phage display platforms are used to synthesize well-defined gold nano-aggregates and their efficiency in formation of gold nano-aggregates are evaluated. Two different T4 phage templates were designed by displaying cysteine and gold binding A3 peptide (AYSSGAPPMPPF) (A: Alanine, Y: Tyrosine, S: Serine, G: Glycine, P: Proline, M: Methionine, F: Phenylalanine, L: Leucine, K: Lysine, H: Histidine, R: Arginine, V: Valine, D: Aspartic acid) on small outer capsid (SOC) protein of phage through the homologous recombination between T4-Z1 mutant phage and pRH recombination plasmid. Ph.D. Peptide Display Cloning System (Biolabs Inc.) was used to design M13 phage templates. Self assembly of gold NP was directed by displaying gold binding peptide sequence (VSGSSPDS) on p8 major coat protein of phage. M13 phage templates were also engineered to display RGD peptides (ACDCRGDCFC, ACRGDGWC, GRGDSP) (C; Cysteine, W; Tryptophan) on p3 minor coat protein to target cancer cells through integrin protein. Recombinant phages were analyzed by PCR and also verified via DNA sequencing. The affinities of RGD displaying M13 phages were tested against human integrin alpha 5 protein fragment by phage-ELISA and M13 phages displaying cyclic RGD peptides (with two and four cysteines) showed significant binding affinity to integrin. Gold NPs were synthesized according to the protocol of Zahr and Blum and formation of gold nano-aggregates was initially studied by using cysteine bearing T4 phage template [3]. Conjugation of gold NPs to phage capsid was monitored by using UV-Vis spectrometer and the growth of plasmon at 600 nm was recorded. After 3 days of reaction, the samples were visualized by transmission electron microscopy (TEM) and formation of phage-templated gold nano-aggregates was confirmed. Herein, it was shown that cysteine-displaying T4 phage was successfully used as a template to develop well-defined gold nano-aggregates. The number of functional moieties on phage surface can be controlled at gene level and make us to avoid the use of irreproducible functionalization steps. The future work will focus on optimization of gold nano-aggregate formation for each phage design and their efficiency in PNB generation will be investigated. We would like to acknowledge that T4Z1 mutant phage and pRH recombination plasmid were kindly supplied by Prof. Lindsay W. Black (University of Maryland, US).

Genetically Programmed Clusters of Gold Nanoparticles for Cancer Cell-Targeted Photothermal Therapy.

Interpretations of the interactions of nanocarriers with biological cells are often complicated by complex synthesis of materials, broad size distribution, and heterogeneous surface chemistry. Herein, the major capsid proteins of an icosahedral T7 phage (55 nm in diameter) are genetically engineered to display a gold-binding peptide and a prostate cancer cell-binding peptide in a tandem sequence. The genetically modified phage attracts gold nanoparticles (AuNPs) to form a cluster of gold nanoparticles (about 70 nanoparticles per phage). The cluster of AuNPs maintains cell-targeting functionality and exhibits excellent dispersion stability in serum. Under a very low light irradiation (60 mW cm(-2)), only targeted AuNP clusters kill the prostate cancer cells in minutes (not in other cell types), whereas neither nontargeted AuNP clusters nor citrate-stabilized AuNPs cause any significant cell death. The result suggests that the prostate cancer cell-targeted clusters of AuNPs are targeted to only prostate cancer cells and, when illuminated, generate local heating to more efficiently and selectively kill the targeted cancer cells. Our strategy can be generalized to target other types of cells and assemble other kinds of nanoparticles for a broad range of applications.

Functional and Selective Bacterial Interfaces Using Cross-Scaffold Gold Binding Peptides

We investigated the functional and selective activity of three phage-derived gold-binding peptides on the Escherichia coli (E. coli) bacterial cell surface display scaffold (eCPX) for the first time. Gold-binding peptides, p3-Au12 (LKAHLPPSRLPS), p8#9 (VSGSSPDS), and Midas-2 (TGTSVLIATPYV), were compared side-by-side through experiment and simulation. All exhibited strong binding to an evaporated gold film, with approximately a 4-log difference in binding between each peptide and the control sample. The increased affinity for gold was also confirmed by direct visualization of samples using Scanning Electron Microscopy (SEM). Peptide dynamics in solution were performed to analyze innate structure, and all three were found to have a high degree of flexibility. Preferential binding to gold over silicon for all three peptides was demonstrated, with up to four orders of magnitude selectivity exhibited by p3-Au12. The selectivity was also clearly evident through SEM analysis of the boundary between the gold film and silicon substrate. Functional activity of bound E. coli cells was further demonstrated by stimulating filamentation and all three peptides were characterized as prolific relative to control samples. This work shows great promise towards functional and active bacterial–hybrid gold surfaces and the potential to enable the next generation living material interfaces.

Influence of Solvent in Controlling Peptide-Surface Interactions.

Protein binding to surfaces is an important phenomenon in biology and in modern technological applications. Extensive experimental and theoretical research has been focused in recent years on revealing the factors that govern binding affinity to surfaces. Theoretical studies mainly focus on examining the contribution of the individual amino acids or, alternatively, the binding potential energies of the full peptide, which are unable to capture entropic contributions and neglect the dynamic nature of the system. We present here a methodology that involves the combination of nonequilibrium dynamics simulations with strategic mutation of polar residues to reveal the different factors governing the binding free energy of a peptide to a surface. Using a gold-binding peptide as an example, we show that relative binding free energies are a consequence of the balance between strong interactions of the peptide with the surface and the ability for the bulk solvent to stabilize the peptide.

Adjusting the Metrics of 1-D Helical Gold Nanoparticle Superstructures Using Multivalent Peptide Conjugates.

The properties of nanoparticle superstructures depend on many factors, including the structural metrics of the nanoparticle superstructure (particle diameter, interparticle distances, etc.). Here, we introduce a family of gold-binding peptide conjugate molecules that can direct nanoparticle assembly, and we describe how these molecules can be systematically modified to adjust the structural metrics of linear double-helical nanoparticle superstructures. Twelve new peptide conjugates are prepared via linking a gold-binding peptide, AYSSGAPPMPPF (PEP(Au)), to a hydrophobic aliphatic tail. The peptide conjugates have 1, 2, or 3 PEP(Au) headgroups and a C12, C14, C16, or C18 aliphatic tail. The soft assembly of these peptide conjugates was studied using transmission electron microscopy (TEM), atomic force microscopy (AFM), and infrared (IR) spectroscopy. Several peptide conjugates assemble into 1-D twisted fibers having measurable structural parameters such as fiber width, thickness, and pitch that can be systematically varied by adjusting the aliphatic tail length and number of peptide headgroups. The linear soft assemblies serve as structural scaffolds for arranging gold nanoparticles into double-helical superstructures, which are examined via TEM. The pitch and interparticle distances of the gold nanoparticle double helices correspond to the underlying metrics of the peptide conjugate soft assemblies, illustrating that designed peptide conjugate molecules can be used to not only direct the assembly of gold nanoparticles but also control the metrics of the assembled structure.

Macromol. Rapid Commun. 14/2015

Front Cover: A method for mass production of biologically functional polymer nanofibers combining controlled flow and gyration is invented. In a one-pot process, integration of gold nanoparticles into the nanofiber assembly is achieved using a gold-binding peptide tag genetically conjugated to red fluorescence protein. The stability and biological functionality of the nanofibers created in this way are demonstrated by fluorescence spectrophotometry including a copper binding assay. Further details can be found in the article by S. Zhang, B. T. Karaca, S. K. VanOosten, E. Yuca, S. Mahalingam, M. Edirisinghe,* and C. Tamerler* on page 1322.

Macromol. Rapid Commun. 14/2015

Front Cover: A method for mass production of biologically functional polymer nanofibers combining controlled flow and gyration is invented. In a one-pot process, integration of gold nanoparticles into the nanofiber assembly is achieved using a gold-binding peptide tag genetically conjugated to red fluorescence protein. The stability and biological functionality of the nanofibers created in this way are demonstrated by fluorescence spectrophotometry including a copper binding assay. Further details can be found in the article by S. Zhang, B. T. Karaca, S. K. VanOosten, E. Yuca, S. Mahalingam, M. Edirisinghe,* and C. Tamerler* on page 1322.

Yeast dual-affinity biobricks: Progress towards renewable whole-cell biosensors.

Point-of-care (POC) diagnostic biosensors offer a promising solution to improve healthcare, not only in developed parts of the world, but also in resource limited areas that lack adequate medical infrastructure and trained technicians. However, in remote and resource limited settings, cost and storage of traditional POC immunoassays often limit actual deployment. Synthetically engineered biological components ("BioBricks") provide an avenue to reduce costs and simplify assay procedures. In this article, the design and development of an ultra-low cost, whole-cell "renewable" capture reagent for use in POC diagnostic applications is described. Yeast cells were genetically modified to display both single chain variable fragment (scFv) antibodies and gold-binding peptide (GBP) on their surfaces for simple one step enrichment and surface functionalization. Electrochemical impedance spectroscopy (EIS) and fluorescent imaging were used to verify and characterize the binding of cells to gold electrodes. A complete electrochemical detection assay was then performed on screen-printed electrodes fixed with yeast displaying scFv directed to Salmonella outer membrane protein D (OmpD). Electrochemical assays were optimized and cross-validated with established fluorescence techniques. Nanomolar detection limits were observed for both formats.

Direct bioelectrocatalysis at the interfaces by genetically engineered dehydrogenase

There is an emerging interest in developing bio-functionalisation routes serving as platforms for assembling diverse enzymes onto material surfaces. Specifically, the fabrication of next-generation, laboratory-on-a-chip-based sensing and energy-harvesting systems requires controlled orientation and organisation of the proteins at the inorganic interfaces. Herein, the authors take the initial steps towards designing multifunctional, enzyme-based platforms by genetically integrating the engineered material-selective peptide tags for tethering redox enzymes onto electrode surfaces. The authors engineered a fusion protein that genetically conjugates gold-binding peptide to formate dehydrogenase derived from Candida methylica. The expressed proteins were tested for both enzyme activity and self-directed gold-surface functionalisation ability. Their findings demonstrate the successful self-immobilisation of the engineered enzyme onto different gold electrodes while retaining the catalytic activity. Building on the functionalisation by the peptides, a fusion enzyme-integrated circuit-based biosensor system was designed. The catalytic conversion of the formate by the engineered dehydrogenase was successfully monitored on the electrode surface at subsequent intervals. The engineered peptide-mediated self-integrated electrode systems can be extended to develop a wide range of biosensing and energy-harvesting platforms using different combinations of materials and biomolecules. This paper contains supporting information that will be made available online once the issue is published.

Facet selectivity in gold binding peptides: exploiting interfacial water structure.

Peptide sequences that can discriminate between gold facets under aqueous conditions offer a promising route to control the growth and organisation of biomimetically-synthesised gold nanoparticles. Knowledge of the interplay between sequence, conformations and interfacial properties is essential for predictable manipulation of these biointerfaces, but the structural connections between a given peptide sequence and its binding affinity remain unclear, impeding practical advances in the field. These structural insights, at atomic-scale resolution, are not easily accessed with experimental approaches, but can be delivered via molecular simulation. A current unmet challenge lies in forging links between predicted adsorption free energies derived from enhanced sampling simulations with the conformational ensemble of the peptide and the water structure at the surface. To meet this challenge, here we use an in situ combination of Replica Exchange with Solute Tempering with Metadynamics simulations to predict the adsorption free energy of a gold-binding peptide sequence, AuBP1, at the aqueous Au(111), Au(100)(1 × 1) and Au(100)(5 × 1) interfaces. We find adsorption to the Au(111) surface is stronger than to Au(100), irrespective of the reconstruction status of the latter. Our predicted free energies agree with experiment, and correlate with trends in interfacial water structuring. For gold, surface hydration is predicted as a chief determining factor in peptide-surface recognition. Our findings can be used to suggest how shaped seed-nanocrystals of Au, in partnership with AuBP1, could be used to control AuNP nanoparticle morphology.

Thermodynamics of engineered gold binding peptides: establishing the structure-activity relationships.

Adsorption behavior of a gold binding peptide was experimentally studied to achieve kinetics and thermodynamics parameters toward understanding of the binding of an engineered peptide onto a solid metal surface. The gold-binding peptide, GBP1, was originally selected using a cell surface display library and contains 14 amino acid residues. In this work, single- and three-repeats of GBP1 were used to assess the effects of two parameters: molecular architecture versus secondary structure on adsorption on to gold substrate. The adsorption measurements were carried out using surface plasmon resonance (SPR) spectroscopy at temperatures ranging from 10 to 55 °C. At all temperatures, two different regimes of peptide adsorption were observed, which, based on the model, correspond to two sets of thermodynamics values. The values of enthalpy, ΔH(ads), and entropy, ΔS(ads), in these two regimes were determined using the van't Hoff approach and Gibbs-Helmholtz relationship. In general, the values of enthalpy for both peptides are negative indicating GBP1 binding to gold is an exothermic phenomenon and that the binding of three repeat gold binding peptide (3l-GBP1) is almost 5 times tighter than that for the single repeat (l-GBP1). More intriguing result is that the entropy of adsorption for the 3l-GBP1 is negative (-43.4 ± 8.5 cal/(mol K)), while that for the l-GBP1 is positive (10.90 ± 1.3 cal/(mol K)). Among a number of factors that synergistically contribute to the decrease of entropy, long-range ordered self-assembly of the 3l-GBP1 on gold surface is the most effective, probably through both peptide-solid and peptide-peptide intermolecular interactions. Additional adsorption experiments were conducted in the presence of 2,2,2-trifluoroethanol (TFE) to determine how the conformational structures of the biomolecules responded to the environmental perturbation. We found that the peptides differ in their conformational responses to the change in solution conditions; while l-GBP does not fold in the presence of TFE, 3l-GBP1 adopted two types of secondary structure (β-strand, α-helix) and that peptide's binding to the solid is enhanced by the presence of low percentages of TFE solvent. Not only do these kinetics and thermodynamics results provide adsorption behavior and binding of genetically engineered peptides for inorganics (GEPI), but they could also provide considerable insights into fundamental understanding peptide molecular recognition and their selective specificity for the solids. Moreover, comprehensive work described herein suggests that multiple repeat forms of the solid binding peptides possess a conformational component that can be exploited to further tailor affinity and binding of a given sequence to a solid material followed by ordered assembly as a convenient tool in future practical applications.

Highly sensitive hydrogen sulfide (H2 S) gas sensors from viral-templated nanocrystalline gold nanowires

A facile, site-specific viral-templated assembly method was used to fabricate sensitive hydrogen sulfide (H2S) gas sensors at room temperature. A gold-binding M13 bacteriophage served to organize gold nanoparticles into linear arrays which were used as seeds for subsequent nanowire formation through electroless deposition. Nanowire widths and densities within the sensors were modified by electroless deposition time and phage concentration, respectively, to tune device resistance. Chemiresistive H2S gas sensors with superior room temperature sensing performance were produced with sensitivity of 654%/ppmv, theoretical lowest detection limit of 2 ppbv, and 70% recovery within 9 min for 0.025 ppmv. The role of the viral template and associated gold-binding peptide was elucidated by removing organics using a short O2 plasma treatment followed by an ethanol dip. The template and gold-binding peptide were crucial to electrical and sensor performance. Without surface organics, the resistance fell by several orders of magnitude, the sensitivity dropped by more than a factor of 100 to 6%/ppmv, the lower limit of detection increased, and no recovery was detected with dry air flow. Viral templates provide a novel, alternative fabrication route for highly sensitive, nanostructured H2S gas sensors.

Engineered Peptides for Nanohybrid Assemblies

Inspired by biological material synthesis, synthetic biomineralization peptides have been screened through a laboratory evolution using biocombinatorial techniques. In this study, using the fine examples in nature, silica binding peptides and gold binding peptides were fused together to form a hybrid peptide. We designed fusion peptides with different gold binding and silica binding parts. First, we have tested the binding capability of the fusion peptides using quartz crystal microbalance on gold surface and silica surface. Second, S1G1 hybrid peptide enabled assembly of gold nanoparticles on a silica surface was achieved. Finally, nanomaterial synthesis ability of the S1G1 peptide was presented by the formation of a silica film on a gold surface. In this study, we are presenting a hybrid peptide tool for nanohybrid assembly as a promising route for nanotechnology applications.

Modular Peptide-Based Hybrid Nanoprobes for Bio-Imaging and Bio-Sensing

ABSTRACTThe self-organization of functional proteins directly onto solid materials is attractive to a wide range of biomaterials and systems that need to accommodate a biological recognition element. In such systems, inorganic binding peptides may be an essential component due to their high affinity and selective binding features onto different types of solid surfaces. This study demonstrates a peptide-enabled self-assembly technique for designing well-defined protein arrays over a metal surface. To illustrate this concept, we designed a fusion protein that simultaneously displays a red fluorescence protein (DsRed-monomer), which is highly selective for copper ions, and a gold binding peptide AuBP. The peptide tag, AuBP, self-directs the organization of DsRed-monomer protein onto a gold surface and forms arrays built upon an efficient control of the organic/inorganic interface at the molecular level. The peptide-assisted design offers a modular approach for fabrication of fluorescent-based protein arrays with copper ion sensing ability.