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.

Spectral-Phase Interferometry Detection of Ochratoxin A via Aptamer-Functionalized Graphene Coated Glass.

In this work, we report a novel method of label-free detection of small molecules based on direct observation of interferometric signal change in graphene-modified glasses. The interferometric sensor chips are fabricated via a conventional wet transfer method of CVD-grown graphene onto the glass coverslips, lowering the device cost and allowing for upscaling the sensor fabrication. For the first time, we report the use of graphene functionalized by the aptamer as the bioreceptor, in conjunction with Spectral-Phase Interferometry (SPI) for detection of ochratoxin A (OTA). In a direct assay with an OTA-specific aptamer, we demonstrated a quick and significant change of the optical signal in response to the maximum tolerable level of OTA concentration. The sensor regeneration is possible in urea solution. The developed platform enables a direct method of kinetic analysis of small molecules using a low-cost optical chip with a graphene-aptamer sensing layer.

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.