In fluorescence‐based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre‐ and post‐photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high‐sensitivity LX‐200 system using photobleached magnetic beads, human interleukin‐8 is detected with a threefold improvement in detection limit and signal‐to‐noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence‐based assays.

Detection of biomarkers at low concentrations is essential for early diagnosis of numerous diseases. In many sensitive assays, the target molecules are tagged using fluorescently labeled probes and captured using magnetic beads. Magnetic beads facilitate washing and separation steps, are well suited for automation, and improve the assay sensitivity. Current devices rely on quantifying the target molecules by detecting the fluorescence signal from individual beads. Thus, to detect low concentrations of target molecules, these devices require sophisticated optical detectors, making them bulky and expensive. Here, we propose a compact fluorescence-based system that simply uses a small permanent magnet with a conic tip to aggregate the magnetic beads, forming a cluster of fluorescently labeled probes whose fluorescence signal is much greater than that of a single bead. Using the magnetically aggregated biosensors to detect human Interleukin-8, we demonstrated a limit of detection of 0.1 ng/l and a 4-log dynamic range performance, which is on par with the most sensitive devices but is achieved without their bulk and cost.

We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a "sandwich" complex.An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam. A synchronous detection enables the removal of background signal from the oscillating target signal without complicated sample preparation. The high sensitivity of the MMB system, combined with the specificity of a sandwich hybridization assay, enables detection of DNA fragments without enzymatic signal amplification. Here, we demonstrate the sensitivity of the assay by directly detecting the EML4-ALK oncogenic translocation sequence spiked in human serum. The calculated limit of detection is 1.4 pM, which is approximately 150 times better than a conventional plate reader. In general, the MMB-assisted SHA can be implemented in many other applications for which enzymatic amplification, such as PCR, is not applicable and where rapid detection of specific nucleic acid targets is required.

Rapid and precise manipulation of magnetic beads on the nano and micro scales is essential in many biosensing applications, such as separating target molecules from background molecules and detecting specific proteins and DNA sequences in plasma. Accurately moving magnetic beads back and forth requires at least two adjustable magnetic field gradients. Unlike permanent magnets, electromagnets are easy to design and can produce strong and adjustable magnetic field gradients without mechanical motion, making them desirable for use in robust and safe medical devices. However, using multiple magnetic field sources to manipulate magnetic beads presents several challenges, including overlapping magnetic fields, added bulk, increased cost, and reduced durability. Here, we provide a thorough analysis, including analytical calculations, numerical simulations, and experimental measurements, of using two electromagnets to manipulate magnetic beads inside a miniature glass cell. We analyze and experimentally demonstrate different aspects of the electromagnets’ design, such as their mutual influence, the advantages and disadvantages of different pole tip geometries, and the correlation between the electromagnets’ positions and the beads’ aggregation during movement. Finally, we have devised a protocol to maximize the magnetic forces acting on magnetic beads in a two-electromagnet setup while minimizing the electromagnets’ size. We used two such electromagnets in a small footprint magnetic modulation biosensing system and detected as little as 13 ng/L of recombinant Zika virus antibodies, which enables detection of Zika IgM antibodies as early as 5 days and as late as 180 days post symptoms onset, significantly extending the number of days that the antibodies are detectable.

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