Alexey V. Orlov

Magnetic Immunoassay for Detection of Staphylococcal Toxins in Complex Media

Method of highly sensitive registration of magnetic nanoparticles by their nonlinear magnetization is used in a novel sandwich-type immunoassay for detection of staphylococcal toxins in complex media of virtually any volume, with increasing sensitivity at higher sample volume. The signal is read out from the entire volume of a nontransparent 3D fiber structure employed as a solid phase, which provides large reaction surface, quick reagent mixing, as well as antigen immunofiltration directly in the course of the assay. The method has demonstrated near-linear dose-response curves within a wide range of ~3 decades, while detection of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST) in neat milk without sample preparation. The limits of detection (LOD) as low as 4 and 10 pg/mL for TSST and SEA, respectively, were obtained in 2-h format using 30-mL samples. The second, 25-min format, showed the LOD of 0.1 and 0.3 ng/mL for the same toxins in a 150 μL sample. The developed immunoassay can be applied in food safety control, in vitro diagnostics, and veterinary for a variety of research from express tests in the field to highly sensitive laboratory tests.

Rapid dry-reagent immunomagnetic biosensing platform based on volumetric detection of nanoparticles on 3D structures

A dry-reagent immunomagnetic (DRIM) biosensing platform is developed for rapid high-precision quantitative analyses for in vitro diagnostics. The platform combines the advantages of immunochromatography with highly sensitive quantification of 200-nm magnetic nanoparticles (MP) from the entire volume of lateral flow membranes. The MP are registered by their non-linear magnetization at combinatorial frequencies by a portable reader that offers the detection limit of 60 zeptomoles or 0.4 ng of magnetic nanolabels and extremely wide linear dynamic range of 7 orders. The efficient combination of small MP with large reaction surface of the 3D membranes has permitted the detection in human serum of as low as 25 pg/ml total prostate specific antigen (PSA) during 30-min assay. The featured 3-fold signal increase per every order of concentration within 3.5 orders of magnitude allows precise analysis of antigen concentrations in a wide range. The system also provides the first tool for quantitative MP mapping along the lateral flow strip for easy development and optimization of assays. The DRIM detection can be used for simple, rapid and sensitive quantification of protein biomarkers for in vitro diagnostics both in laboratory and near-patient conditions, for food analysis, environmental monitoring, security, and safety applications.

Reversible Conformational Transitions of a Polymer Brush Containing Boronic Acid and its Interaction with Mucin Glycoprotein.

Reversible changes of the height of a polymer brush containing phenylboronic acid were studied. The polymer brush thickness underwent reversible changes of 0.5-1 nm, in response to the changes in composition of the contacting aqueous phase from deionized water to bicarbonate buffer and vice versa, apparently due to the conformational transition of the weak polyelectrolyte to the more extended electrically charged state. Adsorption of mucin glycoprotein to the polymer brush took place due to boronate/sugar interactions between the glycoprotein and the graft copolymer and resulted in further increase of the brush height by ca. 1.5 nm, as observed by means of spectral correlation spectroscopy and ellipsometry.

Real-time sensitive detection of low molecular weight compounds by optical immunosensors

Highly sensitive label-free methods have been developed for detection of low molecular weight compounds. The methods are based on real-time detection of biomolecular interactions on the surface of standard microscopic cover glass slips used as affordable single-used sensor chips. The assay performance was successfully validated for detection of antibiotic chloramphenicol which is used in medicine and veterinary as well as for determination of natural toxins in real samples. The proposed methods are an attractive solution for medical monitoring of antibiotics in the organism and for toxicological control of food.

Biosensors based on magnetic nanolabels: Optimization with spectral interferometry and highly-sensitive electronic registration

A new rapid method based on immunochromatographic assay in combination with the electronic registration of nanolabels by their non-linear magnetization has been developed. The method is designed to provide highly accurate measurements of the concentration of protein molecules (e.g. markers, which indicate the onset or development of a disease) in various samples, including opaque solutions or strongly colored liquids. The optimization of the assay parameters is carried out using real-time monitoring of all the immunoassay steps with the spectral-correlation interferometry.

Biosensors based on spectral correlation interferometry for biomedical research and diagnostics

Biosensors based on the spectral correlation interferometry (SCI) with assistance of magnetic nanoparticles (MP) have been developed for medical diagnostics. The SCI has been adapted for studies of affinity constants of MP covered by antibodies and for detection of protein molecules in human serum. The 50-nm MP employed as labels yield 100-fold amplification of the SCI signals to meet the requirements for myocardial infarction diagnostics.