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.

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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.

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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.

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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.