Application of high-performance magnetic nanobeads to biological sensing devices

Abstract

Nanomaterials have extensive applications in the life sciences and in clinical diagnosis. We have developed magnetic nanoparticles with high dispersibility and extremely low nonspecific binding to biomolecules and have demonstrated their application in chemical biology (e.g., for the screening of drug receptor proteins). Recently, the excellent properties of nanobeads have made possible the development of novel rapid immunoassay systems and high-precision technologies for exosome detection. For immunoassays, we developed a technology to encapsulate a fluorescent substance in magnetic nanobeads. The fluorescent nanobeads allow the rapid detection of a specific antigen in solution or in tissue specimens. Exosomes, which are released into the blood, are expected to become markers for several diseases, including cancer, but techniques for measuring the absolute quantity of exosomes in biological fluids are lacking. By integrating magnetic nanobead technology with an optical disc system, we developed a novel method for precisely quantifying exosomes in human serum with high sensitivity and high linearity without requiring enrichment procedures. This review focuses on the properties of our magnetic nanobeads, the development of novel biosensors using these nanobeads, and their broad practical applications. Graphical abstract ᅟ.

Keywords: Biosensor; Exosome; Immunoassay; Liquid biopsy; Magnetic nanobeads.

Graphical abstract

Fig. 1

Construction of high-performance nanobeads. Flowchart of the construction of functionalized nanoparticles. SG beads are prepared by polymerization with styrene and glycidyl methacrylate (GMA) (left). FG beads are prepared with surface-modified ferrite particles, styrene, and GMA (middle). After ferrite particles have been covered by polymerization with styrene and GMA, the polymer-coated ferrite particles are further coated with GMA. Fluorescent FG beads (FF beads) are prepared from FG beads by incorporation with fluorescent molecules (europium complexes) in an organic solvent (right). Transmission electron microscopy images of SG beads and FG beads are shown. FF beads emit red fluorescence under ultraviolet light in solution. By the magnetic collection of FF beads, red fluorescence is collected at the bottom of the vial

Fig. 2

Development of a rapid immunoassay system using magnetic nanobeads. a A standard sandwich immunoassay (top) and a magnetically promoted sandwich immunoassay using antibody-conjugated fluorescent FG beads (FF beads) (bottom). b Detection of brain natriuretic peptide (BNP) by the magnetically promoted sandwich immunoassay using anti-BNP-conjugated FF beads. The graph shows the fluorescence intensity for the detection signal in the presence of BNP at concentrations of 0, 2.0, 20, and 200 pg/mL at the indicated time. All data are presented as the mean ± the standard deviation (n = 4). c Detection of prostate-specific antigen (PSA) by the magnetically promoted sandwich immunoassay using anti-PSA-conjugated FF beads. The graph shows the fluorescence intensity for the detection signal in the presence of PSA at concentrations of 0, 0.020, 0.064, 0.20, 0.64, 2.0, and 6.3 ng/mL at the indicated time. All data are presented as the mean ± the standard deviation (n = 4). d Schemes for standard immunostaining (top) and magnetically prompted immunostaining of cancer cells using antibody-conjugated FF beads (bottom). e The magnetically promoted immunostaining of cancer cells using anti-epidermal growth factor receptor (EGFR)-antibody-conjugated FF beads. Left: Immunostaining of A431 cells (human epidermoid cancer cells, high EGFR expression). Right: Immunostaining of H69 cells (small-cell lung cancer cells, low EGFR expression)

Fig. 2

Development of a rapid immunoassay system using magnetic nanobeads. a A standard sandwich immunoassay (top) and a magnetically promoted sandwich immunoassay using antibody-conjugated fluorescent FG beads (FF beads) (bottom). b Detection of brain natriuretic peptide (BNP) by the magnetically promoted sandwich immunoassay using anti-BNP-conjugated FF beads. The graph shows the fluorescence intensity for the detection signal in the presence of BNP at concentrations of 0, 2.0, 20, and 200 pg/mL at the indicated time. All data are presented as the mean ± the standard deviation (n = 4). c Detection of prostate-specific antigen (PSA) by the magnetically promoted sandwich immunoassay using anti-PSA-conjugated FF beads. The graph shows the fluorescence intensity for the detection signal in the presence of PSA at concentrations of 0, 0.020, 0.064, 0.20, 0.64, 2.0, and 6.3 ng/mL at the indicated time. All data are presented as the mean ± the standard deviation (n = 4). d Schemes for standard immunostaining (top) and magnetically prompted immunostaining of cancer cells using antibody-conjugated FF beads (bottom). e The magnetically promoted immunostaining of cancer cells using anti-epidermal growth factor receptor (EGFR)-antibody-conjugated FF beads. Left: Immunostaining of A431 cells (human epidermoid cancer cells, high EGFR expression). Right: Immunostaining of H69 cells (small-cell lung cancer cells, low EGFR expression)

Fig. 3

Development of a novel counting system using magnetic nanobeads. Overview of exosomes and the ExoCounter system. a Overview of exosomes. When a multivesicular body (MVB) in the cell is fused to the plasma membrane, exosomes are secreted from the cell, delivering genetic material, such as RNAs or proteins, in the membrane to recipient cells. b Illustration of exosomes labeled with nanobeads on an optical disc using the ExoCounter system. Each exosome is isolated in the groove of an optical disc coated with a specific antibody (Ab) for exosomes and covered with an antibody-conjugated single magnetic nanobead (FG bead) that contains ferrite particles. The optical disc has periodic grooves of 260 nm (width) at the top and 160 nm at the bottom, which is suitable for the binding of a single exosome (50–150 nm) or FG bead (200 nm). The width of the convex region was 60 nm to prevent the immobilization of exosomes and FG beads outside the groove. c Optical disc drive of the ExoCounter system. The captured FG beads on the disc are detected by an optical pickup composed of a laser diode and a photodetector. The detection pulses are transferred to the pulse counter circuit to quantify the number of exosomes. d Comparison of Colo1 exosome quantification using the ExoCounter system, enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FCM). e Serum samples were incubated on discs coated with anti-CD9 antibody or a control antibody, then treated with FG beads conjugated to an anti-human epidermal growth factor receptor 2 antibody, and analyzed with the ExoCounter system. Serum samples (12.5 μL) from healthy donors or patients with noncancer diseases (glaucoma or interstitial lung disease/pulmonary fibrosis) or cancer (colorectal, lung, breast, or ovarian cancer) were used in the assay. The data are presented in box plots that represent the first quartile (25%), median (50%), and third quartile (75%). The averages for each group are presented below the graph. Data were analyzed statistically by ANOVA with the Tukey–Kramer test. One asterisk p < 0.05, two asterisks p < 0.01. FITC fluorescein isothiocyanate. (b–e Reproduced from [13], with permission from the American Association for Clinical Chemistry)

Fig. 3

Development of a novel counting system using magnetic nanobeads. Overview of exosomes and the ExoCounter system. a Overview of exosomes. When a multivesicular body (MVB) in the cell is fused to the plasma membrane, exosomes are secreted from the cell, delivering genetic material, such as RNAs or proteins, in the membrane to recipient cells. b Illustration of exosomes labeled with nanobeads on an optical disc using the ExoCounter system. Each exosome is isolated in the groove of an optical disc coated with a specific antibody (Ab) for exosomes and covered with an antibody-conjugated single magnetic nanobead (FG bead) that contains ferrite particles. The optical disc has periodic grooves of 260 nm (width) at the top and 160 nm at the bottom, which is suitable for the binding of a single exosome (50–150 nm) or FG bead (200 nm). The width of the convex region was 60 nm to prevent the immobilization of exosomes and FG beads outside the groove. c Optical disc drive of the ExoCounter system. The captured FG beads on the disc are detected by an optical pickup composed of a laser diode and a photodetector. The detection pulses are transferred to the pulse counter circuit to quantify the number of exosomes. d Comparison of Colo1 exosome quantification using the ExoCounter system, enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FCM). e Serum samples were incubated on discs coated with anti-CD9 antibody or a control antibody, then treated with FG beads conjugated to an anti-human epidermal growth factor receptor 2 antibody, and analyzed with the ExoCounter system. Serum samples (12.5 μL) from healthy donors or patients with noncancer diseases (glaucoma or interstitial lung disease/pulmonary fibrosis) or cancer (colorectal, lung, breast, or ovarian cancer) were used in the assay. The data are presented in box plots that represent the first quartile (25%), median (50%), and third quartile (75%). The averages for each group are presented below the graph. Data were analyzed statistically by ANOVA with the Tukey–Kramer test. One asterisk p < 0.05, two asterisks p < 0.01. FITC fluorescein isothiocyanate. (b–e Reproduced from [13], with permission from the American Association for Clinical Chemistry)