Fatma Yılmaz

Quartz crystal microbalance based biosensors for detecting highly metastatic breast cancer cells via their transferrin receptors

A quartz crystal microbalance (QCM) biosensor was developed to detect highly metastatic breast cancer cells by functionalizing the gold sensor surface with transferrin attachment. MDA-MB 231 breast cancer cells with high and MCF 7 cells with low metastatic potential and transferrin expression were used. Serum starved MDA-MB-231 cells were used as control cells. First, poly(2-hydroxyethyl methacrylate) (PHEMA) nanoparticles were prepared by mini-emulsion polymerization of hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA). Nanoparticles were characterized with a zeta-sizer and then their suspension is dropped on the surface of the QCM and the dried QCM surface was modified further by activation with carbodiimide and transferrin attachment. The QCM biosensor was analyzed by using atomic force microscopy (AFM), ellipsometry, Fourier transform infrared spectrophotometry (FTIR) and contact angle measurements. The cells were applied to the derivatized QCM sensor to investigate the affinity and binding kinetics. The nanoparticles and transferrin were found to form a monolayer on the QCM surface. Binding kinetics were best fitted to the Langmuir–Freundlich adsorption model. The QCM signal was correlated well with the number of transferrin receptors on cells. It is concluded that the QCM biosensor functioning via transferrin receptor interactions may be used to detect breast cancer cells with high metastatic potential.

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector, N-methacryloyl-l-histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d,l‐phenylalanine, d,l‐tyrosine, and d,l‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l‐enantiomers of aromatic amino acids migrated before d‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.

Preparation and evaluation of open tubular C18-silica monolithic microcartridges for preconcentration of peptides by on-line solid phase extraction capillary electrophoresis.

In this study, C18-silica monoliths were synthesized as a porous layer in open tubular capillary columns, to be cut later into microcartridges for the analysis of neuropeptides by on-line solid-phase extraction capillary electrophoresis with UV and MS detection (SPE-CE-UV and SPE-CE-MS). First, several types of C18-silica monolithic (MtC18) microcartridges were used to analyse standard solutions of five neuropeptides (i.e. dynorphin A (1-7), substance P (7-11), endomorphin 1, methionine enkephalin and [Ala]-methionine enkephalin). The MtC18 sorbents were especially selective against endomorphin 1 and substance P (7-11)). The best results in terms of sensitivity and inter-microcartridge reproducibility were achieved with the microcartridges obtained from a 10-cm open tubular capillary column with a thin monolithic coating with large through-pores (1-5μm). Run-to-run repeatability, microcartridge durability, linearity ranges and LODs were studied by MtC18-SPE-CE-MS. As expected due to their greater selectivity, the best LOD enhancement was obtained for End1 and SP (7-11) (50 times with regard to CE-MS). Finally, the suitability of the methodology for analysing biological fluids was tested with plasma samples spiked with End1 and SP (7-11). Results obtained were promising because both neuropeptides could be detected at 0.05μgmL(-1), which was almost the same concentration level as for the standard solutions (0.01μgmL(-1)).

Cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins by nano liquid chromatography†

In this study, vinyl phenyl boronic acid modified lauryl methacrylate-based monolithic column was successfully prepared for cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins in nano LC. The polymeric mixture consisted of lauryl methacrylate, vinyl phenyl boronic acid as cation exchanger, ethylene dimethacrylate as cross-linker, polyethylene glycol and methanol as binary porogenic solvent, and azobisisobutyronitrile as initiator. The resulting monolith showed good permeability and mechanical stability. Different ratios of monomer and porogens were used for optimizing the properties of the column. The monolithic column performance with respect to hydrophobic and cation exchange interactions was assessed by the separation a series of alkyl benzenes and anilines, respectively. cis-Diol-containing compounds such as phenols were also utilized to evaluate the retention behaviors of the vinyl phenyl boronic acid modified monolithic column. The monolithic column showed cation exchange interactions in the separation of aniline compounds. Theoretical plate number up to 52,000 plates/m was successfully achieved. The prepared monolith was further applied to the proteins with different acetonitrile content.

Molecularly imprinted hydrophobic polymers as a tool for separation in capillary electrochromatography

The use of molecular imprinted polymers (MIPs), which provides a means for preparing stationary phases with predetermined selectivity for a target molecule in capillary electrochromatography (CEC), is attractive because it combines selectivity, higher separation efficiency and shorter analysis time. A bisphenol A (BPA)-imprinted monolithic capillary BPA/PMAPA column was synthesized for the selective separation of bisphenol A (BPA) from aqueous solutions containing the competitor molecule phenol (PH), which is similar in size and shape to the template molecule. BPA-imprinted monolithic column was prepared in the presence of the template molecule, BPA, which results in the formation of recognition cavities complementary to the template molecule, after the removal of template molecule. An amino acid based monomer, N-methacryloyl-L-phenyl alanine (MAPA), was used as the functional monomer. The new stationary phase contains both charged and hydrophobic groups originating from MAPA monomer, which behaves as both an electroosmotic flow (EOF) supplier and a hydrophobic matrix. The MAPA containing BPA imprinted column behaves as a mixed mode stationary phase, as ion exchanger and hydrophobic matrix depending on the pH of the medium. Scanning electron microscopy was used to identify the structural features of the molecular imprinted column. MIP column performance was evaluated by the electrochromatographic separation of alkylbenzenes. The novelty of this work originated from dual separation mechanism shown by MAPA, which has the ability to form both hydrophobic and electrostatic interactions by the charged and hydrophobic groups of phenylalanine amino acid. This new column with mixed-mode characteristics was then used successfully as the stationary phase in CEC for the selective separation of BPA in MIP systems.

Surface plasmon resonance sensors for real-time detection of cyclic citrullinated peptide antibodies

ABSTRACT Surface plasmon resonance (SPR) sensors have been used for detection of various biomolecules because of their simplicity, high specificity and sensitivity, real-time detection, low cost, and no requirement of labeling. Recently, molecularly imprinted polymers that are easy to prepare, less expensive, stable, have talent for molecular recognition and also are used for creation selective binding sites for target molecule on the SPR sensors. Here, we show that preparation of cyclic citrullinated peptide antibody (anti-CCP) imprinted SPR sensor to detect CCP antibodies. For this purpose, anti-CCP/AAm pre-complex was synthesized by interacting acrylamide (AAm) monomer with anti-CCP. Then, anti-CCP imprinted (anti-CCP/PAAm) SPR sensor was obtained by reacting with anti-CCP/AAm pre-complex in the presence of the crosslinker, and initiator/activator pair. Besides this, non-imprinted (PAAm) SPR sensor was also prepared without using anti-CCP template. The SPR sensors were characterized and then adsorption-desorption studies were performed with pH 7.0 phosphate buffer (10 mM) and acetic acid (10%) with Tween 20 (1%) in pH 7.0 phosphate buffer. Selectivitiy of sensors was investigated by using immunoglobulin M (IgM) and bovine serum albumin (BSA). To determine the adsorption model of interactions between anti-CCP solutions and anti-CCP/PAAm SPR sensor, different adsorption models were performed. The calculated maximum reflection, detection limit, association and dissociation constants were 1.079 RU/mL, 0.177 RU/mL, 0.589 RU/mL and 1.697 mL/RU, respectively. Repeatability experiments of anti-CCP/PAAm SPR sensor was performed four times with adsorption-desorption-regeneration cycles without any performance losing. Results showed that anti-CCP/PAAm SPR sensor had high selectivity and sensitivity for detection of CCP antibodies.

Preparation of cryogel columns for depletion of hemoglobin from human blood

Abstract In this study, we aimed to prepare the metal chelate affinity cryogels for the hemoglobin (Hb) depletion. Poly(2-hydroxyethyl methacrylate) (PHEMA) cryogels were selected as base matrix because of their blood compatibility, osmotic, chemical, and mechanical stability. Cryogels are also useful when working with the viscous samples such as blood, because of their interconnected macroporous structure. Iminodiacetic acid (IDA), the chelating agent, was covalently coupled with PHEMA cryogels after activation with the epichlorohydrin and then the Ni(II) ions were chelated to the IDA-bound cryogels. The depletion of the Hb from hemolysate was shown by SDS-PAGE.

Triazine herbicide imprinted monolithic column for capillary electrochromatography

Trietazine was selectively separated from aqueous solution containing the competitor molecule cyanazine, which is similar in size and shape to the template molecule. Structural features of the molecularly imprinted column were figured out by SEM. The influence of the mobile‐phase composition, applied electrical field, and pH of the mobile phase on the recognition of trietazine by the imprinted monolithic polymer has been evaluated, and the imprint effect in the trietazine‐imprinted monolithic polymer was demonstrated by an imprinting factor. The optimized monolithic column resulted in separation of trietazine from a structurally related competitor molecule, cyanazine. In addition, fast separation was obtained within 6 min by applying higher electrical field, with the electrophoretic mobility of 2.97 × 10−8 m2V−1s−1 at pH 11.0.

Synthesis of hydrophobic nanoparticles for real-time lysozyme detection using surface plasmon resonance sensor

Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR‐based sensor is developed to detect lysozyme with hydrophobic poly (N‐methacryloyl‐(L)‐phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection‐Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM−1, and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real‐time, label‐free, and a low‐detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.

Dopamine-imprinted monolithic column for capillary electrochromatography

A dopamine‐imprinted monolithic column was prepared and used in capillary electrochromatography as stationary phase for the first time. Dopamine was selectively separated from aqueous solution containing the competitor molecule norepinephrine, which is similar in size and shape to the template molecule. Morphology of the dopamine‐imprinted column was observed by scanning electron microscopy. The influence of the organic solvent content of mobile phase, applied pressure and pH of the mobile phase on the recognition of dopamine by the imprinted monolithic column has been evaluated, and the imprinting effect in the dopamine‐imprinted monolithic polymer was verified. Developed dopamine‐imprinted monolithic column resulted in excellent separation of dopamine from structurally related competitor molecule, norepinephrine. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 5.81 × 10–5 m2V–1s–1 at pH 5.0 and 500 mbar pressure.