Mikaela Nichkova

Magnetic/luminescent core/shell particles synthesized by spray pyrolysis and their application in immunoassays with internal standard

Many types of fluorescent nanoparticles have been investigated as alternatives to conventional organic dyes in biochemistry; magnetic beads also have a long history of biological applications. In this work we apply flame spray pyrolysis in order to engineer a novel type of nanoparticle that has both luminescent and magnetic properties. The particles have magnetic cores of iron oxide doped with cobalt and neodymium and luminescent shells of europium-doped gadolinium oxide (Eu:Gd(2)O(3)). Measurements by vibrating sample magnetometry showed an overall paramagnetic response of these composite particles. Luminescence spectroscopy showed spectra typical of the Eu ion in a Gd(2)O(3) host-a narrow emission peak centred near 615 nm. Our synthesis method offers a low-cost, high-rate synthesis route that enables a wide range of biological applications of magnetic/luminescent core/shell particles. Using these particles we demonstrate a novel immunoassay format with internal luminescent calibration for more precise measurements.

Application of luminescent Eu:Gd2O3 nanoparticles to the visualization of protein micropatterns

Nanoparticle phosphors made of lanthanide oxides are a promising new class of tags in biochemistry because of their large Stokes shift, sharp emission spectra, long luminescence lifetime, and good photostability. We demonstrate the application of these nanoparticles to the visualization of protein micropatterns. Luminescent europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles are synthesized by spray pyrolysis. The size distribution is from 5 to 200 nm. The particles are characterized by means of laser-induced fluorescent spectroscopy and transmission electron microscopy (TEM). The main emission peak is at 612 nm. The nanoparticles are coated with avidin through physical adsorption. biotinylated bovine serum albumin (BSA-b) is patterned on a silicon wafer using a microcontact printing technique. The wafer is then incubated in a solution of avidin-coated nanoparticles. Fluorescent microscopic images reveal that the nanoparticles are organized onto designated area, as defined by the microcontact printing process. The luminescent nanoparticles do not suffer photobleaching during the observation, which demonstrates their suitability as luminescent labels for fluorescence microscopy studies. More detailed studies are preformed using atomic-force microscopy (AFM) at a single nanoparticle level. The specific and the nonspecific binding densities of the particles are qualitatively evaluated.

Immunochemical determination of dioxins in sediment and serum samples

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are considered highly toxic contaminants and the environmental and biological monitoring of these compounds is of great concern. Immunoassays may be used as screening methods to satisfy the growing demand for rapid and low cost analysis. In this work, we describe the application of an immunoassay that uses 2,3,7-trichloro-8-methyldibenzo-p-dioxin (TMDD) as a surrogate standard for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to sediment and human serum samples. Sample extraction and preparation methods were developed with the aim to establish the simplest, cost-effective and efficient removal of the matrix interferences in the enzyme-linked immunosorbent assay (ELISA). The overall method for sediments is based on a hexane extraction; clean up by a multilayered silica gel column and an activated carbon column; an organic solvent exchange with DMSO-Triton X-100 and ELISA measurement. The gas chromatography-high resolution mass spectrometry (GC-HRMS) validation studies (n = 13) revealed that the method is suitable for the toxic equivalents (TEQ) screening of dioxin in sediments with a method detection limit of about 100pgg(-1) dry sediment with a precision of 13-33% R.S.D. The analysis of a large number of samples originating from different sources would be required to establish more precisely the screening level, as well as the number of false positives and negatives of dioxin TEQ by the immunoassay for sediments. The immunoassay method for sediment analysis offers improvement in speed, sample throughput, and cost in comparison to GC-HRMS. Dioxins were determined in serum samples after a simple liquid-liquid extraction and solvent exchange into DMSO-Triton X-100 without further dilution. The current method (approximate method LOQ of 200pgml(-1) serum) is not sufficiently sensitive for the determination of dioxins in serum to measure acceptable exposure limit.

Quantum Dots as Reporters in Multiplexed Immunoassays for Biomarkers of Exposure to Agrochemicals

Abstract The application of quantum dots (QDs) as labels in immunoassay microarrays for the multiplex detection of 3‐phenoxybenzoic acid (PBA) and atrazine‐mercapturate (AM) has been demonstrated. PBA and AM are biomarkers of exposure to the pyrethroid insecticides and to the herbicide atrazine, respectively. Microarrays were fabricated by microcontact printing of the coating antigens in line patterns onto glass substrates. Competitive immunoassays were successfully performed using QDs (QD560 and QD620) as reporters. The multiplexed immunoassays were characterized by fluorescence microscopy and SEM. The application of QD fluorophores facilitates multiplex assays and therefore can contribute to enhanced throughput in biomonitoring.

An enzyme-linked immunosorbent assay for the determination of dioxins in contaminated sediment and soil samples.

A 96-microwell enzyme-linked immunosorbent assay (ELISA) method was evaluated to determine PCDDs/PCDFs in sediment and soil samples from an EPA Superfund site. Samples were prepared and analyzed by both the ELISA and a gas chromatography/high resolution mass spectrometry (GC/HRMS) method. Comparable method precision, accuracy, and detection level (8 ng kg(-1)) were achieved by the ELISA method with respect to GC/HRMS. However, the extraction and cleanup method developed for the ELISA requires refinement for the soil type that yielded a waxy residue after sample processing. Four types of statistical analyses (Pearson correlation coefficient, paired t-test, nonparametric tests, and McNemars test of association) were performed to determine whether the two methods produced statistically different results. The log-transformed ELISA-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin values and log-transformed GC/HRMS-derived TEQ values were significantly correlated (r=0.79) at the 0.05 level. The median difference in values between ELISA and GC/HRMS was not significant at the 0.05 level. Low false negative and false positive rates (<10%) were observed for the ELISA when compared to the GC/HRMS at 1,000 ng TEQ kg(-1). The findings suggest that immunochemical technology could be a complementary monitoring tool for determining concentrations at the 1,000 ng TEQ kg(-1) action level for contaminated sediment and soil. The ELISA could also be used in an analytical triage approach to screen and rank samples prior to instrumental analysis.

Validation of an ELISA for urinary dopamine: applications in monitoring treatment of dopamine-related disorders.

Dopamine is a catecholamine that serves as a neurotransmitter in the central and peripheral nervous system. Non‐invasive, reliable, and high‐throughput techniques for its quantification are needed to assess dysfunctions of the dopaminergic system and monitor therapies. We developed and validated a competitive ELISA for direct determination of dopamine in urine samples. The method provides high specificity, good accuracy, and precision (average inter‐assay variation < 12%). The analysis is not affected by general urinary components and structurally related drugs and metabolites. The correlation between ELISA and LC‐MS/MS analyses was very good (r = 0.986, n = 28). The reference range was 64–261 μg/g Cr (n = 64). Week‐to‐week biological variations of second morning urinary dopamine under free‐living conditions were 23.9% for within‐ and 35.5% for between‐subject variation (n = 10). The assay is applied in monitoring Parkinsons disease patients under different treatments. Urinary dopamine levels significantly increase in a dose‐dependent manner for Parkinsons disease patients under l‐DOPA treatment. The present ELISA provides a cost‐effective alternative to chromatographic methods to monitor patients receiving dopamine restoring treatment to ensure appropriate dosing and clinical efficacy. The method can be used in pathological research for the assessment of possible peripheral biological markers for disorders related to the dopaminergic system.

Novel ELISAs for Screening of the Biogenic Amines GABA, Glycine, β-Phenylethylamine, Agmatine, and Taurine Using One Derivatization Procedure of Whole Urine Samples

The inhibitory neurotransmitters GABA, glycine and agmatine and neuromodulators beta-phenylethylamine (beta-PEA) and taurine are important biogenic amines of the sympathetic and parasympathetic nervous systems in the body. Abnormalities in the metabolism of these biomarkers have been implicated in a vast number of neurological diseases. Novel competitive immunoassays, using one unique whole urine derivatization procedure applicable for all five biomarkers, have been developed. The determination of these biomarkers was highly reproducible: the coefficient of variance of inter- and intra-assay variation is between 3.9% and 9.8% for all assays. The assays show a good linearity in urine samples within the range of 100-400 mg Cr/dL and specificity when urine samples are spiked with biogenic amines. The recoveries are between 76 and 154%. The correlation between HPLC and ELISA for glycine and taurine (n = 10) showed regression coefficients of 0.97 and 0.98, respectively. An in vivo study on the urinary clearance of beta-PEA, agmatine and taurine after oral intake by healthy individuals demonstrated the specificity and clinical significance of these new immunoassays. The immunoassays are useful for clinical and basic research where a fast and accurate assay for the screening of biogenic amines in urine is required, without preclearance of the sample.

Immunochemical screening of pesticides (simazine and cypermethrin) in orange oil.

Pesticide residue analysis in citrus oils is very important for their quality and marketing. This study assessed the reliability and sensitivity of enzyme-linked immunosorbent assays (ELISA) for simazine and cypermethrin screening in orange oil. Simazine was analyzed after extraction of the oil with methanolic phosphate buffer with a limit of quantitation (LOQ) of 40 microg/L for 1-fold and approximately 100 microg/L for 10-fold oils. Due to matrix effects the immunoanalysis of cypermethrin required hexane-acetonitrile partitioning followed by silica solid phase extraction. The method detected levels higher than 0.5 ppm (mg/L). This LOQ is lower than the U.S. EPA tolerance level (0.9 ppm) for cypermethrin in citrus oils. A good correlation (r(2) 0.99) between ELISA and LC-MS/MS was observed for the analysis of both analytes in 1-fold orange oil. Immunochemical screening can be used to reduce instrumental analysis costs by its use in preliminary orange oil screening.

Enzyme-linked immunosorbent assay for screening dioxin soil contamination by uncontrolled combustion during informal recycling in slums.

Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELISA) as the basis for a sustainable, simple, and low-cost monitoring program to assess the toxicological impact of uncontrolled combustion in slums. A panel of 16 samples was analyzed by GC-HRMS and ELISA on split extracts. Close to 20% of the analyzed samples showed dioxin concentrations up to almost twice the guidance level for residential soil in several countries, pointing out the need for performing a large-scale monitoring program. Despite the potential for variations in dioxin congener distribution due to the mixed nature of the incinerated material, there was a good correlation between the toxic equivalents as determined by GC-HRMS and ELISA. Furthermore, an interlaboratory ELISA validation showed that the capacity to perform the dioxin ELISA was successfully transferred between laboratories. It was concluded that the ELISA method performed very well as a screening tool to prioritize samples for instrumental analysis, which allows cutting down costs significantly.

Fast and precise detection of ricin with microcapillary sensor system

Ricin is an easily available toxin which can be used as a bio-terror agent. Fast and inexpensive methods for its detection in different samples are needed. Recently we have developed a novel fluorescent sandwich immunoassay for ricin using magnetic-luminescent nanoparticles (MLNPs) as carriers in a microcapillary system for incubation and detection. Antiricin antibody coated MLNPs that were dispersed in buffer solution were introduced in the capillary tube and immobilized inside using an external electromagnet. Then the sample containing ricin was injected while the MLNPs were mixed by an alternating magnetic field. After the incubation, washing solution and secondary antibody conjugated with Alexa-fluorescent were injected into the capillary while the MLNPs were constantly mixed. After the final wash, the particles were immobilized for detection. The total analysis time was reduced to less than forty minutes which is about 8-10 fold improvement in comparison with the plate-based protocols. This system is promising for the development of a portable biosensor and can be used for the detection of other analytes of interest.