Application of high-performance magnetic nanobeads to biological sensing devices
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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.
KeywordsMagnetic nanobeads Biosensor Immunoassay Exosome Liquid biopsy
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, 8, 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.
Development of high-performance magnetic affinity nanobeads
To develop an automated system for affinity purification, we further developed highly functionalized magnetic beads (FG beads) with characteristics superior to those of SG beads. We found that compounds containing carboxyl or thiol groups strongly bind to magnetic iron oxide (ferrite); therefore, we used these compounds as “adapter molecules” for the modification of the ferrite surface. We prepared ferrite particles with diameters of 35–40 nm and coupled them to adapter molecules [20, 21]. The particles were coated with copolymer of styrene and GMA, and then the core beads were further coated by seed polymerization with GMA . We confirmed that the FG beads were polymer microspheres encapsulating several ferrite nanoparticles with a diameter of approximately 200 nm by transmission electron microscopy (Fig. 1, middle). FG beads were further modified by introducing a spacer molecle ethylene glycol diglycidyl ether on the surface of beads (FGNEGDE). FGNEGDE beads exhibited high dispersion in various organic solvents, such as N,N-dimethylformamide, tetrahydrofuran, ethyl acetate, 1,4-dioxane, toluene, and dichloromethane, without the structural destruction of beads. Thus, FG beads have magnetic properties in addition to superior stability in organic solvents compared with SG beads. Using FG beads, we have identified target proteins for several low molecular weight compounds, including drugs [22, 23], toxins [24, 25, 26], metabolites (amino acids or heme) [27, 28, 29], and natural products . Notably, FG beads allowed us to identify the target protein for thalidomide as cereblon, providing insight into the mechanism of action of thalidomide in teratogenicity and its anticancer effect against multiple myeloma [31, 32, 33]. Thus, FG beads could be a powerful tool for the identification of unknown target proteins for several ligands. In addition, we developed an automated system for affinity purification based on magnetic separation using FG beads. This system allows the systematic identification of target proteins by the simultaneous analysis of multiple samples . FG beads and the automated screening equipment are commercially available from Tamagawa Seiki Co.,Ltd. (http://www.magneticnanoparticle.jp/jp/htdocs/index.html).
Development of fluorescence-encapsulated magnetic nanobeads
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, 35, 36, 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.
To prepare fluorescent particles, fluorescent substances are generally immobilized on the surface of polymer particles by covalent or affinity binding, such as biotin–avidin coupling . However, in this case, there were several issues, such as impaired dispersibility of the particles or increased nonspecific adsorption because the surface was covered with fluorescent molecules. Furthermore, it was difficult to functionalize the fluorescent particles because of a decrease in the number of surface functional groups. Ando and Kawaguchi  synthesized fluorescent particles by adding a fluorescent substance to a monomer solution before polymerization. The fluorescent particles were able to immobilize proteins electrostatically, but it was difficult to include functional groups on the surface of particles because the fluorescent substance leaked from the inside of particles in organic, acidic, or alkaline solutions. A method to enclose fluorescent molecules in polymer particles by regulating environmental conditions in solution has recently been evaluated . Some polymer structures exhibit expansion and shrinkage depending on the environmental parameters, such as temperature, pH, light, or solvent exchange . Therefore, we attempted to incorporate fluorescent substances into FG beads by altering the environmental conditions in the solution.
FG beads are tolerant to several organic solvents; however, we found that the FG bead expands or shrinks according to the type of solvent. In particular, the polymer structure of FG beads swells in acetone and returns to its original configuration in water. The fluorescent substances yttrium (Y), cerium (Ce), europium (Eu), and terbium (Tb), which are rare earth elements, each have peculiar fluorescence wavelengths and can control excitation wavelengths by forming chelate complexes. Eu has been used for labeling with nucleic acids or antibodies . We used a fluorescent Eu derivative, tris(4,4,4-trifluoro-1-(2-thienyl)-1,3-butanediono) europium (Eu(TTA)3(TOPO)2), for incorporation into FG beads. Eu(TTA)3(TOPO)2 was mixed with FG beads in an acetone solution for swelling, and the solvent was removed by quick vacuum drying and added to an aqueous solution for shrinking. In this way, we successfully incorporated Eu(TTA)3(TOPO)2 into the polymer layer of FG beads  (Fig. 1, right).
The fluorescent FG beads (FF beads) emitted a strong red-orange color under ultraviolet light; when the beads were collected with a magnet, the fluorescence was drawn to the magnet (Fig. 1, right). FF beads emit fluorescent light at 618 nm with excitation at 340 nm. Additionally, FF beads maintained good dispersibility in aqueous solution. The fluorescence intensity of Eu(TTA)3(TOPO)2 is shifted by changes in conditions, such as the pH and buffer, and is suppressed by the addition of quenchers, such as ethylenediamine tetraacetic acid (EDTA). However, the dispersion of FF beads in micellar solution with various pH values and EDTA did not affect the fluorescence intensity of FF beads . There was little nonspecific adsorption of the protein by FF beads owing to polyGMA on the surface. FF beads contained approximately 4.5 × 105 molecules of Eu(TTA)3(TOPO)2 in a single particle, as determined by inductivity coupled plasma optical emission spectroscopy, and this enabled us to observe a single particle using a normal fluorescence microscope. In addition, FF beads did not cause density extinction, as is typical of fluorescent molecules. These findings indicated that FF beads can be used to detect time-resolved fluorescence with high sensitivity and low background noise. Furthermore, several ligands can be conjugated to the surface of the beads because the surface is not covered with fluorescent molecules. On the basis of these properties, we developed an immunoassay using FF beads.
Development of a novel rapid immunoassay using FF beads
Using this system, we attempted to detect brain natriuretic peptide (BNP) , which is a hormone secreted by the heart and a representative disease marker of heart failure [51, 52]. The concentration of BNP in plasma derived from healthy donors is usually less than 20 pg/mL, BNP concentrations of 20–40 pg/mL are associated with heart disease, and concentrations exceeding 100 pg/mL are associated with heart failure [53, 54]. For the detection of BNP using FF beads, we immobilized an anti-BNP antibody on the plate and another anti-BNP antibody on the surface of the FF beads. All operations for the sandwich immunoassay using antibody-coated FF beads were performed by prompt collection under a magnetic field; thereby, the assay, from the addition of the sample to the measurement of fluorescence, could be completed in approximately 5 min. The detection signal of BNP reached a maximum within 1.5 min by the magnetic collection of FF beads, in a dose-dependent manner (Fig. 2b). In contrast, when the sandwich immunoassay was performed without magnetic collection, a stable BNP signal was not detected within a few minutes. Thus, our prompt sandwich immunoassay using FF beads allowed the detection of BNP at a concertation of 2.0 pg/mL with high fidelity within 5 min .
We also verified the magnetically promoted sandwich immunoassay system for the detection of a 33–34-kDa glycoprotein enzyme, prostate-specific antigen (PSA) , which is a widely used diagnostic biomarker for prostate cancer . Healthy individuals typically have a low concentration of PSA (less than 0.1 ng/mL), and the probability of prostate cancer is about 30% when the PSA concentration is between 4.0 and 10 ng/mL. Therefore, for the clinical examination of prostate cancer, the sensitive detection of PSA at a concentration of 0.1–10 ng/mL is required [56, 57]. Using the magnetically promoted sandwich immunoassay, we detected PSA with 1 min of magnetic collection of FF beads in a dose-dependent manner, and signal saturation was reached within 10 min (Fig. 2c). Furthermore, PSA at a concentration of 0.02 ng/mL was successfully detected within 5 min. These results indicated that the performance of the magnetically promoted sandwich immunoassay system using antibody-coated FF beads was superior to that of conventional immunoassays. Furthermore, with control PSA samples of known concentrations, detection was successful. These results indicate that the magnetic force accelerates specific interactions between antigens and antibodies, and our system could dramatically reduce both the total assay time and the number of steps required for the assay .
Development of a rapid immunostaining method using FF beads
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.
Development of a novel method to quantify exosomes using magnetic nanobeads
Methods for exosome quantification
Analysis of the Brownian motion of particles by laser imaging
Isolated exosomes 1 × 107/mL
Electrical detection of particles passing through nanopores
Fluorescent, chemical luminescent
Enzyme-linked immunosorbent assay
Flow cytometry with microbeads
Exosomes captured by microbeads are labeled with fluorescently labeled antibodies for detection by flow cytometry
Serum 14 μL 
nuclear magnetic resonance detection
Exosomes labeled with magnetic nanoparticles are detected by nuclear magnetic resonance
Nanoparticle capture by microfluidics
Exosomes captured by magnetic nanoparticles are labeled with fluorescently labeled antibodies for detection by fluorescence microscopy
Plasma <50 μL
Exosomal proteins are captured around nanopores and labeled with Au nanoparticles to generate surface plasmon resonance for detection by spectroscopy
Exosomes captured on an optical disc are labeled with FG beads for detection using an optical disc drive by diffraction
Serum 0.39 μL
For the accurate detection of the number of exosomes expressing specific marker proteins, we have developed a novel device to quantify specific exosomes with high accuracy by combining optical disc technology with nanobead (FG bead) technology, named “ExoCounter” . In this system, the entire area of optical discs has concentric nanogrooves 260 nm wide at the top and 160 nm wide at the bottom; these are suitable for binding by a single exosome (50–100 nm in diameter) or a single FG bead (200 nm in diameter). A convex region between grooves was designed with a width of 60 nm to prevent the immobilization of exosomes or FG beads outside the groove (Fig. 3b). In addition, FG beads with a diameter of 200 nm are optimal for detection by a laser spot of an optical disc drive (wavelength of 405 nm) and generate significant modulation of the amplitude of the detection signal compared with background noise. With this system, exosomes are captured on antibody-coated optical discs and then labeled with antibody-conjugated FG beads. The number of exosomes can be measured by detection of the FG bead–exosome complexes on the optical disc using an optical disc drive (Fig. 3c). Furthermore, by use of different sets of antibodies, several exosomes expressing specific antigens can be detected. Thus, the ExoCounter system exhibits the advantages of both particle count methods and labeling methods.
To evaluate the properties of the ExoCounter system, we analyzed exosomes isolated from the colorectal cancer cell line Colo1. Exosomes were detected only when exosome-specific antibodies were used, indicating that the ExoCounter system has high selectivity (Fig. 3d). In addition, with anti-CD9-antibody-coated discs and anti-CD63-antibody-conjugated FG beads, exosomes were detected in a dose-dependent manner by the ExoCounter system. Compared with conventional labeling methods, such as ELISA and flow cytometry, the detection sensitivity based on the limit of detection for the ExoCounter system was 800-fold higher than that for ELISA (ExoTest™) and 420-fold higher than that for the flow cytometry system. Furthermore, the linearity, as calculated by the coefficients of determination for the linear regression (R2), for the ExoCounter system was superior to that for ELISA or flow cytometry. The ExoCounter system allows quantification of exosomes derived from clinical samples that include colorectal cancer, breast cancer, or lung cancer cells using FG beads conjugated to antibodies against exosomal or cancer-related proteins. In particular, CD9/HER2 double-positive exosomes were observed in the sera of patients with colorectal cancer and breast cancer cells to a greater extent than in those with other malignancies.
Since it is difficult to directly quantify exosomes in crude samples, such as sera, by conventional methods, a preenrichment procedure is generally necessary for reliable results. We found that the ExoCounter system can detect the exact number of exosomes in human serum without any enrichment procedures. On the basis of this result, we attempted to detect disease-specific exosomes in the sera of patients with and without cancer obtained from the Biobank Japan Project and the sera of healthy donors obtained from the Tohoku University Tohoku Megabank Organization using the ExoCounter system. The numbers of CD9/HER2 double-positive exosomes were significantly increased in the sera of patients with breast cancer or ovarian cancer compared with other samples, suggesting that HER2-expressing exosomes were specifically released from the lesions of breast or ovarian cancer (Fig. 3e). Despite qualitative studies reporting a higher number of HER2-carrying exosomes in the sera of patients with cancer, our results  provided the first evidence for a specific increase in the number of HER2-positive exosomes in the sera of patients with cancer based on the accurate quantification of exosomes.
In addition to exosomes secreted in cancer patients, other types of specific exosomes are found in patients with various diseases. For instance, exosomes expressing L1 cell adhesion molecule or neural cell adhesion molecule include disease-associated proteins, such as β-amyloid and α-synuclein, and may be related to neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease [82, 83]. In addition, other researchers reported unique surface proteins of exosomes related to autoimmune diseases  or depression . Many studies have also suggested the importance of specific exosomes for physiological analyses or therapeutic applications [86, 87]. We have recently developed an automated pathological diagnosis system for cancer tissues based on an enforced-learning platform . The combination of imaging technology for on-tissue cancer diagnosis and the ExoCounter system for quantitative exosome analyses may allow comprehensive and objective approaches for the precise diagnosis of malignancies. The ExoCounter system is commercially available from JVCKENWOOD Corporation (http://healthcare.jvc.com/exosome/).
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.
This research was supported in part by the Program of Development of Advanced Measurement and Analysis Systems of the Japan Science and Technology Agency. The analysis of clinical serum samples was supported by an institutional research grant from the Tailor-made Medical Treatment Program (BioBank Japan Project) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to M.S.) until March 2015 and supported was then transferred to the Japan Agency for Medical Research and Development (AMED) from April 2015 (grant no. JP17ek0109166, to Y.K.). This study was supported partly by the Japan Science and Technology Agency ERATO Suematsu Gas Biology Project (until March 2015). It was also supported by AMED Core Research for Evolutional Science and Technology (grant no. JP17gm0710010, to Y.K.), the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (KAKENHI) (grant no. 18K06921, to Y.K.; grant no. 17H06112, to H.H.), AMED (grant no. JP18he0902008h0004, to S.S. and H.H.), and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities S1411011 (H.H.).
Compliance with ethical standards
Conflict of interest
M.H. is a full-time employee of Tamagawa Seiki Co. Ltd. M.I. is a full-time employee of JVCKENWOOD Corporation. H.H. is a technical advisor at JVCKENWOOD Corporation. The authors have no other competing financial and nonfinancial interests regarding the content of this article.
- 3.Colombo M, Raposo G, Thery C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30:255–89. https://doi.org/10.1146/annurev-cellbio-101512-122326.CrossRefPubMedGoogle Scholar
- 7.Chen G, Roy I, Yang C, Prasad PN. Nanochemistry and nanomedicine for nanoparticle-based diagnostics and therapy. Chem Rev. 2016;116(5):2826–85. doi: https://doi.org/10.1021/acs.chemrev.5b00148.
- 13.Kabe Y, Suematsu M, Sakamoto S, Hirai M, Koike I, Hishiki T, et al. Development of a highly sensitive device for counting the number of disease-specific exosomes in human sera. Clin Chem. 2018. https://doi.org/10.1373/clinchem.2018.291963.
- 20.Nishio K, Masaike Y, Ikeda M, Narimatsu H, Gokon N, Tsubouchi S, et al. Development of novel magnetic nano-carriers for high-performance affinity purification. Colloids Surf B. 2008;64(2):162–9. doi: https://doi.org/10.1016/j.colsurfb.2008.01.013.
- 40.Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971;8(9):871–4.Google Scholar
- 47.Elmas B, Senel S, Tuncel A. A new thermosensitive fluorescent probe for diol sensing: poly(N-isopropylacrylamide-co-vinylphenylboronic acid)-alizarin red S complex. React Funct Polym. 2007;67(2):87–96. https://doi.org/10.1016/j.reactfunctpolym.2006.09.006.CrossRefGoogle Scholar
- 49.Harma H, Soukka T, Lonnberg S, Paukkunen J, Tarkkinen P, Lovgren T. Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence. Luminescence. 2000;15(6):351–5. https://doi.org/10.1002/1522-7243(200011/12)15:6<351::aid-bio624>3.0.co;2-3.CrossRefPubMedGoogle Scholar
- 50.Hatakeyama M, Mochizuki Y, Kita Y, Kishi H, Nishio K, Sakamoto S, et al. Characterization of a magnetic carrier encapsulating europium and ferrite nanoparticles for biomolecular recognition and imaging. J Magn Magn Mater. 2009;321(10):1364–7. https://doi.org/10.1016/j.jmmm.2009.02.039.CrossRefGoogle Scholar
- 54.Cowie MR, Struthers AD, Wood DA, Coats AJ, Thompson SG, Poole-Wilson PA et al. Value of natriuretic peptides in assessment of patients with possible new heart failure in primary care. Lancet. 1997;350(9088):1349–53. doi: https://doi.org/10.1016/s0140-6736(97)06031-5.
- 56.Finne P, Auvinen A, Maattanen L, Tammela TL, Ruutu M, Juusela H, et al. Diagnostic value of free prostate-specific antigen among men with a prostate-specific antigen level of <3.0 microg per liter. Eur Urol. 2008;54(2):362–70. https://doi.org/10.1016/j.eururo.2007.10.056.CrossRefPubMedGoogle Scholar
- 61.Belov L, Matic KJ, Hallal S, Best OG, Mulligan SP, Christopherson RI. Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples. J Extracell Vesicles. 2016;5:25355. https://doi.org/10.3402/jev.v5.25355.CrossRefPubMedGoogle Scholar
- 67.Marleau AM, Chen CS, Joyce JA, Tullis RH. Exosome removal as a therapeutic adjuvant in cancer. J Transl Med. 2012;10:–134. https://doi.org/10.1186/1479-5876-10-134.
- 69.Tatischeff I, Larquet E, Falcón-Pérez JM, Turpin P-Y, Kruglik SG. Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy. J Extracell Vesicles. 2012;1:19179. https://doi.org/10.3402/jev.v1i0.19179.CrossRefGoogle Scholar
- 71.Maas SL, De Vrij J, Broekman ML. Quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing. J Vis Exp. 2014;(92):e51623. https://doi.org/10.3791/51623.
- 76.Fang S, Tian H, Li X, Jin D, Li X, Kong J, et al. Clinical application of a microfluidic chip for immunocapture and quantification of circulating exosomes to assist breast cancer diagnosis and molecular classification. PLoS One. 2017;12(4):e0175050. https://doi.org/10.1371/journal.pone.0175050.CrossRefPubMedPubMedCentralGoogle Scholar
- 88.Shiota M, Naya M, Yamamoto T, Hishiki T, Tani T, Takahashi H, et al. Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival. Nat Commun. 2018;9(1):1561. https://doi.org/10.1038/s41467-018-03899-1.CrossRefPubMedPubMedCentralGoogle Scholar
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