A Straightforward Method for the Development of Positively Charged Gold Nanoparticle-Based Vectors for Effective siRNA Delivery.

The therapeutic potential of short interfering RNA (siRNA) to treat many diseases that are incurable with traditional preparations is limited by the extensive metabolism of serum nucleases, low permeability through biological membrane barriers because of a negative charge, and endosomal trapping. Effective delivery vectors are required to overcome these challenges without causing unwanted side effects. Here, we present a relatively simple synthetic protocol to obtain positively charged gold nanoparticles (AuNPs) with narrow size distribution and the surface modified with Tat-related cell-penetrating peptide. The AuNPs were characterized using TEM and the localized surface plasmon resonance technique. The synthesized AuNPs showed low toxicity in experiments in vitro and were able to effectively form complexes with double-stranded siRNA. The obtained delivery vehicles were used for intracellular delivery of siRNA in an ARPE-19 cell line transfected with secreted embryonic alkaline phosphatase (SEAP). The delivered oligonucleotide remained intact and caused a significant knockdown effect on SEAP cell production. The developed material could be useful for delivery of negatively charged macromolecules, such as antisense oligonucleotides and various RNAs, particularly for retinal pigment epithelial cell drug delivery.

Quantitative Rapid Magnetic Immunoassay for Sensitive Toxin Detection in Food: Non-Covalent Functionalization of Nanolabels vs. Covalent Immobilization.

In this study, we present a novel and ultrasensitive magnetic lateral flow immunoassay (LFIA) tailored for the precise detection of zearalenone, a mycotoxin with significant implications for human and animal health. A versatile and straightforward method for creating non-covalent magnetic labels is proposed and comprehensively compared with a covalent immobilization strategy. We employ the magnetic particle quantification (MPQ) technique for precise detection of the labels and characterization of their functionality, including measuring the antibody sorption density on the particle surface. Through kinetic studies using the label-free spectral phase interferometry, the rate and equilibrium constants for the binding of monoclonal antibodies with free (not bound with carrier protein) zearalenone were determined to be kon = 3.42 × 105 M-1s-1, koff = 7.05 × 10-4 s-1, and KD = 2.06 × 10-9 M. The proposed MPQ-LFIA method exhibits detection limits of 2.3 pg/mL and 7.6 pg/mL when employing magnetic labels based on covalent immobilization and non-covalent sorption, with dynamic ranges of 5.5 and 5 orders, correspondingly. We have successfully demonstrated the effective determination of zearalenone in barley flour samples contaminated with Fusarium graminearum. The ease of use and effectiveness of developed test systems further enhances their value as practical tools for addressing mycotoxin contamination challenges.

Method of kinetic characterization of immunoreagents for development of express high-sensitive assays for detection of ochratoxin A and heart fatty acids binding protein.

Development of rapid and sensitive immunoassays is a task of great importance in a variety of fields ranging from clinical practice and urgent diagnostics to food quality control and environmental monitoring. High attention of researches is paid to methods of screening, selection, and kinetic characterization of antibodies that enable fast, specific, and effective formation of immunocomplexes. Herein, we present a method for direct investigation of kinetics of immunoreagents during developments of express high sensitive lateral flow assays. As model biomolecules to be detected, the following substances were tested: ochratoxin A (OTA), which is one of the most dangerous mycotoxins naturally present in many vegetable raw materials; and heart fatty acids binding protein (hFABP), which is a cardiac marker used in differential diagnosis of acute myocardial infarction. The kinetic constants of association (kon) and dissociation (koff) with monoclonal antibodies are determined along with the corresponding equilibrium constants (KA and KD). The obtained values are as follows: for the anti-OTA antibodies - kon = 4.54*103 M-1s-1; koff  = 3.32*10-4 s-1; KA = 1.37*107 M-1; KD = 7.31*10-8 M; and for the anti-hFABP antibodies - kon = 7.28*103 M-1s-1; koff = 1.97*10-4 s-1; KA = 3.70*107 M-1; KD = 2.70*10-8 M. The proposed method can be employed in combination with the immunochromatographic assays based on magnetic biolabels.•Investigation of immunoreagent kinetics for development of express high sensitive lateral flow assays•Kinetic characterization of monoclonal antibodies against OTA and hFABP for their rapid and sensitive detection•Both kinetic and equilibrium constants of association and dissociation are determined.

Multiplex Label-Free Kinetic Characterization of Antibodies for Rapid Sensitive Cardiac Troponin I Detection Based on Functionalized Magnetic Nanotags.

Express and highly sensitive immunoassays for the quantitative registration of cardiac troponin I (cTnI) are in high demand for early point-of-care differential diagnosis of acute myocardial infarction. The selection of antibodies that feature rapid and tight binding with antigens is crucial for immunoassay rate and sensitivity. A method is presented for the selection of the most promising clones for advanced immunoassays via simultaneous characterization of interaction kinetics of different monoclonal antibodies (mAb) using a direct label-free method of multiplex spectral correlation interferometry. mAb-cTnI interactions were real-time registered on an epoxy-modified microarray glass sensor chip that did not require activation. The covalent immobilization of mAb microdots on its surface provided versatility, convenience, and virtually unlimited multiplexing potential. The kinetics of tracer antibody interaction with the "cTnI­capture antibody" complex was characterized. Algorithms are shown for excluding mutual competition of the tracer/capture antibodies and selecting the optimal pairs for different assay formats. Using the selected mAbs, a lateral flow assay was developed for rapid quantitative cTnI determination based on electronic detection of functionalized magnetic nanoparticles applied as labels (detection limit­0.08 ng/mL, dynamic range > 3 orders). The method can be extended to other molecular biomarkers for high-throughput screening of mAbs and rational development of immunoassays.

Express high-sensitive detection of ochratoxin A in food by a lateral flow immunoassay based on magnetic biolabels.

We present an easy-to-use lateral flow immunoassay for rapid, precise and sensitive quantification of one of the most hazardous mycotoxins - ochratoxin A (OTA), which is widely present in food and agricultural commodities. The achieved limit of detection during the 20-min OTA registration is 11 pg/mL. The assay provides accurate results in both low- and high-concentration ranges. That is due to the extraordinary steepness of the linear calibration plot: 5-order dynamic range of concentrations causes almost a 1000-fold change in the signal obtained by electronic detection of magnetic biolabels using their non-linear magnetization. High specificity, repeatability, and reproducibility of the assay have been verified, including measuring OTA in real samples of contaminated corn flour. The developed assay is a promising analytical tool for food and feed safety control; it may become an express, convenient and high-precision alternative to the traditional sophisticated laboratory techniques based on liquid chromatography.

Nanobiosensing based on optically selected antibodies and superparamagnetic labels for rapid and highly sensitive quantification of polyvalent hepatitis B surface antigen.

Hepatitis B surface antigen (HBsAg) is the most clinically relevant serological marker of hepatitis B virus (HBV) infection. Its detection in blood is extremely important for identification of asymptomatic individuals or chronic HBV carriers, screening blood donors, and early seroconversion. Rapid point-of-care HBsAg tests are predominantly qualitative, and their analytical sensitivity does not meet the requirements of regulatory agencies. We present a highly sensitive lateral flow assay based on superparamagnetic nanoparticles for rapid quantification (within 30 min) of polyvalent HBsAg in serum. The demonstrated limit of detection (LOD) of 80 pg mL-1 in human serum is better than both the FDA recommendations for HBsAg assays (which is 0.5 ng mL-1) and the sensitivity of traditional laboratory-based methods such as enzyme linked immunosorbent assays. Along with the attractive LOD at lower concentrations and the wide linear dynamic range of more than 2.5 orders, the assay features rapidity, user-friendliness, on-site operation and effective performance in the complex biological medium. These are due to the combination of the immunochromatographic approach with a highly sensitive electronic registration of superparamagnetic nanolabels over the entire volume of a 3D test structure by their non-linear magnetization and selection of optimal antibodies by original optical label-free methods. The developed cost-efficient bioanalytical technology can be used in many socially important fields such as out-of-lab screening and diagnosis of HBV infection at a point-of-demand, especially in hard-to-reach or sparsely populated areas, as well as highly endemic regions.