Improving the Sensitivity of Fluorescence‐Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads

Abstract

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.

Rare Earth‐Free Luminescent Materials for WLEDs: Recent Progress and Perspectives

“…However, compared with rare earth luminescent materials, perovskites still have poor stability. [ 157 ] Therefore, in order to broaden the applicability of these materials, it is still necessary to continue developing perovskite modification and packaging technology.…”

“…However, compared with rare earth luminescent materials, perovskites still have poor stability. [157] Therefore, in order to broaden the applicability of these materials, it is still necessary to continue developing perovskite modification and packaging technology. Additionally, many rare earth-free white light-emitting materials have much shorter excited-state lifetimes compared with rare-earth fluorescent materials, which gives them advantages in the field of optical communications.…”

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay

“…After the hybridization, 10 5 of streptavidin‐coupled magnetic beads (M280 StreptAvidin, Invitrogen) were added to each reaction mix and incubated for one hour on a rotator (40 RPM) at room temperature. To reduce background noise caused by autofluorescence of the streptavidin‐coupled magnetic beads, the beads were photobleached for 18 hours prior to their use in the assay . Assuming homogenous conjugation of the sandwich molecules to the magnetic beads, the concentration of target molecules per bead in the assay ranged from 10 2 to 10 8 .…”

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

“…To evaluate the MMB-based assay's ability to detect the S1-ACE2 interaction and to determine its LoD, dynamic range, and analytical sensitivity, we coated tosylactivated magnetic beads (0.5 mg, Dynabeads M-280, 14203, Thermo Fisher Scientific, Waltham, MA, USA) with anti His antibodies (10 µg, 70796-3, Novagen, Madison, WI, USA) according to the manufacturer's protocol, and photobleached them for~20 h [27]. The conjugated magnetic beads (~1.2 • 10 6 beads) were then mixed with a recombinant S1 protein (1.2 µg, S1N-C52H3 (His tag), Acro Biosystems, Newark, CA, USA) and incubated overnight at 4 • C. The conjugated beads were then washed by placing the samples on a MagJET separation rack (MR02, Thermo Fisher Scientific) for 2 min, taking out the solution, and pipetting the beads with 1 mL of a Tris buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1% w/v BSA, 0.05% v/v Tween-20).…”