Multiplex label-free biosensor for detection of autoantibodies in human serum: Tool for new kinetics-based diagnostics of autoimmune diseases.

A multiplex label-free biosensor is developed for diagnostics of autoimmune diseases by highly sensitive measuring in human serum both critical characteristics of autoantibody: concentration and native kinetic parameters that reflect autoantibody aggressiveness to the organism's tissues. The biosensor is based on the spectral-correlation interferometry and image processing of a microarray glass biochip, affordable to be single-used in medical applications. Simultaneous 25-min detection and activity characterization of several autoantibodies in the same serum sample have been demonstrated for anti-thyroglobulin (anti-TG) and anti-thyroid peroxidase (anti-TPO) as models. The biosensor offers extremely high sensitivity: limits of detection in serum are 1.7 IU/mL and 6 IU/mL for anti-TPO and anti-TG, respectively. The dynamic range covers the whole range of clinically relevant concentrations of the autoantibodies up to 1000 IU/mL. The developed method of characterization of autoantibody activity by recording the kinetics of their binding with free native antigens is based on autoantibody polyvalency. The measurements in clinical serum samples have shown that the native kinetic parameters are independent of concentration. The proposed biosensor and method of native kinetic registration can be used to develop new criteria for comprehensive diagnostics of autoimmune diseases, based not only on traditional measurements of concentration but also on quantitative evaluation of autoantibody aggressiveness. The developed method can be adapted to other label-free sensors such as those based on the surface plasmon resonance, optical waveguides, etc.

Data on characterization of glass biochips and validation of the label-free biosensor for detection of autoantibodies in human serum.

The data represent in-depth characterization of a novel method for highly sensitive simultaneous measuring in human serum of both critical parameters of autoantibodies: concentration and native kinetics. The latter refers to autoantibody interaction with free, not immobilized, antigen. The method and related biosensors are based on the spectral-correlation and spectral-phase interferometry. The data cover: multi-factor optimization and quantitative characterization of the developed affordable single-used biochips, including X-ray photoelectron spectroscopy (XPS) control of chemical modifications of the surface during fabrication; antibody screening; optimization and verification of protocols for label-free biosensing in human serum; mathematical model for fitting experimental data and calculation of kinetic constants of interaction of autoantibodies with free antigen; comprehensive verification of the method specificity; correlation between the data obtained with the developed biosensor and with enzyme linked immunosorbent assay (ELISA); comparison of analytical characteristics of the developed biosensor with the most advanced label-based methods. The data importance is confirmed by a companion paper (DOI 10.1016/j.bios.2020.112187), which shows that the combination of mentioned autoantibody parameters is promising for more accurate criteria for early diagnostics and efficient therapy of autoimmune disorders. The obtained data can be used in development of a wide range of biosensors, both label-free and based on various labels.

Dynamic light scattering biosensing based on analyte-induced inhibition of nanoparticle aggregation.

A new approach to direct quantitative detection of small molecules (haptens) by dynamic light scattering biosensing is presented. The proposed technique implements a homogeneous competitive immunoassay and is based on optical detection of specific inhibition of nanoparticle aggregation induced by the analyte in a sample. The technique performance was tested both in buffer and milk for detection of chloramphenicol - antibiotic relevant to food safety diagnostics. Good specificity, sensitivity (LOD in milk is 2.4 ng/ml), precision (4.0 ± 1.2%), ruggedness (8.3%), and 96% recovery in conjunction with a record wide dynamic range (3 orders of magnitude) of the nanosensing technique were demonstrated. Such characteristics complemented by the assay simplicity (no washing step) and a short assay time make the approach attractive for application as an analytical platform for point-of-care and field-oriented diagnostics. Graphical abstract.

Nanoparticle-based drug delivery via RBC-hitchhiking for the inhibition of lung metastases growth.

Delivery of particle-based theranostic agents via their transportation on the surfaces of red blood cells, commonly referred to as RBC-hitchhiking, has historically been developed as a promising strategy for increasing the extremely poor blood circulation lifetime, primarily, of the large-sized sub-micron agents. Here, we show for the first time that RBC-hitchhiking can be extremely efficient for nanoparticle delivery and tumor treatment even in those cases when no circulation prolongation is observed. Specifically, we demonstrate that RBC-hitchhiking of certain small 100 nm particles, unlike that of the conventional sub-micron ones, can boost the delivery of non-targeted particles to lungs up to a record high value of 120-fold (and up to 40% of the injected dose). To achieve this remarkable result, we screened sub-200 nm nanoparticles of different sizes, polymer coatings and ζ-potentials and identified particles with the optimal RBC adsorption/desorption behavior. Furthermore, we demonstrated that such RBC-mediated rerouting of particles to lungs can be used to fight pulmonary metastases of aggressive melanoma B16-F1. Our findings could change the general paradigm of drug delivery for cancer treatment with RBC-hitchhiking. It is not the blood circulation lifetime that is the key factor for nanoparticle efficiency, but rather the complexation of nanoparticles with the RBC. The demonstrated technology could become a valuable tool for development of new strategies based on small nanoparticles for the treatment of aggressive and small-cell types of cancer as well as other lung diseases.

Magnetometry based method for investigation of nanoparticle clearance from circulation in a liver perfusion model.

Nanoparticles (NPs) are among the most promising agents for advanced theranostics. However, their functioning in vivo is severely inhibited by the mononuclear phagocyte system (MPS), which rapidly removes all foreign entities from blood circulation. Little is known about the sequestration mechanisms and the ways to counteract them. New methods are highly demanded for investigation with high scrutiny of each aspect of NP clearance from blood. For example, while liver macrophages capture the majority of the administered particles, reliable investigation of this process in absence of other MPS components is hard to implement in vivo. Here, we demonstrate a novel method for real-time investigation hepatic uptake of NPs in an isolated perfused liver based on an extremely accurate magnetometric registration technique. The signal is obtained solely from the magnetic NPs without any 'background' from blood or tissues, which is a significant advantage over other techniques, e.g. optical ones. We illustrate the method capacity by investigation of behavior of different particles and show good correlation with in vivo studies. We also demonstrate notable suitability of the method for studying the NP clearance from the flow in the user-defined mediums, e.g. those containing specific serum components. Finally, the method was applied to reveal an interesting effect of short-term decrease of liver macrophage activity after the first interaction with small amounts of NPs. The developed perfusion model based on the high-performance magnetometry can be used for finding new mechanisms of NP sequestration and for development of novel 'stealth' nanoagents.

Data on characterization and validation of assays for ultrasensitive quantitative detection of small molecules: Determination of free thyroxine with magnetic and interferometric methods.

The presented data refer to optimization and quantitative characterization of a rapid lateral flow assay based on high-affinity bifunctional ligand and magnetic nanolabels, which was developed for detection of small molecules of thyroid hormones. The results were obtained by several techniques, including the magnetic particle quantification method, spectral-correlation interferometry and spectral-phase interferometry, dynamic light scattering, enzyme linked immunosorbent assay. The long-term stability of "antibody - magnetic nanoparticle" conjugates is shown. The assay specificity is confirmed, and verification of successful combination of magnetic particles and antibodies is demonstrated. The kinetic and equilibrium dissociation constants are determined for interactions between thyroxine and monoclonal antibodies. The obtained data could be used for design of other platforms for detection of small molecules.

Ultrasensitive detection enabled by nonlinear magnetization of nanomagnetic labels.

Geometrically confined magnetic particles due to their unique response to external magnetic fields find a variety of applications, including magnetic guidance, heat and drug delivery, magneto-mechanical actuation, and contrast enhancement. Highly sensitive detection and imaging techniques based on the nonlinear properties of nanomagnets were recently proposed as innovative strong-translational potential methods applicable in complex, often opaque, biological systems. Here we report on the significant enhancement of the detection capability using optical-lithography-defined, ferromagnetic iron-nickel alloy disk-shaped particles. We show that an irreversible transition between strongly non-collinear (vortex) and single domain states, driven by an alternating magnetic field, translates into a nonlinear magnetic response that enables ultrasensitive detection of these particles. The record sensitivity of ∼3.5 × 10-9 emu, which is equivalent to ∼39 pg of magnetic material is demonstrated at room temperature for arrays of patterned disks. We also show that unbound disks suspended in the aqueous buffer can be successfully detected and quantified in real-time when administered into a live animal allowing for tracing of their biodistribution. The use of nanoscale ferromagnetic particles with engineered nonlinear properties opens prospects for further enhancing the sensitivity, scalability, and tunability of noise-free magnetic tag detection in high-background environments for various applications spanning from biosensing and medical imaging to anti-counterfeiting technologies.

Synthesis and Characterization of Hybrid Core-Shell Fe3O4/SiO2 Nanoparticles for Biomedical Applications.

The creation of markers that provide both visual and quantitative information is of considerable importance for the mapping of tissue macrophages and other cells. We synthesized magnetic and magneto-fluorescent nanomarkers for the labeling of cells which can be detected with high sensitivity by the magnetic particle quantification (MPQ) technique. For stabilization under physiological conditions, the markers were coated with a dense silica shell. In this case, the size and zeta-potential of nanoparticles were controlled by a modified Stober reaction. Also, we developed a novel facile two-step synthesis of carboxylic acid-functionalized magnetic SiO2 nanoparticles, with a carboxyl polymer shell forming on the nanoparticles before the initiation of the Stober reaction. We extensively characterized the nanomarkers by transmission electron microscopy, electron microdiffraction, and dynamic and electrophoretic light scattering. We also studied the nanoparticle cellular uptake by various eukaryotic cell lines.

Synthesis of magnetic silica nanomarkers with controlled physicochemical properties.

Magnetic markers which can be detected with an extremely high sensitivity with the method of magnetic particle quantification (MPQ) were synthesized. Using a controlled Stober reaction, a set of magnetic silica markers of different sizes and zeta potentials was obtained. The use of a carboxymethyl dextran polymer to stabilize the magnetite particles during the synthesis made it possible to substantially reduce the detection limit of the obtained construct, which opens up new opportunities for creating effective diagnostic nanoagents.

MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions.

Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.

A comprehensive study of interactions between lectins and glycoproteins for the development of effective theranostic nanoagents.

A comprehensive study of the interactions between lectins and glycoproteins possessing different glycosylation profiles in the composition of nanoparticles was carried out in order to find specifically interacting protein pairs for the creation of novel classes of multifunctional nanoagets that based on protein-assisted selfassembly. We obtained information about specific interactions of certain lectins with selected glycoproteins as well as about the ability of certain monosaccharides to competitively inhibit binding of glycoproteins with lectins. These protein-mediated interactions may be involved in the formulation of self-assembled nanoparticles for therapy and diagnostics of various diseases.

Development of immunoassays using interferometric real-time registration of their kinetics.

A method for effective development of solid-phase immunoassays on a glass surface and for optimization of related protocols by highly sensitive quantitative monitoring of each assay step has been proposed and experimentally implemented. The method is based on the spectral correlation interferometry (SCI) that allows real-time measuring of the thickness of a biomolecular layer bound to the recognition molecular receptors on the sensor chip surface. The method is realized with compact 3-channel SCI-biosensors that employ as the sensor chips standard cover glass slips without deposition of any additional films. Different schemes for antibody immobilization on a glass surface have been experimentally compared and optimized toward a higher sorption capacity of the sensor chips. Comparative characterization of the kinetics of each immunoassay stage has been implemented with the optimized protocols: i) covalent immobilization of antibody on an epoxylated surface and ii) biotinylated antibody sorption on a biotinylated surface via a high-affinity biotin-streptavidin bond. We have shown that magnetic nanoparticles employed as labels with model detection of cardiac troponin I further amplify the SCI signal, resulting in 100-fold improvement of the detection limit. The developed protocols can also be used with the alternative immunoassay platforms, including the label methods based on registration of only the final assay result, which is the quantity of bound labels.

[Detection of pyrethroids by spectral correlation interferometry].

A label-free method based on spectral correlation interferometry has been developed for highly sensitive detection of pyrethroids by competitive immunoassay on the surface of sensor chips made of widely available microscopy glass cover slips. It is shown that the method allows independent optimization of each step of the sensor surface modification. This fact may be used to increase the efficiency of development of protocols for a wide spectrum of immunoassays that employ glass surface as a solid phase. Detection of 3-phenoxybenzoic acid, which is one of the most stable metabolites of a large number of pyrethroids, on the surface of the optimized sensor chips has been demonstrated on the level of 15 pg/ml. That is 50 times better than the sensitivity of the enzyme-linked immunosorbent assay (ELISA).

Synthetic peptide fragment (65-76) of monocyte chemotactic protein-1 (MCP-1) inhibits MCP-1 binding to heparin and possesses anti-inflammatory activity in stable angina patients after coronary stenting.

OBJECTIVE AND DESIGN: The peptide from C-terminal domain of MCP-1 (Ingramon) has been shown to inhibit monocyte migration and possess anti-inflammatory activity in animal models of inflammation and post-angioplasty restenosis. Here, we investigate the effect of Ingramon treatment on blood levels of acute-phase reactants and chemokines in patients after coronary stenting and the mechanisms of Ingramon anti-inflammatory activity. SUBJECTS: Eighty-seven patients with ischemic heart disease (IHD) who faced the necessity of coronary angiography (CA) were enrolled. In 67 patients, one-stage coronary stenting was performed; 33 of them were treated with Ingramon in addition to standard therapy. Twenty patients underwent CA only. METHODS: High-sensitivity C-reactive protein (hsCRP) and fibrinogen blood levels were detected routinely. The chemokine concentration in plasma was measured by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array-based immunoassay. Intracellular Ca(2+) levels and cell surface integrin exposure were assayed by flow cytometry. MCP-1 dimerization was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). MCP-1-heparin binding was assessed with a biosensor and ELISA. RESULTS AND CONCLUSIONS: Ingramon treatment was accompanied by less pronounced elevation of hsCRP and fibrinogen levels and decreased MCP-1 concentration in plasma in patients after coronary stenting. Ingramon had no effect on MCP-1 interaction with cell receptors or MCP-1 dimerization, but inhibited MCP-1 binding to heparin. The anti-inflammatory activity of the peptide may be mediated by an impaired chemokine interaction with glycosaminoglycans.

[Embolization of cerebral aneurysms in acute period of hemorrhage complicated by the development of vasospasm].

Results of endovascular treatment of cerebral aneurysms in acute period of hemorrhage complicated by the development of vasospasm in 80 patients were analyzed. The results of treatment of patients with vasospasm were worse. The appearance of vasospasm did not depend of the severity of the patient's state, so all the patients independent on their state need prophylactic measures against this complication. Based on the analysis a strategy of embolization of aneurysms in acute period of hemorrhage complicated by the development of vasospasm was worked out.

The intracranial B-waves' amplitude as prognostication criterion of neurological complications in neuroendovascular interventions.

The purpose of this study was to evaluate dynamics of B-waves' amplitudes (BWA) of blood flow velocity (BFV) in patients with cerebrovascular diseases during endovascular operations. We examined 12 patients with neurovascular pathology during neuroendovascular interventions. Patients were divided into two groups: 1st group (6 cases)--without intraoperative neurological complications, 2nd group (6 cases)--with complications. Bilateral monitoring of BFV in middle cerebral arteries was carried out applying Multi Dop X. To estimate BWA Fourier analysis was used. In the 1st group preoperative BWA on the affected side was 3.9 +/- 0.6 cm/s. Intraoperative (during an access to pathologic formation and its embolisation) BWA increased up to 7.7 +/- 1.1 cm/s (p < 0.05). Postoperative BWA decreased to 4.2 +/- 0.8 cm/s. In the 2nd group the preoperative BWA on the affected side was 9.6 +/- 1.1 cm/s (p < 0.05), thus higher than in the 1st group. Intraoperatively we observed further increase of BWA up to 12.1 +/- 2.6 cm/s, accompanied by occurrence or increase of neurological symptoms. Postoperative BWA decreased to 10.4 +/- 2.9 cm/s, whereas we didn't observe regression of neurological symptoms.

[Racemose arteriovenous angioma (malformation) of the brain]. / Ratsemoznye arteriovenoznye angiomy (mal'formatsii) golovnogo mozga.

242 cases of brain arterio-venous malformations (AVM) are analysed clinically and anatomically. Their pathology, histological, ultrastructural and immunohistochemical characteristics, therapeutic pathomorphosis are described. The concept is suggested according to which AVM is a collective term including true developmental defects, dysembryogenetic tumors and outcomes of various pathologic processes (trauma, brain circulation and compensatory-adaptive disturbances, etc.). The necessity of therapeutic methods corrections in agreement with the disease pathogenesis is outlined.

Phase-polarisation contrast for surface plasmon resonance biosensors.

A technique of phase-polarisation contrast (PPC) for the enhancement of the contrast of a surface plasmon resonance (SPR) intensity profile is proposed and experimentally realised. The technique exploits the peculiarities of light phase and polarisation behaviour under SPR. It applies to non-optimum SPR coupling conditions and enables one to lower the resonant minimum of reflected intensity nearly to zero, and hence to increase substantially the ratio of the intensity from the resonance to that at the minimum. We observed the contrast enhancement by more than one order of magnitude when we applied the PPC scheme. The PPC can be efficiently employed in commercial SPR sensors, as it significantly reduces restrictions on allowable parameters of SPR-supporting metal films and biomolecular layers immobilised on them, facilitates SPR observation, and increases the accuracy of SPR shift measurements.

[The choice of the methods for the surgical treatment of patients with cerebral arteriovenous malformations]. / Vybor metodov khirurgicheskogo lecheniia bol'nykh s arteriovenoznymi mal'formatsiiami golovnogo mozga.

The paper analyzes the outcomes of surgical treatment made in 431 patients in 1971-1994. Multimodality treatment for this abnormality was used, which reduced postoperative mortality.