RESUMO
The application of a potential to deposit a monolayer of 3-mercaptopropionic acid-histidinyl-histidinyl-histidinyl-aspartyl-aspartyl (3-MPA-HHHDD-OH) controls the density and molecular structure of the peptide monolayer, which results in different wettabilities of the surface, surface density, orientation of the molecule (extended or bent), and nonspecific adsorption of serum proteins. 3-MPA-HHHDD-OH must be deposited at 200 mV to maintain an extended configuration, which promoted low biofouling properties.
Assuntos
Ouro/química , Peptídeos/química , Adsorção , Proteínas Sanguíneas/química , Conformação Proteica , Ressonância de Plasmônio de Superfície , Propriedades de Superfície , MolhabilidadeRESUMO
Nanomaterials developed for localized surface plasmon resonance (LSPR) are increasingly integrated to classical prism-based SPR sensors, providing enhanced sensitivity and lower detection limits. The unique properties of these novel nanomaterials in addition to novel surface chemistry to minimize nonspecific adsorption and surface plasmon-coupled techniques with other spectroscopic or mass spectrometry techniques are highlighted in this article.
RESUMO
A peptide self-assembled monolayer (SAM) was designed to bind His-tagged biomolecules for surface plasmon resonance (SPR) bioanalysis, which was applied for the determination of K(d) for small ligand screening against CD36. Nonspecific adsorption could be minimized using penta- and hexa-peptide monolayers. In particular, monolayers consisting of 3-mercaptopropionyl-leucinyl-histidinyl-aspartyl-leucinyl-histidinyl-aspartic acid (3-Mpa-LHDLHD) exhibited little (12 ng cm(-2)) nonspecific adsorption in crude serum. Modification of this peptide monolayer with Nα,Nα-bis(carboxymethyl)-L-lysine gave a surface competent for binding His-tagged proteins, as demonstrated using enzyme (human dihydrofolate reductase), protein/antibody and receptor (CD36) examples. Immobilization featured chelation of copper and the His-tagged protein by the peptide monolayer, which could be recycled by removing the copper using imidazole washes prior to reuse.
Assuntos
Antígenos CD36/metabolismo , Histidina/química , Peptídeos/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ressonância de Plasmônio de Superfície/métodos , Adsorção , Sequência de Aminoácidos , Animais , Bovinos , Avaliação Pré-Clínica de Medicamentos/métodos , Histidina/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Ligantes , Peptídeos/metabolismo , Ligação Proteica , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
The properties of a surface modified with an ionic liquid self-assembled monolayer (IL-SAM) can be tuned by simply changing the deposition temperature. Mid-IR, SERS, and molecular modelling demonstrated that 1-(12-mercaptododecyl)-3-methylimidazolium bromide (MDMIBr) exhibited a crystalline monolayer for deposition temperatures below 25 °C. Above 25 °C, the aliphatic chain collapsed into a disordered conformation. At 40 °C, another phase transition occurs due to the imidazolium group tilting parallel to the surface. Consequently, the wettability of IL-SAM was tuned over a broad range of contact angle (from 20° to nearly 40°) by varying the deposition temperature. Permeation of redox mediators to a Au electrode coated with MDMIBr strongly depends on the net charge of the redox mediator. Electron transfer was excellent for neutral and negatively charged redox mediators on electrodes coated with IL-SAM regardless of deposition temperature.
RESUMO
Near-zero fouling monolayers based on binary patterned peptides allow low nanomolar detection of the matrix metalloproteinase-3 (MMP-3) directly in crude bovine serum, without sample pretreatment, secondary antibody detection or signal amplification. The peptide 3-MPA-HHHDD-OH (3-MPA, 3-mercaptopropionic acid) was found optimal compared to other binary patterned peptides based on 3-MPA-A(x)-B(y)-OH, where 0 Assuntos
Técnicas Biossensoriais
, Proteínas Sanguíneas/análise
, Metaloproteinase 3 da Matriz/análise
, Peptídeos/química
, Ressonância de Plasmônio de Superfície
, Sequência de Aminoácidos
, Animais
, Proteínas Sanguíneas/química
, Bovinos
, Enzimas Imobilizadas/química
, Metaloproteinase 3 da Matriz/química
, Dados de Sequência Molecular
, Estrutura Molecular
RESUMO
Metallic thin films patterned with micrometer size triangle or hole arrays present plasmonic properties when excited in the Kretschmann configuration, that are improved in comparison to conventional thin film surface plasmon resonance (SPR). These optical properties can be tuned by varying the physical aspects of the microplasmonic structures. Triangles and microhole arrays were prepared with modified nanosphere lithography (NSL) using latex spheres of 0.65, 0.82, 1.0, 1.5, or 3.2 microm in diameter. This allowed the preparation of triangles with edge lengths between 275 to 2000 nm and microhole arrays of various periodicities, diameters, and hole depths. These microstructures were studied to understand the relationship between the physical aspects and the optical properties, such as the sensitivity, working refractive index range, spectral width of the plasmonic peaks, spectral noise, and refractive index resolution. Microhole arrays with a hole diameter equal to half the periodicity were found to combine the advantages of both localized surface plasmon resonance (LSPR) on nanoparticles and SPR on a thin film. These microhole arrays exhibited high sensitivity to refractive index (>3000 nm/RIU), sensitivity to monolayer formation (2-fold improvement compared to thin films), and excellent refractive index resolution (10(-6) RIU). Finally, a biosensor for the detection of 10 nM of immunoglobulin G (IgG) exhibited a greater response with microplasmonic materials compared to conventional thin Au films. Hence, these novel plasmonic materials exhibit a strong potential as an SPR sensing platform. They can be implemented on existing instrumentation and use detection protocols developed for current SPR sensors.
Assuntos
Técnicas Biossensoriais , Análise em Microsséries , Ressonância de Plasmônio de Superfície , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Microscopia de Força Atômica , Propriedades de SuperfícieRESUMO
Short peptides, composed of polar or ionic amino acids, derived with a short organic thiol, significantly reduce nonspecific adsorption of proteins in complex biological matrices such as serum and crude cell lysate, which have nonspecific protein concentrations of 76 and 30-60 mg/mL, respectively. Minimizing these nonspecific interactions has allowed rapid and direct quantification of beta-lactamase in a crude cell lysate using a surface plasmon resonance (SPR) biosensor. A library of short peptides with varying chain length and amino acid composition were synthesized using a solid-phase approach. A 3-mercaptopropionic acid (3-MPA) linker was covalently attached to the amino terminus of the peptides to subsequently form a monolayer on gold in the form of 3-MPA-(AA)(n)-OH, where n is the length of the amino acid chain (n = 2-5). Leu, Phe, Ser, Asp, and His were selected to investigate the effect on nonspecific adsorption with different physicochemical properties of the sidechains; aliphatic, aromatic, polar, acid, and base. Advancing contact angles measured the hydrophobicity of each peptidic self-assembled monolayer (SAM) and showed that hydrophilicity of the gold surface improved as the chain length of the polar or ionic peptides increased, while aromatic and aliphatic peptides decreased the hydrophilicity as the chain length increased. The nonspecific adsorption of undiluted bovine serum on SPR sensors prepared with the library of 3-MPA-(AA)(n)-OH showed that the lowest nonspecific adsorption occurred with polar or ionic amino acids with a chain length of n = 5. We demonstrate that a monolayer composed of 3-MPA-(Ser)(5)-OH has significant advantages, including the following: (1) it minimizes nonspecific adsorption in undiluted bovine serum; (2) it provides a high surface concentration of immobilized antibodies; (3) it shows a great retention of activity for the antibodies; (4) it improves the response from beta-lactamase by approximately 1 order of magnitude, compared to previous experiments; and (5) it allows direct quantification of submicromolar beta-lactamase concentration in a crude cell lysate with a nonspecific protein concentration of 30-60 mg/mL. The use of this peptide-based monolayer offers great advantages for quantitative SPR biosensing in complex biological media.
Assuntos
Técnicas Biossensoriais , Peptídeos/química , Ressonância de Plasmônio de Superfície/métodos , Adsorção , Proteínas Sanguíneas/química , Calibragem , Enzimas Imobilizadas/química , beta-Lactamases/químicaRESUMO
Biorecognition-element labeled liposomes are simple and versatile tools used to amplify signals for the detection of analytes of environmental, clinical, food safety, and national security interest. Relying on measurement of encapsulated species via electrochemical or spectroscopic techniques, or properties inherent to liposomes themselves (such as mass, refractive index, or charge), many advances have been made in both bench-scale and microfluidic applications. Some of these measurement techniques are inherently sensitivity limited, but through the inclusion of liposomes, reduced limits of detection potentially broaden the utility towards otherwise challenging levels of analytes. Other advances took advantage of the hydrophobic environment required by many biorecognition elements to expand the target selectivity range or utilized the amphipathic nature of the lipid bilayer to provide enhanced separation capabilities. Novel handling approaches included wavelength-specific release of contents encapsulated within thermosensitive liposomes or application of electric fields to move, concentrate, and strategically lyse liposomes. These and other topics are discussed in terms of either present incorporation or adaptation to microfluidic devices.
Assuntos
Técnicas de Química Analítica/métodos , Lipossomos , Miniaturização/métodos , Bioensaio , Humanos , Lipossomos/química , Lipossomos/metabolismoRESUMO
Wavelength interrogation surface plasmon resonance (SPR) spectroscopy using a dove prism combines a simple and inexpensive optical design with high-resolution refractive index monitoring and biosensing. A BK7 dove prism inverts an optical image with a total internal reflection angle of 72.8 degrees , an angle active in SPR. Hence, a unique system can accomplish SPR biosensing using wavelength interrogation and also perform SPR imaging. This optical configuration advantageously uses a single axis optical path between each optical component, simplifying the optical design of SPR instruments without compromise of the analytical performance. Fluidics were also incorporated to the instrument design for efficient sample delivery. The SPR instrument is characterized in terms of refractive index (RI) sensitivity, RI resolution, reproducibility, and application for monitoring low concentration biological events. Data analysis methodologies are compared for improved resolution of the measured response. Raw data analyzed using a minimum hunting procedure results in RI resolution in the 10(-6) range, while pre-treating data with singular value decomposition improves the resolution by one order of magnitude. Depending on the spectrophotometer employed, the RI range accessible can be easily tuned; examples with a 550-850 nm and a 550-1100 nm spectrophotometers are shown and results respectively in RI ranges of 1.32-1.39 RIU and 1.32-1.42 RIU. Monitoring of microM concentration of beta-lactamase is performed using the wavelength interrogation configuration of the biosensor. Finally, a SPR image of a surface with a water droplet (volume=500 nL) was obtained using the dove prism SPR with a band pass filter and a CCD camera. SPR using a dove prism configuration combines advantages of portable SPR instruments, SPR imagers and research-grade SPR instruments in a unique platform.
Assuntos
Ressonância de Plasmônio de Superfície/instrumentação , Desenho de EquipamentoRESUMO
Monolayers prepared with polar or ionic amino acids with short side chains have a reduced nonspecific adsorption of serum proteins compared to that of hydrophobic amino acids and organic monolayers immobilized on the gold surface of surface plasmon resonance (SPR) biosensors. Proteins contained in biological samples adsorb on most surfaces, which in the case of biosensors causes a nonspecific response that hinders the quantification of biomarkers in these biological samples. To circumvent this problem, self-assembled monolayers (SAM) of N-3-mercaptopropyl-amino acids (3-MPA-amino acids) were prepared from 19 natural amino acids. These SAM were investigated to limit the nonspecific adsorption of proteins contained in biological fluids and to immobilize molecular receptors (i.e., antibodies) that are necessary in the construction of biosensors. SPR and Ge attenuated total reflection (GATR) FTIR spectroscopy were employed to characterize the formation of the amino acid SAMs. Monolayers of 3-MPA-amino acids densely packed on the surface of the SPR biosensors result in a surface concentration of approximately 10 (15) molecules/cm (2). SPR also quantifies the surface concentration of serum proteins nonspecifically adsorbed on 3-MPA-amino acids following the exposure of the biosensor to undiluted bovine serum. The concentration of nonspecifically bound proteins ranged from approximately 400 ng/cm (2) with polar and ionic amino acids to approximately 800 ng/cm (2) with amino acids of increased hydrophobicity. The nonspecific adsorption of serum proteins on the 3-MPA-amino acids increases in the following order: Asp < Asn < Ser < Met < Glu < Gln < Thr < Gly < His < Cys < Arg < Phe < Trp < Val < Pro < Ile < Leu < Ala < Tyr. The analysis of the adsorption and desorption curves for serum proteins on the SPR sensorgram has demonstrated the strong irreversibility of the protein adsorption on each surface. The effective hydrophilicity of the SAMs was measured from the contact angle with a saline buffer and has demonstrated that surfaces minimizing the contact angle with PBS performed better in serum. The antibody for beta-lactamase was immobilized on a 3-MPA-glycine SAM, and beta-lactamase was detected in the nanomolar range. The presence of beta-lactamase is an indicator of antibiotic resistance.