Date Published: January 9, 2019
Publisher: Springer Berlin Heidelberg
Author(s): Yasuaki Kabe, Satoshi Sakamoto, Mamoru Hatakeyama, Yuki Yamaguchi, Makoto Suematsu, Makoto Itonaga, Hiroshi Handa.
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.
Magnetic materials have been used as magnetic storage systems, sensors, and shielding systems. Ferrite particles are frequently used as magnetic materials in the life sciences owing to their biocompatibility . For instance, ferrite particles have been used as carriers for hyperthermia treatment and as MRI contrast agents coated with biocompatible materials, such as dextran . Furthermore, they are widely used for the separation of bioactive substances (e.g., proteins, enzymes, and nucleic acids), blood screening, cell separation, and immunoassays [3, 4]. For these applications, magnetic materials should be subjected to coating and conjugation with appropriate materials on the surfaces of particles [5, 6]. With use of functionalized magnetic particles, a broad range of biosensing systems (e.g., immunoassays) for bioimaging or drug delivery have been developed [7–9]. Several types of magnetic particles have been developed for various biotechnological applications [10, 11]. For effective biosensor application, low nonspecific adsorptivity, high dispersibility, and high stability of magnetic particles are most important. We have focused on the development of high-performance nanoparticles for analyses in chemical biology, such as drug receptor protein screening . Furthermore, we have recently demonstrated their applications as biosensors, for example, in novel immunofluorescent assays or exosome detection systems, utilizing the excellent properties of our magnetic nanobeads [13, 14]. This review describes the preparation and modification of nanobeads and their application in immunoassays.
Various types of chromatography, such as ion-exchange or gel-filtration chromatography, have been used for protein purification. Affinity chromatography is an effective technique, but efficient purification is limited by the nonspecific binding of proteins, insufficient chemical stability, and physical properties, such as dispersibility. We initially developed affinity latex beads (SG beads) as a carrier for affinity chromatography . As shown in Fig. 1 (left), SG beads have polystyrene as a core and poly (glycidyl methacrylate) (polyGMA) on the surface, and their particle diameter is approximately 200 nm. SG beads have several beneficial characteristics, including (1) low nonspecific adsorption owing to their moderately hydrophilic and nonporous surface, (2) high dispersibility and mobility in various solvents owing to their stability, and (3) the potential to covalently immobilize several ligands, such as proteins, nucleic acids, or low molecular weight compounds, via epoxy groups derived from polyGMA on the surface. Therefore, SG beads allow the efficient purification of target proteins from crude extracts, such as tissues or cell lysates, with high recovery and purity. These excellent properties of SG beads have been used to successfully purify and identify several target proteins, such as transcription factors targeting specific DNA sequences and drug receptors [15–19].Fig. 1Construction 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
In addition to their use for affinity purification, we applied FG beads in the development of novel biosensors. Biomarkers in biological fluids provide important information regarding disease progression and prognosis [34–37]. Various types of bioassays, including immunoassays, are used for measurement of biomarkers [38, 39], but these assays are often time-consuming and insufficient to obtain reliable results. For instance, although enzyme-linked immunosorbent assay (ELISA) is widely used as a standard diagnostic test, the completion of the reaction takes a long time [40, 41]. Various approaches have been evaluated to resolve these intrinsic issues. In the biomedical research field, appropriate functional materials or particles allow efficient diagnosis and treatment . Magnetic particles are frequently used as efficient and sensitive functional materials [43, 44]. We developed a novel type of fluorescence-encapsulated magnetic nanobead for immunoassays.
In conventional immunoassays, several processes are time-consuming, such as the antigen–antibody reaction or signal amplification step by an enzymatic reaction. When antibody-labeled fluorescent magnetic beads are collected or concentrated magnetically on an antigen-coated plate, the antigen–antibody reaction will be accelerated. Furthermore, the target antigen can be detected by the direct measurement of the fluorescence of beads. Therefore, we expected FF beads to be suitable for a rapid fluorescence-based immunoassay. Accordingly, using FF beads, we evaluated a sandwich immunoassay system with magnetic collection. Figure 2a illustrates the schemes for conventional sandwich immunoassay systems using enzyme-modified antibodies and a method for introducing magnetic collection using antibody-labeled FF beads. Antigens were immobilized on the antibody-coated plate, and the antibody-labeled FF beads were allowed to react and collected under a magnetic field. Thus, the fluorescence of the FF beads bound to the antigen on the plate can be directly detected .Fig. 2Development 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)
We further applied the magnetically promoted immunoreaction system to the analysis of pathological tissue sections . For tissue diagnosis, immunostaining is frequently used for the detection of pathological regions, such as tumors; however, conventional immunostaining often requires several hours for a definitive diagnosis. We performed an immunostaining assay of cancer cell lines using FF beads immobilized with an antibody against epidermal growth factor receptor (EGFR), which is a cancer marker involved in cancer cell survival and proliferation [58, 59]. As illustrated schematically in Fig, 2d, anti-EGFR-antibody-coated FF beads were added to fixed cancer cell samples. In this step, a permanent magnet was attached beneath the fixed samples to promote the immunoreaction between antigens expressed on the surface of carcinoma cells and antibodies immobilized on FF beads. After the magnet was removed, the samples were washed to remove unbound anti-EGFR-antibody-coated FF beads and were directly observed by fluorescence microscopy. When the immunostaining assay was performed under a magnetic field, high EGFR expression was observed as red fluorescence by FF beads in the epidermoid cancer cell line A341, which is known to express high levels of EGFR (Fig. 2e). This assay was completed within 15 min. In contrast, red fluorescence derived from FF beads was minimal in the lung cancer cell line H69, which is known to express low levels of EGFR. Thus, our rapid immunostaining system using FF beads allows the selective detection of target proteins on cultured cells or tissues, such as pathological sections, with short time requirements.
In addition to the development of immunoassays using FF beads, we recently developed a novel system for quantifying the absolute number of exosomes by integrating the magnetic nanobead technology with the optical disc system. Exosomes are cell-secreted membranous vesicles of around 100-nm diameter [3, 60]. When multivesicular bodies in cells fuse to the plasma membrane, the cells release exosomes into the extracellular space  (Fig. 3a). Exosomes contain proteins or genetic material such as messenger RNA or microRNA inside a vesicle . Exosomes are present in body fluids, such as blood, urine, saliva, breast milk, semen, ascites fluid, and cerebrospinal fluid, and contribute to intercellular communication by carrying proteins  and genetic material through the circulation from parent cells to recipient cells . Through intercellular communication, exosomes play important roles in regulating physiological processes and mediating the systemic dissemination of various types of cancer . On the surface, exosomes ubiquitously express membrane proteins, such as tetraspanin, CD9, CD63, and CD81, called “exosomal proteins.” Furthermore, various specific surface proteins from the parent cell are expressed in exosomes, for instance, CD147 from colorectal cancer cells [65, 66], human epidermal growth factor receptor 2 (HER2) from breast cancer cells , and CD91 from lung cancer cells , suggesting that exosomes containing specific antigens could be disease biomarkers. Therefore, the detection of exosomes has important practical applications.Fig. 3Development 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 , with permission from the American Association for Clinical Chemistry) In conclusion, by applying our magnetic nanobead technology, we successfully developed a rapid immunoassay system and a quantification system for the absolute number of exosomes, beginning with affinity purification. The development of these systems was possible because of the excellent properties of the beads, such as their strong paramagnetism, physical stability, low nonspecific adsorption, and uniform dispersibility with a nanosized diameter. Further analyses are expected to lead to the development of novel diagnostic systems based on our high-performance magnetic nanobeads. Source: http://doi.org/10.1007/s00216-018-1548-y