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1.
ACS Appl Mater Interfaces ; 15(9): 11391-11402, 2023 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-36847552

RESUMO

Discovery of microorganisms and their relevant surface peptides that specifically bind to target materials of interest can be achieved through iterative biopanning-based screening of cellular libraries having high diversity. Recently, microfluidics-based biopanning methods have been developed and exploited to overcome the limitations of conventional methods where controlling the shear stress applied to remove cells that do not bind or only weakly bind to target surfaces is difficult and the overall experimental procedure is labor-intensive. Despite the advantages of such microfluidic methods and successful demonstration of their utility, these methods still require several rounds of iterative biopanning. In this work, a magnetophoretic microfluidic biopanning platform was developed to isolate microorganisms that bind to target materials of interest, which is gold in this case. To achieve this, gold-coated magnetic nanobeads, which only attached to microorganisms that exhibit high affinity to gold, were used. The platform was first utilized to screen a bacterial peptide display library, where only the cells with surface peptides that specifically bind to gold could be isolated by the high-gradient magnetic field generated within the microchannel, resulting in enrichment and isolation of many isolates with high affinity and high specificity toward gold even after only a single round of separation. The amino acid profile of the resulting isolates was analyzed to provide a better understanding of the distinctive attributes of peptides that contribute to their specific material-binding capabilities. Next, the microfluidic system was utilized to screen soil microbes, a rich source of extremely diverse microorganisms, successfully isolating many naturally occurring microorganisms that show strong and specific binding to gold. The results show that the developed microfluidic platform is a powerful screening tool for identifying microorganisms that specifically bind to a target material surface of interest, which can greatly accelerate the development of new peptide-driven biological materials and hybrid organic-inorganic materials.


Assuntos
Microfluídica , Biblioteca de Peptídeos , Microfluídica/métodos , Peptídeos/química , Magnetismo , Ouro
2.
Colloids Surf B Biointerfaces ; 203: 111730, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33853002

RESUMO

Biocompatible approaches to labeling bacteria with fluorescent nanoparticles are essential in order to create living bacterial bioconjugates for imaging, biosensors, medicine, and other applications. Herein we report the direct conjugation of carboxyl quantum dots (QDs) with E. coli outer membrane via surface-displayed binding peptides. The histidine-containing peptide H6G9 was displayed at the N-terminus of membrane-embedded enhanced circularly permuted outer membrane protein X (eCPX) scaffold, which was expressed upon chemical induction. The presence of the binding peptide creates an environment distinct from the negatively charged E. coli surface and provides strong binding affinity to carboxyl quantum dots (QDs). Transmission electron microscopy (TEM) analysis of E. coli-QD bioconjugates revealed high loading densities of these QDs immobilized on the cell surface, even when adding a very low concentration (10 µg/mL) of QDs in order to reduce the cell exposure. These hybrid cells strongly fluoresce with each of the distinct colors of loaded QDs with different emission wavelengths, which can be easily visualized by fluorescence microscopy or differentiated using flow cytometry. Importantly, the E. coli-QD bioconjugates were highly viable and maintained the ability to grow and divide. This study demonstrates a simple, direct, and highly efficient method for labelling bacteria with QDs, without significantly compromising the vitality of the cells.


Assuntos
Pontos Quânticos , Escherichia coli/genética , Histidina , Microscopia de Fluorescência , Peptídeos
3.
Nat Nanotechnol ; 16(6): 688-697, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33782589

RESUMO

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.


Assuntos
Eletrônica/métodos , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Células Imobilizadas/química , Eletrodos , Desenho de Equipamento , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação Bacteriana da Expressão Gênica , Ouro/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Microbiota , Microrganismos Geneticamente Modificados/genética , Oxirredução , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , beta-Galactosidase/metabolismo
4.
BMC Biotechnol ; 19(1): 100, 2019 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-31864334

RESUMO

BACKGROUND: Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest. RESULTS: In this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity. CONCLUSIONS: We demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.


Assuntos
Bioprospecção/métodos , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biblioteca de Peptídeos , Peptídeos/genética , Escherichia coli/química , Escherichia coli/metabolismo , Peptídeos/metabolismo
5.
Methods ; 158: 12-16, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30707950

RESUMO

The need for the functionalization of magnetic, water-soluble dyed microspheres with peptides is apparent with the ever-growing biointeraction capabilities and the increased use of dyed microspheres in multiplex, microsphere-based detection assays. This method describes the attachment of any peptide to dyed magnetic microspheres regardless of peptide length, size, or sequence. The method exploits 'click' chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching. All optimization studies were performed using a Luminex 200 assay platform, but the functionalized microspheres are capable of use in any similar multiplex format.


Assuntos
Química Click/métodos , Corantes/química , Ensaios de Triagem em Larga Escala/instrumentação , Microesferas , Peptídeos/química , Ensaios de Triagem em Larga Escala/métodos , Fenômenos Magnéticos , Polietilenoglicóis/química
6.
ACS Appl Bio Mater ; 2(7): 2937-2945, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35030787

RESUMO

In this study, naturally derived cellulose nanofibrils (CNFs), a renewable and easily modified nanomaterial with low cytotoxicity, were rendered bioactive via one-step functionalization with mannopyranoside (CNFs-mannose) for use as a new glyconanomaterial platform for control of bacterial pathogenesis. The recognition affinity of the bioactive surfaces toward fimbriated Escherichia coli was assessed using genetically engineered strains as well as wild-type (WT) MG1655 bacteria. The results revealed high surface coverages of FimH+ (with overexpressed FimH) and WT bacteria on the films of CNFs-mannose due to specific interaction between prevalent mannose on nanofibrils and FimH receptors on E. coli fimbriae. The CNFs-mannose nanofibrils were capable of capturing E. coli from a liquid suspension, as demonstrated either by the nanofibril clusters or by the cellulose filter papers impregnated with CNFs-mannose. More importantly, CNFs-mannose efficiently inhibited adhesion of both FimH+ and WT E. coli to mannosylated surfaces even at a very low concentration, resulting in over 95% reduction of bacterial adhesion. Furthermore, the bioactive nanofibrils showed effective disruption of nonspecific binding of bacteria to abiotic surfaces in flow channel tests. These findings highlight the potential of cellulose nanofibrils as a biocompatible polyvalent nanoscale scaffold and exemplify sugar grafted nanofibrils as novel and effective tools in control of bacterial pathogenesis, bacterial removal, as well as in many other applications.

7.
Nat Microbiol ; 3(9): 1043-1053, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30127494

RESUMO

Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10-1 to 10-7 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-ß-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.


Assuntos
Bacillus subtilis/genética , Conjugação Genética/genética , DNA Bacteriano/genética , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Sequências Repetitivas Dispersas/genética , DNA Bacteriano/metabolismo , Microbioma Gastrointestinal/genética , Fixação de Nitrogênio/genética , Pele/microbiologia , Microbiologia do Solo
9.
Langmuir ; 34(20): 5837-5848, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29692178

RESUMO

In this study, we investigated the preparation of living bacteria-nanoparticle hybrids mediated by surface-displayed peptides. The assembly of metallic nanoparticles on living bacteria has been achieved under mild conditions utilizing metal-peptide interactions, whereas the viability of the bacterial cells was greatly preserved. Escherichia coli was engineered with inducible gene circuits to control the display of peptides with desired sequences. Several designed peptide sequences as well as known gold-binding peptides were expressed on the cell surface using enhanced circularly permuted outer membrane protein X (eCPX) scaffolds. Driven by metal-peptide affinity, "biofriendly" citrate-stabilized gold nanoparticles were self-assembled onto the surface of bacteria with displayed peptides, which required overcoming the repulsive force between negatively charged nanoparticles and negatively charged cells. The bacteria/Au nanoparticle hybrids were highly viable and maintained the ability to grow and divide, which is a crucial step toward the creation of living material systems. Further activity and preservation of the bacterial hybrid assembly was demonstrated. The method described herein enables the conjugation of bacterial surfaces with diverse metal-rich nanoparticles in an inducible, and therefore easily controlled, manner. The expressed peptide sequences can be easily modified to alter the binding affinity and specificity for a wide variety of materials to form on-demand, high-density living biohybrids.


Assuntos
Nanopartículas Metálicas/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Escherichia coli , Ouro/química , Peptídeos/química
10.
J Vis Exp ; (130)2017 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-29286465

RESUMO

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.


Assuntos
Técnicas Biossensoriais/métodos , Biblioteca de Peptídeos , Peptídeos/química
11.
Biointerphases ; 12(2): 02C410, 2017 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-28490179

RESUMO

The potential advantages of cell-based biohybrid devices over conventional nonliving systems drive the interest to control the behavior of the underlying biological cells in microdevices. Here, the authors studied how shear influenced the geometry and elongation of fimbriated filaments on affinity substrates. The cells were engineered to express FimH, which binds to mannose with a high affinity. A microfluidic channel was functionalized with RNAse B, which is rich in mannose residues, and the device was used to control the hydrodynamic force on live Escherichia coli under filamentous growth. It was discovered that filamentous E. coli cells adopt buckled geometry when the shear rate is low, but assume an extended geometry at high shear and align with the flow direction. The extension moves from bidirectional to preferentially downstream as the shear rate increases. Furthermore, living filaments slide easily on the substrate, and detach from the substrates at a rate nearly ten times greater than unfilamented live E. coli at high shear conditions (1000-4000 s-1). The hydrodynamic force and binding force experienced by the cells are further analyzed by COMSOL simulation and atomic force microscopy measurements, respectively, to explore the mechanism behind the living cell dynamics. Knowledge from this work helps guide design of interfacial properties and shear environments to control the geometry of living filamentous bacteria.


Assuntos
Adesinas de Escherichia coli , Engenharia Celular , Escherichia coli , Proteínas de Fímbrias , Hidrodinâmica , Resistência ao Cisalhamento , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Ribonucleases/química , Ribonucleases/genética , Ribonucleases/metabolismo
12.
Biopolymers ; 108(2)2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27539157

RESUMO

We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T1/2 ) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.


Assuntos
Química Click/métodos , Biblioteca de Peptídeos , Peptídeos/química , Fator A de Crescimento do Endotélio Vascular/química , Sequência de Aminoácidos , Animais , Anticorpos/administração & dosagem , Anticorpos/química , Anticorpos/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Varredura Diferencial de Calorimetria , Catálise , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/metabolismo , Feminino , Células HT29 , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Ligantes , Masculino , Espectrometria de Massas , Camundongos , Microssomos Hepáticos/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacocinética , Ligação Proteica , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Molecules ; 21(11)2016 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-27834872

RESUMO

Peptide capture agents have become increasingly useful tools for a variety of sensing applications due to their ease of discovery, stability, and robustness. Despite the ability to rapidly discover candidates through biopanning bacterial display libraries and easily mature them to Protein Catalyzed Capture (PCC) agents with even higher affinity and selectivity, an ongoing challenge and critical selection criteria is that the peptide candidates and final reagent be selective enough to replace antibodies, the gold-standard across immunoassay platforms. Here, we have discovered peptide affinity reagents against abrax, a derivative of abrin with reduced toxicity. Using on-cell Fluorescence Activated Cell Sorting (FACS) assays, we show that the peptides are highly selective for abrax over RiVax, a similar derivative of ricin originally designed as a vaccine, with significant structural homology to abrax. We rank the newly discovered peptides for strongest affinity and analyze three observed consensus sequences with varying affinity and specificity. The strongest (Tier 1) consensus was FWDTWF, which is highly aromatic and hydrophobic. To better understand the observed selectivity, we use the XPairIt peptide-protein docking protocol to analyze binding location predictions of the individual Tier 1 peptides and consensus on abrax and RiVax. The binding location profiles on the two proteins are quite distinct, which we determine is due to differences in pocket size, pocket environment (including hydrophobicity and electronegativity), and steric hindrance. This study provides a model system to show that peptide capture candidates can be quite selective for a structurally similar protein system, even without further maturation, and offers an in silico method of analysis for understanding binding and down-selecting candidates.


Assuntos
Abrina/antagonistas & inibidores , Abrina/química , Simulação de Acoplamento Molecular , Peptídeos/química , Ricina/antagonistas & inibidores , Ricina/química , Homologia Estrutural de Proteína
14.
Molecules ; 21(8)2016 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-27483214

RESUMO

Microfabricated devices have increasingly incorporated bacterial cells for microscale studies and exploiting cell-based functions in situ. However, the role of surface interactions in controlling the bacterial cell behavior is not well understood. In this study, microfluidic substrates of varied bacterial-binding affinity were used to probe the interaction-driven behavior of filamentous Escherichia coli. In particular, cell alignment under controlled shear flow as well as subsequent orientation and filamentation were compared between cells presenting distinct outer membrane phenotypes. We demonstrated that filaments retained position under flow, which allowed for dynamic single-cell monitoring with in situ elongation of over 100 µm for adherent cells. This maximum was not reached by planktonic cells and was, therefore, adhesion-dependent. The bound filaments initially aligned with flow under a range of flow rates and their continual elongation was traced in terms of length and growth path; analysis demonstrated that fimbriae-mediated adhesion increased growth rate, increased terminal length, as well as dramatically changed the adherent geometry, particularly buckling behavior. The effects to filament length and buckling were further exaggerated by the strongest, specificity-driven adhesion tested. Such surface-guided control of the elongation process may be valuable to yield interesting "living" filamentous structures in microdevices. In addition, this work may offer a biomedically relevant platform for further elucidation of filamentation as an immune-resistant morphology. Overall, this work should inspire broader exploration of microfabricated devices for the study and application of single bacterial cells.


Assuntos
Escherichia coli/fisiologia , Microfluídica/instrumentação , Aderência Bacteriana , Microfluídica/métodos , Estresse Mecânico , Propriedades de Superfície
15.
Angew Chem Int Ed Engl ; 54(45): 13219-24, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26377818

RESUMO

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.


Assuntos
Desenho de Fármacos , Epitopos/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Proteínas/química , Ligantes , Peso Molecular , Peptídeos Cíclicos/química , Proteínas/antagonistas & inibidores
16.
J Mol Recognit ; 27(12): 739-45, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25319622

RESUMO

Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15-mer random library on the outer surface of Escherichia coli (E.coli), high-affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On-cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off-cell, using peptide-ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB-binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The Kd measured by peptide-ELISA off-cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.


Assuntos
Enterotoxinas/metabolismo , Fenômenos Magnéticos , Microfluídica/métodos , Biblioteca de Peptídeos , Peptídeos/análise , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Fluorescência , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica
17.
ACS Nano ; 7(10): 9452-60, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24063758

RESUMO

We report on a robust and sensitive approach for detecting protective antigen (PA) exotoxin from Bacillus anthracis in complex media. A peptide-based capture agent against PA was developed by improving a bacteria display-developed peptide into a highly selective biligand through in situ click screening against a large, chemically synthesized peptide library. This biligand was coupled with an electrochemical enzyme-linked immunosorbent assay utilizing nanostructured gold electrodes. The resultant assay yielded a limit of detection of PA of 170 pg/mL (2.1 pM) in buffer, with minimal sensitivity reduction in 1% serum. The powdered capture agent could be stably stored for several days at 65 °C, and the full electrochemical biosensor showed no loss of performance after extended storage at 40 °C. The engineered stability and specificity of this assay should be extendable to other cases in which biomolecular detection in demanding environments is required.


Assuntos
Antígenos de Bactérias/análise , Toxinas Bacterianas/análise , Técnicas Eletroquímicas/métodos , Ensaio de Imunoadsorção Enzimática , Limite de Detecção
18.
Adv Mater ; 25(33): 4585-91, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-23868808

RESUMO

The first-ever peptide biomaterial discovery using an unconstrained engineered bacterial display technology is reported. Using this approach, we have developed genetically engineered peptide binders for a bulk aluminum alloy and use molecular dynamics simulation of peptide conformational fluctuations to demonstrate sequence-dependent, structure-function relationships for metal and metal oxide interactions.


Assuntos
Ligas/química , Alumínio/química , Escherichia coli/genética , Engenharia Genética , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/química , Propriedades de Superfície
19.
Biotechniques ; 52(2): 95-102, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22313407

RESUMO

Peptide reagents can serve as alternatives or replacements to antibodies in sensing or diagnostic applications. The passive adsorption of peptides onto polystyrene surfaces can limit the target binding capability, especially for short, positively charged, or hydrophobic sequences. In this report, we show that fusing a peptide with a previously characterized 12-amino acid polystyrene binding sequence (PS-tag) improves overall peptide solubility and enzyme-linked immunosorbent assay (ELISA) results using the peptide as a capture agent. Specific improvements for protective antigen (PA; Bacillus anthracis) protein binding peptides selected from bacterial surface display were compared with native or biotinylated peptides. The PS-tag was added to either peptide terminus, using a (Gly)(4) spacer, and comparable binding affinities were obtained. Fusion with the PS-tag did not have any negative impact on peptide secondary structure as measured by circular dichroism. The addition of the PS-tag provides a convenient method to utilize peptide reagents from peptide display libraries as capture agents in an ELISA format without the need for a biotin tag or concerns about passive adsorption of critical residues for target capture.


Assuntos
Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Poliestirenos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática/métodos , Dados de Sequência Molecular , Ligação Proteica/genética , Análise de Sequência de Proteína/métodos , Solubilidade
20.
Nanomaterials (Basel) ; 2(3): 275-285, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-28348308

RESUMO

In the present work, the procedures for the creation of self-assembled DNA nanostructures in aqueous and non-aqueous media are described. DNA-Surfactant complex formation renders the DNA soluble in organic solvents offering an exciting way to bridge the transition of DNA origami materials electronics applications. The DNA retains its structural features, and these unique geometries provide an interesting candidate for future electronics and nanofabrication applications with potential for new properties. The DNA architectures were first assembled under aqueous conditions, and then characterized in solution (using circular dichroism (CD) spectroscopy) and on the surface (using atomic force microscopy (AFM)). Following aqueous assembly, the DNA nanostructures were transitioned to a non-aqueous environment, where butanol was chosen for optical compatibility and thermal properties. The retention of DNA hierarchical structure and thermal stability in non-aqueous conditions were confirmed via CD spectroscopy. The formation and characterization of these higher order DNA-surfactant complexes is described in this paper.

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