Recognition of human hemoglobin with macromolecularly imprinted polymeric nanoparticles using non-covalent interactions.

Hemoglobin (Hb) is the most abundant protein in the blood. It is vital for the living as oxygen carriers. Some of the very pure Hb-containing biological fluids are currently under clinical trial. However, the removal and purification of Hb from the blood are quite difficult, especially when it is at a low concentration level. In this study, the molecularly imprinted polymeric nanoparticles (MIPNs) were prepared using N-methacryloyl-histidine methyl ester (MAH) by mini-emulsion polymerization technique for specific binding of human hemoglobin (HHb). MIPNs in monosize form have a size of 152 ± 4 nm. They also have a high binding capacity (32.33 mg/g) of HHb. MIPNs retain 84% of the re-binding capacity for HHb after 10 cycles. The nanoparticles have 16 and 5 times higher binding capacity of HHb, respectively, in the presence of bovine serum albumin and lysozyme. Thanks to their high binding capacity and selectivity, MIPNs will allow them to be detected selectively for different target molecules. According to molecular docking, the main binding forces depend on hydrogen bonds and Van der Waals forces in the interaction within 5 Å around MAH molecule are observed through the amino acid residues of HHb at ß1 and ß2 subunit. The statistical mechanical analysis of docking showed that the free energy (ΔG) is -2732.14 kcal/mol, which indicates the interaction between MAH and HHb is energetically favorable at 298.15°K.

Fabrication of Plasmonic Nanorod-Embedded Dipeptide Microspheres via the Freeze-Quenching Method for Near-Infrared Laser-Triggered Drug-Delivery Applications.

Control of drug release by an external stimulus may provide remote controllability, low toxicity, and reduced side effects. In this context, varying physical external stimuli, including magnetic and electric fields, ultrasound, light, and pharmacological stimuli, have been employed to control the release rate of drug molecules in a diseased region. However, the design and development of alternative on-demand drug-delivery systems that permit control of the dosage of drug released via an external stimulus are still required. Here, we developed near-infrared laser-activatable microspheres based on Fmoc-diphenylalanine (Phe-Phe) dipeptides and plasmonic gold nanorods (AuNRs) via a simple freeze-quenching approach. These plasmonic nanoparticle-embedded microspheres were then employed as a smart drug-delivery platform for native, continuous, and pulsatile doxorubicin (DOX) release. Remarkable sustained, burst, and on-demand DOX release from the fabricated microspheres were achieved by manipulating the laser exposure time. Our results demonstrate that AuNR-embedded dipeptide microspheres have great potential for controlled drug-delivery systems.

Precision phenylalanine sensing in blood with nanomaterial-enhanced electrodes.

Phenylketonuria (PKU) is a genetic disorder associated with the metabolic dysfunction of the phenylalanine hydroxylase enzyme, which catalyses the conversion of l-phenylalanine (Phe) to l-tyrosine. Elevated levels of phenylalanine disrupt the central nervous system by impairing the myelination process and leading to mental retardation. Currently, commonly used diagnostic methods for PKU include the Guthrie test, liquid chromatography, and tandem mass spectrometry, all of which necessitate sophisticated infrastructure and costly equipment. Conversely, electrochemical detection methods hold promise in clinical diagnosis due to their high accuracy, rapid response time, and user-friendly nature. The choice of electrodes in electrochemical methods significantly influences sensitivity and analytical performance. In this study, we evaluated the performance of various nanomaterial-modified electrodes and compared their responses to the redox reaction of phenylalanine, focusing on detection capabilities in blood samples. Specifically, we examined carbon nanotube-gold nanoparticle modified carbon electrode (C-CNT-GNP), graphene-gold nanoparticle modified carbon electrode (C-GPH-GNP), electrochemically reduced graphene oxide (ERGO) modified carbon electrode (C-ERGO), bare carbon electrode (C-BARE), ERGO modified gold electrode (Au-ERGO), and bare gold electrode (Au-BARE) using amperometric detection. The performance of these electrodes was compared in terms of their limit of detection (LOD), limit of quantification (LOQ), and sensitivity. Among all electrodes, ERGO gold electrode showed the lowest LOD, LOQ, and highest sensitivity. This study highlights the potential of ERGO-modified gold electrodes for enhancing electrocatalytic activity, thus offering promising prospects for further diagnostic applications.

Characterizing the S-layer structure and anti-S-layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X-ray scattering.

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22-nm-thick, 2D crystalline surface (S-) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium-host cross-talk. The S-layer is composed of two intercalating glycoproteins (TfsA-GP, TfsB-GP) that are aligned into a periodic lattice. To characterize this unique S-layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and single-molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild-type and S-layer-deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high-resolution imaging of the outer surface of T. forsythia wild-type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti-TfsA antibody coupled to the AFM tip and the S-layer as present on T. forsythia wild-type and mutant cells, displaying TfsA-GP alone. Unbinding forces between the antibody and wild-type cells were greater than with mutant cells. This indicated that the TfsA-GP is not so strongly attached to the mutant cell surface when the co-assembling TfsB-GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S-layer architecture with two intercalating S-layer glycoproteins and TfsA-GP being mainly outwardly oriented.

Nanomapping of CD1d-glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging.

CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.

Red cabbage extract-mediated colorimetric sensor for swift, sensitive and economic detection of urease-positive bacteria by naked eye and Smartphone platform.

The bacterial pathogens have caused various serious infectious diseases in the human body, and even some threats to human life by leading to deaths. Enterobacteriaceae species especially urease positive ones, Proteus mirabilis (P. mirabilis) and Klebsiella pneumoniae (K. pneumoniae), show resistance to antibiotics and cause respiratory and urinary tract infections. We have developed natural indicator-incorporated colorimetric urease tests with a naked eye and smartphone readout to rapidly, sensitively and economically detect P. mirabilis and K. pneumoniae. We utilized anthocyanin found as a predominant component in red cabbage (Brassica oleracea) extract as a natural pH indicator instead of toxic and synthetic indicators. As a mechanistic explanation for the detection of P. mirabilis and K. pneumoniae, urease enzymes secreted from the P. mirabilis and K. pneumoniae hydrolyze urea to produce ammonia (NH3), which increases the pH value of the reaction environment and leads to deprotonation from anthocyanins. The changes in the molecular structure and electronic structure of anthocyanins are responsible for revealing many different colors. We demonstrated how some reaction parameters including the concentration of the bacteria (colony-forming unit, CFU), the concentration of anthocyanin in the tests, initial color and pH values (pHs) of the tests influence their detection performance. We further developed a 3D-printed smartphone platform with smartphone based digital image processing software to improve the detection limit and shorten the detection time. We claim that natural indicator-incorporated rapid urease tests providing colorimetric readout evaluated by the human eye and smartphone imaging processing has great potential in practical use and they can be implemented in clinics.

Experimental Investigation of Microfluidic Device for Platelet Activation.

OBJECTIVE: Wound healing is accelerated when Platelet Rich Plasma is activated and growth factors are released. In this study, it was aimed to stimulate platelets without using chemical stimulants. METHOD: Two types of mechanical platelet activation methods have been proposed in this study. The first one is a microfluidic chip developed with the shear-induced platelet activation approach. The second one is a piezo-based ultrasound-assisted device which provides platelet activation by stimulating with an ultrasonic wave (0.55 and 1.1 MHz). Three different microfluidic chip designs were worked out to determine the optimal shear stress characteristics; 8-nodes (2789 µs, 288 shear pulses, and 98.3 dyne/cm2), 40-nodes (2765 µs, 1440 shear pulses, and 95.5 dyne/cm2) and pillar-shaped (1030 µs, 1656 shear pulses, and 48.1 dyne/cm2). RESULTS: The highest platelet activation rate (72.7%) was obtained from the chips with 8-nodes. In the ultrasound-assisted device, 32.4% activation rate was obtained from ultrasound waves with 0.55 MHz frequency and 10 Vp-p amplitude. These activation rates, determined by CD62P (P-Selectin) expression, are significantly higher than spontaneous activation of intact platelets (8.5%). In addition, the gradual increase in activation of stimulated platelets with incubation at room temperature showed that activation continued after stimulation. CONCLUSION: The results showed that these microfluidic devices can be used for platelet activation to enhance the effect of PRP treatment and might reduce adverse immune reactions that may happened due to the use of exogenous activator substances. SIGNIFICANCE: Fast-response, low-cost, easy-to-use and controllable biomedical device have been developed for PRP applications.

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

Study on Cost-Efficient Carbon Aerogel to Remove Antibiotics from Water Resources.

Because of pharmaceutical-emerging contaminants in water resources, there has been a significant increase in the antibiotic resistance in bacteria. Therefore, the removal of antibiotics from water resources is essential. Various antibiotics have been greatly studied using many different carbon-based materials including graphene-based hydrogels and aerogels. In this study, carbon aerogels (CAs) were synthesized from waste paper sources and their adsorption behaviors toward three antibiotics (hygromycin B, gentamicin, and vancomycin) were investigated, for which there exist a limited number of reports in the literature. The prepared CAs were characterized with scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and micro-computerized tomography (µ-CT). According to the µ-CT results, total porosity and open porosity were calculated as 90.80 and 90.76%, respectively. The surface area and surface-to-volume ratio were found as 795.15 mm2 and 16.79 mm-1, respectively. The specific surface area of the CAs was found as 104.2 m2/g. A detailed adsorption study was carried out based on different pH values, times, and analyte concentrations. The adsorption capacities were found as 104.16, 81.30, and 107.52 mg/g for Hyg B, Gen, and Van, respectively. For all three antibiotics, the adsorption behavior fits the Langmuir model. The kinetic studies showed that the system fits the pseudo-second-order kinetic model. The production of CAs, within the scope of this study, is safe, facile, and cost-efficient, which makes these green adsorbents a good candidate for the removal of antibiotics from water resources. This study represents the first antibiotic adsorption study based on CAs obtained from waste paper.

Selective binding of antibiotics using magnetic molecular imprint polymer (MMIP) networks prepared from vinyl-functionalized magnetic nanoparticles.

Adverse effects of pharmaceutical emerging contaminants (PECs), including antibiotics, in water supplies has been a global concern in recent years as they threaten fresh water security and lead to serious health problems to human, wildlife and the environment. However, detection of these contaminants in water sources, as well as food products, is difficult due to their low concentration. Here, we prepared a new family of magnetic molecular imprinted polymer (MMIP) networks for binding antibiotics via a microemulsion polymerization technique using vinyl silane modified Fe3O4 magnetic nanoparticles. The cross-linked polymer backbone successfully integrated with 20-30 nm magnetic nanoparticles and generated a novel porous polymeric network structure. These networks showed a high binding capacity for both templates, erythromycin and ciprofloxacin at 70 and 32 mg/g. Both MMIPs were also recyclable, retaining 75 % and 68 % of the binding capacity after 4 cycles. These MMIPs have showed a clear preference for binding the template molecules, with a binding capacity 4- to 7-fold higher than the other antibiotics in the same matrix. These results demonstrate our MMIP networks, which offered high binding capacity and selectivity as well as recyclability, can be used for both removal and monitoring hazardous antibiotic pollutants in different sources/samples and food products.

Synthesis of Conductive Carbon Aerogels Decorated with ß-Tricalcium Phosphate Nanocrystallites.

There has been substantial interest in research aimed at conductive carbon-based supports since the discovery that the electrical stimulus can have dramatic effect on cell behavior. Among these carbon-aerogels decorated with biocompatible polymers were suggested as future materials for tissue engineering. However, high reaction temperatures required for the synthesis of the aerogels tend to impair the stability of the polymeric networks. Herein, we report a synthetic route towards carbon-aerogel scaffolds decorated with biocompatible ceramic nanoparticles of tricalcium phosphate. The composites can be prepared at temperature as high as 1100 °C without significant effect on the morphology of the composite which is comparable with the original aerogel framework. Although the conductivity of the composites tends to decrease with the increasing ceramic content the measured conductivity values are similar to those previously reported on polymer-functionalized carbon-aerogels. The cell culture study revealed that the developed constructs support cell proliferation and provide good cell attachment suggesting them as potentially good candidates for tissue-engineering applications.

ATP scavenging by the intracellular pathogen Porphyromonas gingivalis inhibits P2X7-mediated host-cell apoptosis.

The purinergic receptor P2X(7) is involved in cell death, inhibition of intracellular infection and secretion of inflammatory cytokines. The role of the P2X(7) receptor in bacterial infection has been primarily established in macrophages. Here we show that primary gingival epithelial cells, an important component of the oral innate immune response, also express functional P2X(7) and are sensitive to ATP-induced apoptosis. Porphyromonas gingivalis, an intracellular bacterium and successful colonizer of oral tissues, can inhibit gingival epithelial cell apoptosis induced by ATP ligation of P2X(7) receptors. A P. gingivalis homologue of nucleoside diphosphate kinase (NDK), an ATP-consuming enzyme, is secreted extracellularly and is required for maximal suppression of apoptosis. An ndk-deficient mutant was unable to prevent ATP-induced host-cell death nor plasma membrane permeabilization in the epithelial cells. Treatment with purified recombinant NDK inhibited ATP-mediated host-cell plasma membrane permeabilization in a dose-dependent manner. Therefore, NDK promotes survival of host cells by hydrolysing extracellular ATP and preventing apoptosis-mediated through P2X(7).

Detection of ciprofloxacin through surface plasmon resonance nanosensor with specific recognition sites.

The novel nanosensor based on Surface Plasmon Resonance (SPR) was developed for sensitive and selective detection of ciprofloxacin via molecularly imprinted nanoparticles (MIP/NPs). NPs were synthesized through miniemulsion polymerization technique with methacrylic acid as a functional monomer. FTIR, SEM, zetasizer and contact angle measurements were used for the characterization of MIP/NPs. After modification the SPR chip surface, the nanosensor was used for detection of ciprofloxacin in aqueous solution. According to selected concentration range, the correlation coefficient and limit of detections were obtained as 0.993 (R2) and 3.21 and 7.1 ppb in ultrapure water and SWW, respectively. Association kinetic analysis, Scatchard, Freundlich, Langmuir and Freundlich-Langmuir isotherms were also performed on the data to investigate adsorption behaviour of ciprofloxacin on the surface of nanosensor. Tetracycline and enrofloxacin were used as competitor agents to examine the selectivity of nanosensor. Performance of the SPR nanosensor was also investigated by using synthetic wastewater (SWW) for detection of CPX. The reusability of nanosensor was investigated and good repeatability was obtained with 5.81% RSD. As a result, selective, simple and low-cost method to detect ciprofloxacin in aqueous solution was developed by combining MIP/NPs and SPR.

Preparation and in vitro evaluation of bFGF-loaded chitosan nanoparticles.

The objective of our study was to prepare and characterize basic fibroblast growth factor (bFGF)-loaded nanoparticles. Protein-loaded chitosan nanoparticles were obtained by ionotropic gelation process based on the interaction between chitosan and tripolyphosphate (TPP). The protein-loading capacity and encapsulation efficiency were 0.021% and 27.388%, respectively. The bFGF-loaded nanoparticles have a mean diameter of 424 nm, a narrow size distribution, spherical shape and positive surface charges. In vitro release showed that the extent of release was 68% at 24 hr. The protein integrity was investigated by SDS-PAGE analysis that confirmed protein integrity was not affected by the encapsulation procedure and release conditions.

Molecularly imprinted polymer based micromechanical cantilever sensor system for the selective determination of ciprofloxacin.

The main objective of this study is to develop molecularly imprinted polymer (MIP) based micromechanical cantilever sensor system that has high specificity, fast response time and is easily applicable by user for the detection of ciprofloxacin (CPX) molecule in water resources. Highly specific CPX imprinted nanoparticles were synthesized by miniemulsion polymerization technique. The average size of the synthesized nanoparticles was measured about 160nm with high monodispersivity. Covalent and monolayer binding of the MIP nanoparticles on cantilevers was provided by EDC/NHS activation. Validation of the developed cantilever nanosensor was performed in air with dip-and-dry technique by employing the dynamic sensing mode. According to the results obtained, micromechanical cantilever sensor system worked linearly for the concentration range of 1.5-150.9µM. This concentration range resulted with 18.4-48.9pg mass load on the MIP modified cantilever. The sensitivity of the developed sensor was calculated as 2.6Hz/pg. To control the specificity of MIPs, a different antibiotic enrofloxacin (ENF), with a similar physical and chemical structure with CPX, was used, which showed 7 folds low binding affinity. The developed highly specific microcantilever sensor has a response time of approximately 2min and is reusable up to 4 times. The results indicate that the MIP based AFM nanosensor has high sensitivity for the CPX molecule. This combination of MIP nanoparticles with micromechanical sensors is one of the pioneer studies in the mass sensing applications. This fast, low cost and highly sensitive CPX specific MIP nanoparticle based nanosensor developed in this research have the potential to pave the way for further studies.

Materials specificity and directed assembly of a gold-binding peptide.

Adsorption studies of a genetically engineered gold-binding peptide, GBP1, were carried out using a quartz-crystal microbalance (QCM) to quantify its molecular affinity to noble metals. The peptide showed higher adsorption onto and lower desorption from a gold surface compared to a platinum substrate. The material specificity, that is, the preferential adsorption, of GBP1 was also demonstrated using gold and platinum micropatterned on a silicon wafer containing native oxide. The biotinylated three-repeat units of GBP1 were preferentially adsorbed onto gold regions delineated using streptavidin-conjugated quantum dots (SAQDs). These experiments not only demonstrate that an inorganic-binding peptide could preferentially adsorb onto a metal (Au) rather than an oxide (SiO2) but also onto one noble metal (Au) over another (Pt). This result shows the utility of an engineered peptide as a molecular erector in the directed immobilization of a nanoscale hybrid entity (SAQDs) over selected regions (Au) on a fairly complex substrate (Au and Pt micropatterned regions on silica). The selective and controlled adsorption of inorganic-binding peptides may have significant implications in nano- and nanobiotechnology, where they could be genetically tailored for specific use in the development of self-assembled molecular systems.

Rapid and alternative fabrication method for microfluidic paper based analytical devices.

A major application of microfluidic paper-based analytical devices (µPADs) includes the field of point-of-care (POC) diagnostics. It is important for POC diagnostics to possess properties such as ease-of-use and low cost. However, µPADs need multiple instruments and fabrication steps. In this study, two different chemicals (Hexamethyldisilazane and Tetra-ethylorthosilicate) were used, and three different methods (heating, plasma treatment, and microwave irradiation) were compared to develop µPADs. Additionally, an inkjet-printing technique was used for generating a hydrophilic channel and printing certain chemical agents on different regions of a modified filter paper. A rapid and effective fabrication method to develop µPADs within 10min was introduced using an inkjet-printing technique in conjunction with a microwave irradiation method. Environmental scanning electron microscope (ESEM) and x-ray photoelectron spectroscopy (XPS) were used for morphology characterization and determining the surface chemical compositions of the modified filter paper, respectively. Contact angle measurements were used to fulfill the hydrophobicity of the treated filter paper. The highest contact angle value (141°±1) was obtained using the microwave irradiation method over a period of 7min, when the filter paper was modified by TEOS. Furthermore, by using this method, the XPS results of TEOS-modified filter paper revealed Si2p (23%) and Si-O bounds (81.55%) indicating the presence of Si-O-Si bridges and Si(OEt) groups, respectively. The ESEM results revealed changes in the porous structures of the papers and decreases in the pore sizes. Washburn assay measurements tested the efficiency of the generated hydrophilic channels in which similar water penetration rates were observed in the TEOS-modified filter paper and unmodified (plain) filter paper. The validation of the developed µPADs was performed by utilizing the rapid urease test as a model test system. The detection limit of the developed µPADs was measured as 1unitml(-1) urease enzyme in detection zones within a period of 3min. The study findings suggested that a combination of microwave irradiation with inkjet-printing technique could improve the fabrication method of µPADs, enabling faster production of µPADs that are easy to use and cost-effective with long shelf lives.

Fabrication of surface plasmon resonance nanosensor for the selective determination of erythromycin via molecular imprinted nanoparticles.

The main objective of this study was to develop a novel surface plasmon resonance (SPR) nanosensor method based on a more rapid and selective determination of erythromycin (ERY) in the aqueous solution. This study is a combination of three techniques, which are miniemulsion polymerization, molecular imprinting and surface plasmon resonance techniques. In the first part, nanoparticles prepared with methacryl groups of functional monomer at surface acted as reactive sites for erythromycin as a template molecule. The molecularly imprinted nanoparticles were characterized by FTIR, SEM and zetasizer. After immobilization of nanoparticles on gold surface of SPR chip, nanosensor was characterized with contact angle measurements. This nanosensor was then used for selective determination of erythromycin. The linearity range and detection limit were obtained as 0.99 (r(2)) and 0.29 ppm, respectively. Association kinetic analysis, Scatchard, Langmuir, Freundlich and Freundlich-Langmuir isotherms were applied data. The selectivity of the SPR nanosensor was determined by using competitor agents (kanamycin sulfate, neomycin sulfate, spiramycin). The non-imprinted nanosensor was also used to evaluate the selectivity of ERY imprinted nanosensor. Finally, the nanosensor was tested for repeatability and it gave satisfactory response. These results demonstrate a method which is of low cost, rapid and provide reliable results in order to be used in detection of erythromycin from aqueous solution.

A new approach for immobilization of oligonucleotides onto piezoelectric quartz crystal for preparation of a nucleic acid sensor for following hybridization.

The aim of this study is to develop a nucleic acid sensor based on piezoelectric crystal microbalance system (QCM) for following hybridization. Piezoelectric quartz crystal surfaces were first treated in a glow-discharge apparatus with ethylene diamine (EDA) plasma at 15 W (discharge power), 2.5 min (incubation time) and 35 ml/min (monomer flow rate) to create amino groups on the crystal surfaces. The thickness of the EDA-plasma film formed was about 43+/-24 A. Then, the amino groups on the crystal surfaces were converted to aldehyde groups by reacting the amino groups with glutaraldehyde (GA) at different conditions. A GA concentration of 2.5% and an incubation time of 2 h were selected as optimal values at this step, corresponding to a GA surface concentration of about 270 ng/cm2. A double strand Oligonucleotides, having one extra base on 5'-end of one of the complementary strands, were immobilized through the amino groups of this base onto the GA-modified crystals. Optimal immobilization conditions were as follows: oligonucleotide concentration: 1 microg/ml; time: 3 h; pH: 9.2 carbonate buffer; ionic strength: 0.1; and temperature: 20 degrees C. The QCM sensor carrying the covalently bound strand was used in the hybridization experiments, which showed that equilibrium is achieved in about 5 min, and the frequency shift measured is related to the concentration of the target strand to be measured within the medium.

Probing and mapping the binding sites on streptavidin imprinted polymer surface.

Molecular imprinting is an effective technique for preparing recognition sites which act as synthetic receptors on polymeric surfaces. Herein, we synthesized MIP surfaces with specific binding sites for streptavidin and characterized them at nanoscale by using two different atomic force microscopy (AFM) techniques. While the single molecule force spectroscopy (SMFS) reveals the unbinding kinetics between streptavidin molecule and binding sites, simultaneous topography and recognition imaging (TREC) was employed, for the first time, to directly map the binding sites on streptavidin imprinted polymers. Streptavidin modified AFM cantilever showed specific unbinding events with an unbinding force around 300 pN and the binding probability was calculated as 35.2% at a given loading rate. In order to prove the specificity of the interaction, free streptavidin molecules were added to AFM liquid cell and the binding probability was significantly decreased to 7.6%. Moreover, the recognition maps show that the smallest recognition site with a diameter of around ~21 nm which corresponds to a single streptavidin molecule binding site. We believe that the potential of combining SMFS and TREC opens new possibilities for the characterization of MIP surfaces with single molecule resolution under physiological conditions.