Efficient Chlorostannate Modification of Magnetite Nanoparticles for Their Biofunctionalization.

Magnetite nanoparticles (MNPs) are highly favored materials for a wide range of applications, from smart composite materials and biosensors to targeted drug delivery. These multifunctional applications typically require the biofunctional coating of MNPs that involves various conjugation techniques to form stable MNP-biomolecule complexes. In this study, a cost-effective method is developed for the chlorostannate modification of MNP surfaces that provides efficient one-step conjugation with biomolecules. The proposed method was validated using MNPs obtained via an optimized co-precipitation technique that included the use of degassed water, argon atmosphere, and the pre-filtering of FeCl2 and FeCl3 solutions followed by MNP surface modification using stannous chloride. The resulting chlorostannated nanoparticles were comprehensively characterized, and their efficiency was compared with both carboxylate-modified and unmodified MNPs. The biorecognition performance of MNPs was verified via magnetic immunochromatography. Mouse monoclonal antibodies to folic acid served as model biomolecules conjugated with the MNP to produce nanobioconjugates, while folic acid-gelatin conjugates were immobilized on the test lines of immunochromatography lateral flow test strips. The specific trapping of the obtained nanobioconjugates via antibody-antigen interactions was registered via the highly sensitive magnetic particle quantification technique. The developed chlorostannate modification of MNPs is a versatile, rapid, and convenient tool for creating multifunctional nanobioconjugates with applications that span in vitro diagnostics, magnetic separation, and potential in vivo uses.

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.

Highly Sensitive Nanomagnetic Quantification of Extracellular Vesicles by Immunochromatographic Strips: A Tool for Liquid Biopsy.

Extracellular vesicles (EVs) are promising agents for liquid biopsy-a non-invasive approach for the diagnosis of cancer and evaluation of therapy response. However, EV potential is limited by the lack of sufficiently sensitive, time-, and cost-efficient methods for their registration. This research aimed at developing a highly sensitive and easy-to-use immunochromatographic tool based on magnetic nanoparticles for EV quantification. The tool is demonstrated by detection of EVs isolated from cell culture supernatants and various body fluids using characteristic biomarkers, CD9 and CD81, and a tumor-associated marker-epithelial cell adhesion molecules. The detection limit of 3.7 × 105 EV/µL is one to two orders better than the most sensitive traditional lateral flow system and commercial ELISA kits. The detection specificity is ensured by an isotype control line on the test strip. The tool's advantages are due to the spatial quantification of EV-bound magnetic nanolabels within the strip volume by an original electronic technique. The inexpensive tool, promising for liquid biopsy in daily clinical routines, can be extended to other relevant biomarkers.

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.

Rapid and Easy-to-Use Method for Accurate Characterization of Target Binding and Kinetics of Magnetic Particle Bioconjugates for Biosensing.

The ever-increasing use of magnetic particle bioconjugates (MPB) in biosensors calls for methods of comprehensive characterization of their interaction with targets. Label-free optical sensors commonly used for studying inter-molecular interactions have limited potential for MPB because of their large size and multi-component non-transparent structure. We present an easy-to-use method that requires only three 20-min express measurements to determine the key parameters for selection of optimal MPB for a biosensor: kinetic and equilibrium characteristics, and a fraction of biomolecules on the MPB surface that are capable of active targeting. The method also provides a prognostic dependence of MPB targeting efficiency upon interaction duration and sample volume. These features are possible due to joining a magnetic lateral flow assay, a highly sensitive sensor for MPB detection by the magnetic particle quantification technique, and a novel mathematical model that explicitly describes the MPB-target interactions and does not comprise parameters to be fitted additionally. The method was demonstrated by experiments on MPB targeting of cardiac troponin I and staphylococcal enterotoxin B. The validation by an independent label-free technique of spectral-correlation interferometry showed good correlation between the results obtained by both methods. The presented method can be applied to other targets for faster development and selection of MPB for affinity sensors, analytical technologies, and realization of novel concepts of MPB-based biosensing in vivo.

Designing a magnetic inductive micro-electrode for virus monitoring: modelling and feasibility for hepatitis B virus.

A simple model is designed for an inductive immunosensor in which the magnetic particles are attached to the bioreceptors to form a sandwich on the surface of an inductor. The inductor consists of a coil covered on a silicon oxide wafer. The coil comprises 250 turns of a planar gold wire, which is approximately 200 nm thick and 392 mm long, placed in a circle with a diameter of 2 mm. The model is well characterised by controlling the geometrical and electrical parameters and also the permeability of the magnetic material. To evaluate the feasibility of the model for virus monitoring, a novel inductive immunosensor is designed and for the first time applied for the detection of hepatitis B surface antigen (HBsAg). At first, Fab' segment of primary anti-HBsAg is immobilised on the coil. Then, the coil is exposed to HBsAg and the complex is introduced to a secondary antibody conjugated with magnetic particles to form an immune-sandwich. Finally, the influence of magnetic particles on the coil inductance is recorded and used as a signal for HBsAg detection. The magnetic inductive immunosensor showed specific responses toward HBsAg with the detection limit of 1 ng mL-1, linear range of 1 to 200 ng mL-1, and a sensitivity of 6 × 10-4 mL ng-1. The experimental results showed a very good agreement with simulation data indicating the compatibility of sensor sensitivity to the expected theoretical values. Graphical abstract.

Nanomagnetic lateral flow assay for high-precision quantification of diagnostically relevant concentrations of serum TSH.

Thyroid stimulating hormone (TSH) is the first-line marker for initial evaluation of the thyroid gland function. We present a lateral flow immunoassay based on superparamagnetic nanolabels for rapid (<25 min) quantitative determination of TSH at a point of care. The demonstrated limit of detection (LOD) of 0.017 µIU/mL in human serum is on the level of third-generation TSH laboratory tests. The wide linear dynamic range of more than 3 orders covers the whole range of clinically relevant TSH concentrations for confident quantitative diagnostics of the gland function from hyper- to hypothyroidism, and different states in-between. The attractive values of LOD and linear dynamic range are due to counting of the superparamagnetic nanolabels over the whole reaction volume by their non-linear magnetization at two frequencies of an alternating magnetic field and detecting the response at combinatorial frequencies. The developed cost-efficient and user-friendly immunoassay can be used for express in vitro diagnostics and long-term quantitative monitoring of thyroid dysfunctions, especially in distant regions, developing countries, and sparsely populated areas.

Rapid lateral flow assays based on the quantification of magnetic nanoparticle labels for multiplexed immunodetection of small molecules: application to the determination of drugs of abuse.

A rapid lateral flow immunoassay is presented that uses carboxyl-modified superparamagnetic nanoparticles as labels that can be quantified by highly sensitive multi-channel electronic readers. The approach is generic in that it is likely to be applicable to numerous small molecules. The method permits both single- and multiplex assays at a point-of-need without sample pretreatment. It is user-friendly and offers attractive characteristics demonstrated here for detection of morphine, fentanyl and methamphetamine in urine. The competitive immunoassay uses commercially available reagents that do not require special permissions. After migration of sample, the lateral flow test strips are subjected to an alternating magnetic field at two frequencies. The response from the nanolabels is readout at a combinatorial frequency from the entire volume of a porous immunochromatographic membrane by the magnetic particle quantification technique. Even trace concentrations can be quantified within ≤20 min with the limits of detection (LOD) of 0.20 ng·mL-1, 0.36 ng·mL-1 and 1.30 ng·mL-1 for morphine, fentanyl and methamphetamine, respectively. The second variant presented here features highly sensitive quantification of haptens (LOD for fentanyl - 0.05 ng·mL-1). This is due to high-affinity trapping of magnetic nanolabels in a universal streptavidin-based test strip, which can be also used for detection of virtually any other small molecule. The third variant is of the multiplexed type and intended for rapid and simultaneous detection of the drugs of abuse in human urine with LODs equal to 0.60 ng·mL-1 and 3.0 ng·mL-1 for morphine and methamphetamine, respectively. In addition to the low LODs, the RSDs did not exceed 7%, 9%, and 11% for methamphetamine, morphine and fentanyl, respectively. Graphical abstract Three variants of small molecule detection in competitive format at a point-of-need. Single-plex variants feature antibody and high-affinity streptavidin test lines, while multiplex variant - several antibody test lines. Magnetic nanolabels are quantified from the whole volume of test strip.

Analytical Platform with Selectable Assay Parameters Based on Three Functions of Magnetic Nanoparticles: Demonstration of Highly Sensitive Rapid Quantitation of Staphylococcal Enterotoxin B in Food.

Many immunoassay platforms require time- and labor-consuming tuning of parameters for operation in complex mediums (food, whole blood, etc.), but no universal method has been proposed to accelerate that "trial-and-error" stage. We present a lateral flow platform, applicable to the multitude of assays comprising immunomagnetic separation, as a tool to establish quantitative relationship between analytical characteristics, sample volume, and magnetic enrichment time. The tool permits a user, prior to the analysis, to knowingly select from a "menu" of parameters' values a particular combination that better suits a purpose. Besides, the platform showed quantitative detection in various food of staphylococcal enterotoxin B (SEB) as a model up to 6 pg/mL at the dynamic range of 3.5 orders with minimal sample pretreatment. Such performance is achieved due to using the same magnetic nanoparticles through all stages of analysis in contrast to the traditional approaches that engage these agents either for separation or as labels. The unique combination of broad benefits of magnetic particles, e.g., rapid enrichment and purification of analyte, reduction of matrix effect, extremely high signal-to-noise ratio, etc., are joined in one platform due to the method of their registration by nonlinear magnetization. The platform also retains the advantages of lateral flow principle such as extraordinary simplicity, on-site operation, affordable consumables, and permits samples of virtually any volume. Although tested here for SEB detection, the platform can be extended to other analytes for point-of-care in vitro diagnostics, food analysis, biosafety, environmental applications, etc.

Ultrasensitive quantitative detection of small molecules with rapid lateral-flow assay based on high-affinity bifunctional ligand and magnetic nanolabels.

An ultrasensitive lateral-flow assay is developed for rapid quantitative detection of small molecules on-site. The conceptual novelty, which transfers lateral-flow assays to the category of highly sensitive quantitative systems, is due to employment of a bifunctional ligand combined with volumetric registration of magnetic nanolabels. The ligand provides extremely high affinity for trapping the nanolabels and, simultaneously, efficiently competes with the analyzed molecules for the limited quantity of antigen-binding sites on the nanolabels. The developed assay has been demonstrated as the first express method for measuring in human serum of free thyroxine (fT4). The limit of detection is 20 fМ or 16 fg/ml at the assay time <30 min with the dynamic range of 3 orders. Besides, we present the results of first characterization of kinetic parameters of interaction between free thyroxine and monoclonal antibody, as well as of competitive relationship between fT4 and fT4-biotin. The proposed universal platform can be used for ultrasensitive detection of small molecules in human in vitro diagnostics, veterinary, biosafety and counter-terrorism, food quality control, environmental monitoring, etc., as well as for search of new, previously undetectable, diagnostic markers in medicine.

Multiplex Biosensing Based on Highly Sensitive Magnetic Nanolabel Quantification: Rapid Detection of Botulinum Neurotoxins A, B, and E in Liquids.

We present a multiplex quantitative lateral flow (LF) assay for simultaneous on-site detection of botulinum neurotoxin (BoNT) types A, B, and E in complex matrixes, which is innovative by virtually no sacrifice in performance while transition from the single-plex assays and by characteristics on the level of laboratory quantitative methods. The novel approach to easy multiplexing is realized via joining an on-demand set of single-plex LF strips, which employ magnetic nanolabels, into a miniature cylinder cartridge that mimics LF strip during all assay stages. The cartridge is read out by an original portable multichannel reader based on the magnetic particle quantification technique. The developed reader offers the unmatched 60 zmol detection limit and 7-order linear dynamic range for volumetric registration of magnetic labels inside a cartridge of several millimeters in diameter regardless of its optical transparency. Each of the test strips, developed here as building blocks for the multiplex assay, can be used "as is" for autonomous quantitative single-plex detection with the same measuring setup, exhibiting the limits of detection (LOD) of 0.22, 0.11, and 0.32 ng/mL for BoNT-A, -B, and -E, respectively. The proposed multiplex assay has demonstrated the remarkably similar LOD values of 0.20, 0.12, 0.35 ng/mL under the same conditions. The multiplex assay performance was successfully validated by BoNT detection in milk and apple and orange juices. The developed methods can be extended to other proteins and used for rapid multianalyte tests for point-of-care in vitro diagnostics, food analysis, biosafety and environmental monitoring, forensics, and security, etc.