Fahriye Ceyda Dudak

Rapid and label-free bacteria detection by surface plasmon resonance (SPR) biosensors.

Surface Plasmon Resonance (SPR) biosensor technology has been successfully used for the detection of various analytes such as proteins, drugs, DNA, and microorganisms. SPR‐based immunosensors that coupled with a specific antigen‐antibody reaction, have become a promising tool for the quantification of bacteria as it offers sensitive, specific, rapid, and label‐free detection. In this paper, we review the important issues in the development of SPR‐based immunoassays for bacteria detection, concentrating on instrumentation, surface functionalization, liquid handling, and surface regeneration. In addition, this review touches on the recent advances in SPR biosensing for sensitivity enhancement.

Attomole sensitivity of staphylococcal enterotoxin B detection using an aptamer-modified surface-enhanced Raman scattering probe.

In this report, we present a new homogeneous detection method for staphylococcal enterotoxin B (SEB) utilizing core-shell-structured iron-gold magnetic nanoparticles and a gold nanorod surface-enhanced Raman scattering (SERS) probe in solution. Peptide ligand (aptamer) functionalized magnetic gold nanorod particles were used as scavengers for target SEB. After the SEB molecules were separated from the matrix, the sandwich assay procedure was tested by gold nanorod particles that act as SERS probes. The binding constant between SEB and peptide-nanoparticle complex was determined as 8.0 × 10(7) M(-1). The correlation between the SEB concentration and SERS signal was found to be linear within the range of 2.5 fM to 3.2 nM. The limit of detection for the homogeneous assay was determined as 224 aM (ca. 2697 SEB molecules/20 μL sample volume). Also, gold-coated surfaces were used as capture substrates and performances of the two methods were compared. Furthermore, the developed method was evaluated for investigating the SEB specificity on bovine serum albumin (BSA) and avidin and detecting SEB in artificially contaminated milk, blood, and urine.

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

Abstract In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide–SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody–SEB interaction. The binding constant of the peptide was determined as 4.2±0.7×105 M−1 which indicates a strong binding close to that of antibody.

Glucose determination based on a two component self-assembled monolayer functionalized surface-enhanced Raman spectroscopy (SERS) probe

In this report, we present a new detection method for blood glucose, using gold nanorod SERS, a surface enhanced Raman scattering probe embedded in two component self-assembled monolayers (SAMs). Gold nanorod particles and a gold coated slide surface were modified with the two component SAMs consisting of 3-mercaptophenylboronic acid (3-MBA) and 1-decanethiol (1–DT). The immobilization of 3-MBA/1-DT surface-functionalized gold nanoparticles onto 3-MBA/1-DT modified gold-coated slide surfaces was achieved by the cooperation of hydrophobic forces. Two component SAM functionalized substrates were used as SERS probes, by means of the boronic acid and the alkyl spacer functional groups that serve as the molecular recognition and penetration agents, respectively. The SERS platform surface was characterized by cyclic voltammetry, contact angle measurements, AFM (atomic force microscopy) and Raman spectroscopy. Optimum values of the parameters such as pH, time and (3-MBA/1-DT) molar ratio were also examined for the glucose determination. The analytical performance was evaluated and linear calibration graphs were obtained in the glucose concentration range of 2–16 mM, which is also in the range of the blood glucose levels, and the detection limit was found to be 0.5 mM. As a result, the SERS platform was also used for the determination of glucose in plasma samples.

The Discovery of Small-Molecule Mimicking Peptides through Phage Display

Using peptides to achieve the functional and structural mimicry of small-molecules, especially those with biological activity or clear biotechnological applications, has great potential in overcoming difficulties associated with synthesis, or unfavorable physical properties. Combinatorial techniques like phage display can aid in the discovery of these peptides even if their mechanism of mimicry is not rationally obvious.The major focus of this field has been limited to developing biotin and sugar mimetics. However, the full “mimicry” of these peptides has not yet been fully established as some bind to the target with a different mechanism than that of the natural ligand and some do not share all of the natural ligand’s binding partners. In this article, mimicry of small-molecules by phage display-discovered peptides is reviewed and their potential in biochemical and medical applications is analyzed.

Thermodynamic and structural analysis of interactions between peptide ligands and SEB.

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB‐binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (105 M−1). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications. Copyright

Enhancing the affinity of SEB‐binding peptides by repeating their sequence

The utilization of peptide ligands in biosensors and bioassays is dependent on achieving high affinity of these peptides toward their targets. In a previous report, we identified 12‐mer peptides that could selectively bind to Staphylococcal enterotoxin B (SEB) using a phage‐display library. In this study, we explore for new modification approaches to enhance the affinity of two different SEB‐binding peptides. In order to identify the binding regions of selected peptides, the charged residues and the ones, critical for the structure of peptide, were replaced with alanine. However, a specific binding region could not be suggested as all mutant peptides have lost their affinities toward SEB completely. The modifications for the affinity enhancement were done by repeating the 12‐mer peptide sequences. A 10‐fold increase was observed in the binding affinity of one of the two‐repeated peptides, while this modification did not affect the affinity of the other tested peptide. The peptide, with enhanced affinity, was further modified as three repeats; however the affinity of the peptide decreased. The structural basis of the affinity difference between modified peptides was examined by molecular dynamics simulation. The results showed that the conformational differences hold the key for affinity of peptides modified by repeating the sequence. This high affinity peptide with increased affinity is a promising molecular recognition agent to be used in the detection of SEB to be utilized in biosensing systems.

The investigation of the secondary structure propensities and free-energy landscapes of peptide ligands by replica exchange molecular dynamics simulations

The conformational states of two peptide sequences that bind to staphylococcal enterotoxin B are sampled by replica exchange molecular dynamic (REMD) simulations in explicit water. REMD simulations were treated with 52 replicas in the range of 280–501 K for both peptides. The conformational ensembles of both peptides are dominated by random coil, bend and turn structures with a small amount of helical structures for each temperature. In addition, while an insignificant presence of β-bridge structures were observed for both peptides, the β-sheet structure was observed only for peptide 3. The results obtained from simulations at 300 K are consistent with the experimental results obtained from circular dichroism spectroscopy. From the analysis of REMD results, we also calculated hydrophobic and hydrophilic solvent accessible surface areas for both peptides, and it was observed that the hydrophobic segments of the peptides tend to form bend or turn structures. Moreover, the free-energy landscapes of both peptides were obtained by principal component analysis to understand how the secondary structural properties change according to their complex space. From the free-energy analysis, we have found several minima for both peptides at decreased temperature. For these obvious minima of both peptides, it was observed that the random coil, bend and turn structures are still dominant and the helix, β-bridge or β-sheet structures can appear or disappear with respect to minima. On the other hand, when we compare the results of REMD with conventional MD simulations for these peptides, the configurations of peptide 3 might be trapped in energy minima during the conventional MD simulations. Hence, it can be said that the REMD simulations have provided a sufficiently high sampling efficiency.

Statistical Modeling of β-galactosidase Inhibition During Lactose Hydrolysis

A statistical model approach called response surface methodology was used to describe the product and substrate inhibition effect on β-galactosidase enzyme during lactose hydrolysis. The effect of independent variables, namely the initial concentrations of lactose (73 – 146 mM), galactose (44 – 122 mM) and glucose (83 – 167 mM) on the reaction rate of β-galactosidase was evaluated. The enzymatic reaction rate was influenced by both combined and individual effects of all the substrate and products. Although, glucose acted as an activator at low lactose and low galactose concentrations, glucose caused the inhibition of β-galactosidase at higher concentrations of lactose and galactose. The effect of galactose concentration on β-galactosidase enzyme was in the direction of inhibition. At low lactose concentrations and high glucose concentrations, galactose concentration became more effective on the reaction rate.