Molecularly imprinted polymer films for reflectometric interference spectroscopic sensors

Reflectometric interference spectroscopic measurements were performed on molecularly imprinted polymer (MIP) films with the herbicide atrazine as the template molecule. A conventional imprinting protocol was used relying on non-covalent interactions between the functional monomers and the template. The MIPs were deposited on glass transducers by two different methods: spin-coating followed by in situ polymerization of thin films of monomers containing a sacrificial polymeric porogen, and autoassembly of MIP nanoparticles with the aid of an associative linear polymer. Reproducible results were obtained upon measurements of atrazine solutions in toluene with both films. Atrazine concentrations as low as 1.7 ppm could be detected with the autoassembled particle film. No or very little analyte adsorption was observed onto non-imprinted control films made by spin-coating and by particle assembly, respectively. We believe that these MIP layers in combination with the general reflectrometric transduction scheme could be a versatile sensing tool for the detection of environmentally important and other analytes.     

Molecularly imprinted polymer as storage medium for an analyte

A molecularly imprinted polymer (MIP) able to bind theophylline with a considerable degree of selectivity was prepared through a noncovalent route. The polymer after equilibrating with the template molecule (theophylline) and storage for various periods of time, the amount of theophylline desorbed from the polymer was found to be effectively quantitative indicating that MIPs could be used as a storage matrix for analytes. The stored analyte could be desorbed and analysed conveniently.     

Operating Cooperatively (OC) Sensor for Highly Specific Recognition of Nucleic Acids

Molecular Beacon (MB) probes have been extensively used for nucleic acid analysis because of their ability to produce fluorescent signal in solution instantly after hybridization. The indirect binding of MB probe to a target analyte offers several advantages, including: improved genotyping accuracy and the possibility to analyse folded nucleic acids. Here we report on a new design for MB-based sensor, called ‘Operating Cooperatively’ (OC), which takes advantage of indirect binding of MB probe to a target analyte. The sensor consists of two unmodified DNA strands, which hybridize to a universal MB probe and a nucleic acid analyte to form a fluorescent complex. OC sensors were designed to analyze two human SNPs and E.coli 16S rRNA. High specificity of the approach was demonstrated by the detection of true analyte in over 100 times excess amount of single base substituted analytes. Taking into account the flexibility in the design and the simplicity in optimization, we conclude that OC sensors may become versatile and efficient tools for instant DNA and RNA analysis in homogeneous solution.     

Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein

The activation of peroxisome-proliferator-activated receptor-γ (PPARγ), which plays a central role in adipocyte differentiation, depends on ligand-dependent co-activator recruitment. In this study, we developed a novel method of PPARγ ligand screening by measuring the increase in fluorescent polarization accompanied by the interaction of a fluorescent co-activator and PPARγ. Sterol receptor co-activator-1 (SRC-1), a major PPARγ co-activator, was probed by fluorescent TAMRA by the Amber codon fluorescence probe method. Polarization was increased by adding PPARγ ligands to a solution containing labeled SRC-1 (designated TAMRA-SRC-S) and PPARγ. The disassociation constants (Kd) of the PPARγ synthesized ligands, pioglitazone (221 nM), troglitazone (83.0 nM), and 15-deoxy-Δ12,14-prostaglandin J(2) (15d-ΔPGJ(2)) (156 nM), were determined by this method. Farnesol (2.89 μM) and bixin (21.1 μM), which we have reported to be PPARγ ligands, increased the fluorescent polarization. Their Kd values were in agreement with the ED(50) values obtained in the luciferase assay. The results indicate that the method is valuable for screening natural PPARγ ligands.     

Bio-Rad's Bio-Plex® suspension array system, xMAP technology overview

The Bio-Plex(?) system utilizes xMAP technology to permit the multiplexing of up to 100 different analytes. Multiplex analysis gives researchers the ability to look at analytes simultaneously providing more information from less sample volume in less time than traditional immunoassay methods. Similar to ELISA, xMAP utilizes an antibody sandwich for detection but differs from ELISA in capture substrate and detection method. Rather than a flat surface, Bio-Plex(?) assays make use of differentially detectable bead sets as a substrate capturing analytes in solution and employs fluorescent methods for detection. These bead sets identify the analytes and detection antibodies are used to measure the quantity of analyte. The use of differentially detectable beads enables the simultaneous identification and quantification of many analytes in the same sample.     

Determination of temozolomide in serum and brain tumor with micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatographic (MEKC) with photodiode-array detection was applied to determine temozolomide (TMZ) in human serum and brain tumor. The limit of quantitation (LOQ) was 0.096 μg/mL using 325 nm as detection wavelength. The method made possible that the TMZ could be detected in in vivo serum samples without sample pretreatment. In order to detect TMZ at lower concentration, an extraction with ethyl acetate was applied to preconcentrate the analyte. Small amount of brain tumor tissues (less than 1g) were lyophilized and pretreated using extraction as a clean up and concentrating step. After removing the organic solvent a final sample volume of only 10 μL was analyzed. The obtained peak concentrations (8.2-10.1 μg/mL) and T(max) (44-65 min) of TMZ in serum were similar to the data reported by others, the in vivo TMZ concentrations found in brain tumor ranged between 0.061 and 0.117 μg/g.     

Determination of β-lactam antibiotics in milk based on magnetic molecularly imprinted polymer extraction coupled with liquid chromatography-tandem mass spectrometry

In this work, a rapid and selective method was successfully developed using the magnetic molecularly imprinted polymer (MMIP) as sorbent for the extraction of β-lactam antibiotics (BLAs) from milk samples. The MMIP has been prepared using penicillin V potassium (PENV) as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and Fe(3)O(4) magnetite as magnetic component. The experimental results showed that the MMIP had high affinity and selectivity toward PENV and other structurally related BLAs. The extraction process was carried out in a single step by mixing the extraction solvent, MMIPs and milk samples under ultrasonic action. When the extraction was completed, the MMIPs adsorbing the analytes were separated from the sample matrix by an external magnet. The analytes eluted from the MMIP were analyzed by liquid chromatography-tandem mass spectrometry. For achieving optimal preconcentration and reducing non-specific interactions, various parameters affecting the extraction efficiency such as extraction mode, extraction solvent, the amount of MMIPs, extraction time, washing solution and eluting solution were comprehensively evaluated. Under the optimal conditions, the detection limits of BLAs are in the range of 1.6-2.8 ng mL(-1). The relative standard deviations of intra- and inter-day ranging from 3.2% to 8.3% and from 3.6% to 9.8% are obtained, respectively. The method was applied to determine BLAs including PENV, amoxicillin and oxacillin in five milk samples from different provenances. The recoveries of BLAs in these samples from 71.6% to 90.7% are obtained.     

Visualizing stimulus-responsive dual-ligand fluorescent probes for hypochlorite: A novel strategy for real application in tap water

As an effective ingredient of disinfectants, ClO⁻ inevitably remains in water, which induces potential health hazards such as lung damage and kidney disease. In this study, we synthesized stimulus-responsive dual-ligand luminol-Tb-GMP coordination polymer nanoparticles (luminol-Tb-GMP CPNPs) as highly selective fluorescent probes for the real-time and visual detection of ClO⁻. CPNPs consist of Tb³⁺, a nuclear metal, that coordinates with GMP and luminol, an auxiliary ligand. GMP can be oxidized by ClO⁻ and damage its structure, resulting in fluorescence quenching of CPNPs. The two-ligand CPNPs sensor has a rapid fluorescent response, significant fluorescent color change, and high sensitivity, with a linear range of 2–18 μM and a detection limit of 0.14 μM. It has been successfully used to detect ClO⁻ in tap water, fountain water, and drinking water. Simultaneously, the portable filter paper strip was prepared to expand the range of applications outside the laboratory, which will provide a promising application for the real-time and semiquantitative analysis of ClO⁻.     

New fluorescent macrolide derivatives for studying interactions of antibiotics and their analogs with the ribosomal exit tunnel

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.     

Fast and sensitive liquid chromatography-mass spectrometry assay for seven androgenic and progestagenic steroids in human serum

A fast and sensitive LC-MS/MS method for the quantitative analysis of seven steroid hormones in 150 μl of human serum was developed and validated. The following compounds were included: 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, pregnenolone, and progesterone. Individual stable isotope-labeled analogues were used as internal standards. Sample preparation was performed by liquid-liquid extraction, followed by oxime derivatization to improve the ionization efficiency of the analytes. In contrast to the common derivatization-based methods, the reaction was incorporated into the sample preparation process and the only additional step due to the derivatization was a short heating of the autosampler vials before the sample injection. Chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. For the analyte detection, a triple quadrupole instrument with electrospray ionization was used. Total run time was 7.0 min and the lower limits of quantification were in the range of 0.03-0.34 nM (0.01-0.10 ng/ml), depending on the analyte. The method was validated using human serum samples from both sexes and applied for the serum steroid profiling of endometriosis patients.     

Array biosensor for detection of biohazards

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.     

Optical detection of choline and acetylcholine based on H₂O₂-sensitive quantum dots

In this paper, we have constructed a simple, rapid and sensitive biosensor for detection of choline and acetylcholine (ACh) based on the hydrogen peroxide (H(2)O(2))-sensitive quantum dots (QDs). The detection limit for choline was 0.1 μM and the linear range was 0.1-0.9 μM and 5-150 μM, respectively. The detection limit for ACh was found to be 10 μM and the linear range was 10-5000 μM. The wide linear ranges were shown to be suitable for routine analyses of choline and ACh. Possible mechanism of the fluorescence of QDs quenched by H(2)O(2) was an electron transfer (ET) process. The experimental conditions of biosensors were optimized, and anti-interference ability was also presented. We also detected the choline in milk samples and the linear range was 5-150 μM. The detection linear range of ACh in serum was 10-140 μM. Most importantly, the recovery of choline in milk and ACh in serum samples were both close to 99%. The excellent performance of this biosensor showed that the method can be used in practice detection of choline and ACh.     

Electrochemical and piezoelectric monitoring of taurine via electropolymerized molecularly imprinted films

A molecularly imprinted electrochemical quartz crystal microbalance (EQCM) sensor is fabricated here for taurine, a β ‐amino acid significant for functioning of almost all vital organs. The polymeric film of l ‐methionine was electrochemically deposited on gold‐coated EQCM electrode. Experimental parameters were optimized for controlling the performance of molecularly imprinted polymer (MIP)‐modified sensor such as ratio of monomer and template, number of electropolymerization cycles, mass deposited in each cycle, and pH. Thus, fabricated MIP‐EQCM sensor was successfully applied for estimation of taurine in solutions with varying matrices, such as aqueous, human blood plasma, milk from cow, buffalo, and milk powder. Under optimized parameters, response of MIP sensor to taurine was linearly proportional to its concentration with limit of detection as 0.12μM. Hence, a highly sensitive and selective piezoelectric sensor for taurine has been reported here via imprinting approach.     

Protein detection using biobarcodes

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.     

Capillary electrophoresis with laser-induced fluorescence detection, an adequate alternative to high-performance liquid chromatography, for the determination of ciprofloxacin and its metabolite desethyleneciprofloxacin in human plasma

A method to determine plasma concentrations of ciprofloxacin and its metabolite desethyleneciprofloxacin (M1) by CE with HeCd laser-induced fluorescence detection is described. Following precipitation of proteins and centrifugation supernatant is injected hydrodynamically (10 s, 0.5 p.s.i.) into the capillary. Overall analysis time for the quantification of both analytes was 7 min. The total amount of plasma needed for multiple injections (n>5) was 10–20 μl. Data on accuracy and precision are presented. The assay performance is compared to the specifications of a validated HPLC method, which is routinely used for the quantification of ciprofloxacin and M1 in body fluids. Both methods showed comparable accuracy and precision for both analytes throughout the whole working range (inter-day precision <9%; inter-day accuracy 96–110%). The limit of quantification (LOQ) of 20 μg/l (M1 10 μg/l) for the CE procedure was slightly higher than for the HPLC method, where 10 μg/l (M1 2.5 μg/l) was determined. However, application of the methods to human plasma samples derived from a clinical study proved that comparable results are obtained and that the sensivity of the HPCE method was sufficient to fully describe typical plasma concentration timie profiles of ciprofloxacin and its metabolite M1. Both the adequate sensitivity and the required smaller sample volume compared to HPLC indicate that the method is feasible for clinical studies where sample amounts are limited, e.g., studies to investigate pharmacokinetics in pediatric patients. Preclinical studies form another possible application of this technique.     

Molecularly imprinted polymer diffraction grating as label-free optical bio(mimetic)sensor

Micropatterned molecularly imprinted polymer (MIP) transmissive 2D diffraction gratings (DGs) are fabricated and evaluated as label-free antibiotic bio(mimetic)sensors. Polymeric gratings are prepared by using microtransfer molding based on SiO(2)/Si molds. The morphology of the MIP gratings is studied by optical and atomic force microscopes. MIP 2D-DGs exhibit 2D optical diffraction patterns, and measurement of changes in diffraction efficiency is used as sensor response. The refractive index of the micropatterned MIP material was estimated, via solvent index matching experiments, to be 1.486. Immersion of a MIP 2D-DG in different solutions of target-antibiotic enrofloxacin leads to significant variations in diffraction efficiency, demonstrating target-molecule detection. On the other hand, no significant response is observed for both control experiments: MIP grating exposed to a non-retained analyte and an equivalent non-imprinted polymer grating exposed to the target analyte, showing highly specific antibiotic label-free optical recognition.     

Sensitive detection of cysteine based on fluorescent silver clusters

In this work, we report the application of novel, water-soluble fluorescent Ag clusters in fluorescent sensors for detecting cysteine, an important biological analyte. The fluorescence of poly(methacrylic acid) (PMAA)-templated Ag clusters was found to be quenched effectively by cysteine, but not when the other alpha-amino acids were present. By virtue of the specific response, a new, simple, and sensitive fluorescent method for detecting cysteine has been developed based on Ag clusters. The present assay allows for the selective determination of cysteine in the range of 2.5 x 10(-8) to 6.0 x 10(-6)M with a detection limit of 20 nM at a signal-to-noise ratio of 3. Based on the absorption and fluorescence studies, we suggested that cysteine quenched the emission by the thiol-adsorption-accelerated oxidation of the emissive Ag clusters. The present study shows a promising step toward the application of silver clusters, a new class of attractive fluorescence probes.     

Iron specificity of a biosensor based on fluorescent pyoverdin immobilized in sol-gel glass

Two current technologies used in biosensor development are very promising: 1. The sol-gel process of making microporous glass at room temperature, and 2. Using a fluorescent compound that undergoes fluorescence quenching in response to a specific analyte. These technologies have been combined to produce an iron biosensor. To optimize the iron (II or III) specificity of an iron biosensor, pyoverdin (a fluorescent siderophore produced by Pseudomonas spp.) was immobilized in 3 formulations of porous sol-gel glass. The formulations, A, B, and C, varied in the amount of water added, resulting in respective R values (molar ratio of water:silicon) of 5.6, 8.2, and 10.8. Pyoverdin-doped sol-gel pellets were placed in a flow cell in a fluorometer and the fluorescence quenching was measured as pellets were exposed to 0.28 - 0.56 mM iron (II or III). After 10 minutes of exposure to iron, ferrous ion caused a small fluorescence quenching (89 - 97% of the initial fluorescence, over the range of iron tested) while ferric ion caused much greater quenching (65 - 88%). The most specific and linear response was observed for pyoverdin immobilized in sol-gel C. In contrast, a solution of pyoverdin (3.0 μM) exposed to iron (II or III) for 10 minutes showed an increase in fluorescence (101 - 114%) at low ferrous concentrations (0.45 - 2.18 μM) while exposure to all ferric ion concentrations (0.45 - 3.03 μM) caused quenching. In summary, the iron specificity of pyoverdin was improved by immobilizing it in sol-gel glass C.     

Molecularly imprinted polymer-assisted sample clean-up of ochratoxin A from red wine: merits and limitations

A new analytical method for the determination of the carcinogenic mycotoxin ochratoxin A (OTA) in red wines has been developed involving a two-dimensional solid-phase extraction (SPE) clean-up protocol on C18-silica and a target-selective molecularly imprinted polymer (MIP). Prior removal of the interfering acidic matrix compounds by C18 solid-phase extraction was crucial for a successful clean-up as direct sample loading onto the MIP led to poor recoveries. The combined solid-phase extraction protocol afforded extracts suitable for sensitive ochratoxin A quantification by HPLC-fluorescence detection. Preliminary validation of the method performance with spiked (0.033-1.0 ng OTA/ml) and commercial red wines provided recoveries >90% and < 10%, with limit of detection (LOD) and limit of quantification (LOQ) of 0.01 and 0.033 ng/ml. However, a similarly favorable performance characteristics was observed in control experiments in which the MIP was replaced by the corresponding non-imprinted polymer (NIP). These findings provide evidence that under the employed experimental conditions specific analyte binding to imprinted binding sites plays a minor role in selective OTA retention. In the framework of this study, other problems inherent to MIP-based solid-phase extraction have been addressed. These include the reproducible preparation of MIP materials with consistent molecular recognition characteristics, the potential for repeated use of MIP, unfavorable polymer swelling in application-relevant solvents, potential sample contamination by template bleeding, and slow analyte binding kinetics.     

Effect of antibiotics on viability staining of Escherichia coli in solid phase cytometry

Solid phase cytometry (SPC) has been investigated as a tool to assess the effect of antibiotics on the viability of Escherichia coli. After exposure of the cells to the antibiotic, they are retained on a polyester membrane filter and labelled using a fluorescein derivative as a substrate for intracellular esterases. The number of fluorescent bacteria is automatically counted in an Ar laser scanning device. In the presence of nutrients, all antibiotics tested in concentrations exceeding the MIC inhibited the multiplication of cells but not the labelling per se. However, when no nutrients were added, the cells did not multiply, and inhibition of the fluorescent staining was only observed for membrane permeabilizing antibiotics, even at sub-MIC concentrations. The selective detection by SPC of membrane-permeabilizing antibiotics corroborates the requirement of membrane integrity for viability labelling of bacteria. This selectivity has been exploited to develop a method for the detection of colistin residues in milk.