ARTICLE IN PRESS

Journal of Magnetism and Magnetic Materials 321 (2009) 1686–1688

Contents lists available at ScienceDirect

Journal of Magnetism and Magnetic Materials

0304-88

doi:10.1

� Corr

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journal homepage: www.elsevier.com/locate/jmmm

Application of magnetic nanoparticles in full-automated chemiluminescentenzyme immunoassay

Xiaomao Xie a,�, Noriyuki Ohnishi a, Yuki Takahashi a, Akihiko Kondo b

a Magnabeat Inc., 5-1 Goi-kaigan, Ichihara, Chiba 290-8551, Japanb Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan

a r t i c l e i n f o

Available online 21 February 2009

Keywords:

Magnetic nanoparticle

Full-automation

Chemiluminescent enzyme immunoassay

Thyroid-stimulating hormone

Light emission

53/$ - see front matter & 2009 Elsevier B.V. A

016/j.jmmm.2009.02.115

esponding author. Tel.: 81436 215127; fax: 8

ail address: xiexiaomao@chisso.co.jp (X. Xie).

a b s t r a c t

The magnetic nanoparticles (MNPs) Therma-MaxTM were used as a carrier to develop an automated

sandwich chemiluminescent enzyme immunoassay (CLEIA) to detect thyroid-stimulating hormone

(TSH) in a sensitive and specific way. The Therma-MaxTM particles allow for automation because, unlike

magnetic microspheres, they are completely dispersed in aqueous solution and allow for accurate

automatic handling. Signal intensities detected with MNPs were 8-fold higher than those found with

conventional micron-sized magnetic particles. A reproducibility study suggests that these particles

allow for a stable detection method, as the coefficient of variation (CV) is less than 6% (n ¼ 10).

& 2009 Elsevier B.V. All rights reserved.

0. Introduction

Magnetic particles increasingly attract attention, since theirmagnetic properties enable magnetic particles to be used forbiomagnetic separation and magnetic signal detection [1–3].Immunoassays have become an important tool in clinicaldiagnostics and in fundamental investigations, because of theirsensitivity, specificity, and general applicability. Magnetic parti-cles have been applied increasingly for various immunoassaysthat include colorimetric immunoassays, fluoroimmunoassays,enzyme immunoassays or radioimmunoassay. Magnetic particlesbound with primary or secondary antibodies are often used forseparation and quantification of antigens because of their abilitiesto separate bound and free analyte magnetically. The use ofmagnetically bound antibodies eliminates centrifugation stepsand thus reduces assay time and simplifies operation, while at thesame time increasing efficiency and accuracy of the assay [4–9].

Magnetic particles are typically in the order of microns in size.A larger surface area is expected to show higher specificity for thereaction and shorter reaction times. However, when the size of themagnetic particles becomes less than several hundred nan-ometers, it becomes difficult to magnetically collect the particles.To resolve this dilemma, a magnetic column was used to separatethe nano-sized magnetic particles. Another possible method is tocoat the magnetic particles with thermo-responsive polymers thatreversibly aggregate and disperse through temperature changes[10–15]. We have developed thermo-responsive magnetic nano-particles, the Therma-Max series, and used them successfully to

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1436 23 0381.

separate proteins and cells [11–15]. However, the magneticnanoparticles, Therma-Max, have not been applied in automationbecause a process of changing the temperature is needed toaggregate the nano-sized magnetic particles for magnetic separa-tion. In the present study, we developed a method by which theTherma-Max can be magnetically collected within 1 min byadding a small amount of flocculants at a constant temperature.The Therma-Max has excellent dispersibility in an aqueoussolution thus being easy in liquid handling and suitable forautomation. We used the Therma-Max particles as carriers anddeveloped an automated sandwich chemiluminescent enzymeimmunoassay (CLEIA) for measurement of thyroid-stimulatinghormone (TSH). The objective of the present study is to establish arapid and sensitive fully automated CLEIA system by using thewell-dispersible magnetic nanoparticles (MNPs) as a carrier.

1. Materials and methods

1.1. Preparation of avidin-conjugated MNPs

Solution of MNPs, Therma-Max, was synthesized as follows.A suspension of MNPs coated with oleic acid and sodiumdodecylbenzenesulfonate (ferrofluid) was prepared by using theco-precipitation method [16]. The MNPs in deionized water(25 ml, 2 mg/ml) were covered by thermo-responsive polymersprepared by copolymerization of N-isopropylacrylamide (488 mg,Wako Pure Chemical Indutries, Ltd., Japan) and biotin derivative[N-methacroyl-N0-bioninylpropylenediamine] [17] (16 mg) in thepresence of potassium persulfate (25 mg, Wako Pure ChemicalIndutries, Ltd., Japan) and N,N,N0,N0-tetramethylenediamine

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X. Xie et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1686–1688 1687

(100ml, Wako Pure Chemical Indutries, Ltd., Japan), as the initiatorand the accelerator of the reaction, respectively. After polymer-ization of the mixture at 100 rpm stirring for 6 h at roomtemperature, the MNPs were purified by dialysis. Finally, avidin(Wako Pure Chemical Indutries, Ltd., Japan) was conjugated ontothe surface of the MNPs. The number of avidin moleculesconjugated onto the MNPs was estimated by SDS-PAGE. Theavidin-conjugated magnetic nanoparticle (avidin-MNP) solution,was sterilized with sterile filter unit, Millex-GV, (SLGVJ13 SL,F ¼ 0.22mm, Millipore, USA). Particle size of the avidin-MNPs wasdetermined by a dynamic light-scattering method using a modelof ELS-8000 (Otsuka Electronics Co., Ltd., Japan). The averageparticle size was 95 nm.

Table 1Reproducibility in the measurement of TSH with 80mg Therma-Max magnetic

particles (n ¼ 10).

TSH (mIU/ml) Mean RLU7SD CV (%)

0 545731 5.7

5 136217514 3.8

60 12376673325 2.7

200 26105376316 2.4

Biotin-Ab a: 0.2ml (0.75 mg/ml) and ALP-Ab b: 0.2ml (1.09 mg/ml) were used.

1.2. Procedure of full-automated chemiluminescent enzyme

immunoassay (CLEIA)

A full-automatic liquid handling system with microplatereader, Freedom EVO200 (Tecan Group Ltd., Switzerland) wasemployed for the chemiluminescent enzyme immunoassay inwhich thyroid-stimulating hormone was determined by a sand-wich method using an antibody bound to the avidin-MNPs and anantibody bound to alkaline phosphatase (ALP) (Fig. 1).

Biotinylated anti-TSH-a antibody (Biotin-Aba) (0.2ml) obtainedfrom Asahi Techno Glass by biotinylating a mouse anti-TSH-a(0.75 mg/ml, Leinco Technologies, Inc., USA), alkaline phospha-tase-binding anti-TSH-b antibody (ALP-Abb) (0.2ml) obtained fromAsahi Techno Glass by binding alkaline phosphatase to a mouseanti-TSH-b antibody (1.09 mg/ml, Leinco Technologies, Inc., USA),TBS buffer (12.5ml, 20 mM Tris–HCl, 150 mM NaCl, pH 7.5)and avidin-conjugated magnetic nanoparticles (20ml, 4 mg/ml),Therma-Max, were mixed and a necessary amount thereof wasprepared. The thus prepared mixture was transferred to a 96-wellmicroplate in an amount of 35ml each. To each well of themicroplate, 65ml of TSH solution (Lumipulse TSH-N standard TSHsolution (WHO Standard, 2nd International Standard), FujirebioInc., Japan) was added. The concentration of TSH was 0, 5.0, 60.0 or200.0mIU/ml, followed by mixing by pipetting, and a reaction wasallowed to proceed at 30 1C for 5 min. After completion of thereaction, 30ml of flocculants was added to aggregate the MNPs andmagnetic separation was carried out at 30 1C for 1 min, and thesupernatant was removed. Then, 100ml of Lumipulse washingbuffer (Fujirebio Inc., Japan) was used for removal of non-specificbindings. After washing twice, the magnetic nanoparticles con-jugated with sandwich of antigen and antibodies were added by100ml of a luminescent substrate (Lumigen APS-5, Lumigen Inc.,USA), and the mixture was stirred for 5 s. Then, a reaction wasallowed to proceed for 10 s, and a luminescence intensity wasmeasured for 0.1 s. To compare our magnetic nanoparticles,Therma-Max, with the conventional micron-sized magneticparticles, 15ml (10 mg/ml) Dynabeads Myone Streptavidin andM-280 Streptavidin (Dynal Biotech, Norway) were used for thepresent experiment.

Analyte Substrate

Measurement of chemiluminescent

light emission

Avidin-MNP Biotin-Ab α Antigen ALP-Ab β

Fig. 1. Schematic diagram illustrating CLEIA with magnetic nanoparticles.

2. Results and discussion

A standard series with the TSH concentrations of 0, 5.0, 60.0and 200.0mIU/ml was measured. As shown in Table 1, the lightemission increased with increasing concentrations, indicating thatthe magnetic nanoparticles are specifically available for CLEIA andthis method is sensitive.

Analytical reproducibility was evaluated by comparing thelight emission to the standards. Statistics were calculated from theresults of 10 replicate measurements obtained in 10 different runs(see Table 1). The coefficient of variation was less than 6% for allconcentrations within the working range, suggesting that thisCLEIA with the magnetic Therma-Max nanoparticles is a stablemethod. The high precision in this CLEIA with the Therma-Max isprobably attributable to the good dispersibility of the magneticnanoparticles which allowed an accurate liquid handling byautomation.

A standard series was diluted for measuring the lowerconcentrations of TSH. Although the TSH concentration was 100-fold diluted to 0.05mIU/ml a linear correlation still remainedbetween the light emission and the concentration of TSH(y ¼ 2178.5x+400.56, R2

¼ 0.9983). These findings suggest a lownon-specific binding to the magnetic nanoparticles, Therma-Max,and therefore, a possibly broader measurement range.

The comparison of conventional micron-sized magnetic parti-cles to Therma-Max MNPs is shown in Fig. 2. The light emissionwith magnetic nanoparticles, Therma-Max, was much higher thanthat with micron-sized magnetic particles although amounts ofthe micro-magnetic particles used for the CLEIA are almost doubleof Therma-Max. At a TSH concentration of 200mIU/ml, the lightemission for both magnetic particles with diameter of 1 and2.8mm is about 50,000 relative luminescence units (RLU),however, the light emission for magnetic nanoparticles, Therma-

Fig. 2. Dose-response of the CLEIA with magnetic nanoparticles for a wide range of

TSH concentrations and a sensitivity comparison between 80mg of Therma-Max

and 150mg of micron-sized magnetic particles. Results are expressed as mean7SD,

n ¼ 3.

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Fig. 3. Kinetics of light emission in assays of TSH standards with Therma-Max

magnetic particles. Relative light emissions (RLU) were measured for 0 (K), 5 (’),

60 (m) and 200 (&)mIU/ml TSH. Results are expressed as mean7SD, n ¼ 3.

X. Xie et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1686–16881688

Max, is more than 2,00,000. The amounts of Therma-Max usedwas although only half of micron-sized magnetic particles, thesignal of light emission was 4 times higher, suggesting that thesensitivity of magnetic nanoparticles, Therma-Max, is 8 timeshigher than that of the micron-sized magnetic particles.

The higher sensitivity for the magnetic nanoparticles, Therma-Max, would benefit from the small size and the good dispersity ofthe magnetic nanoparticles. The small size resulted in rapiddiffusion/mixing in the CLEIA reaction system, hence, themagnetic nanoparticles conjugated with ligand could rapidlyrecognize and capture the target molecule, thus enhancing theeffects of the immunoreaction. Time required for an assay is shortdue to a fast requirement for the results of assay in moderndiagnostics. The CLEIA with Therma-Max as a magnetic carrierreaches the needs of rapid results of assay because lowchemiluminescent signals can be detected after even shorterperiods of enzyme reaction, and the magnetic separation of boundand free antigen can be carried out in a short time. In the presentstudy, the immunoreaction time was set to 5 min. According toour experimental data the rate of the immunoreaction almostreached its equilibrium in 5 min when using the magneticnanoparticles, Therma-Max, for CLEIA (Fig. 3). Only a smallfurther increase in signal was observed over the next 25 min.The immunoreaction seemed to not have reached equilibrium in5 min with both micron-sized magnetic particles while it did withthe magnetic nanoparticles. Nishizono et al. [5] have earliercompared 6mm polystyrene beads with ferrite-coated 300 nmparticles used for CLEIA to measure tumor markers and found thatthe ferrite-particle system reached the maximum signal in a muchshorter time than the bead system. The magnetic nanoparticles,Therma-Max, reach the equilibrium faster than the micron-sizedmagnetic particles in the immunoreaction system because of theincrease in the number of immobilized antibodies available forimmunoreaction to be bound to the surface of the solid support.Furthermore, it is reasonably considered, from their less than100 nm particle size, where the magnetic nanoparticles may obeyBrownian motion, as colloids do. The Brownian motion would

enhance the binding kinetics between the antibodies conjugatedon the magnetic nanoparticle surface and the antigens in thesolution phase, supporting a tendency to reach the maximumbinding faster [5].

Additional advantage using the magnetic nanoparticles, Ther-ma-Max, for CLEIA is convenience in liquid handling. It is knownthat magnetic particles sediment when their size becomes morethan 1mm, such as Dynabeads. Unlike the micron-sized magneticparticles, the magnetic Therma-Max MNPs disperse well in anaqueous solution because of their hydrophilic polymer surfacecoating and the possible Brownian motion. This guaranteesaccuracy in liquid handling by both manual and automatedpipetting. This advantage makes it possible that the magneticTherma-Max MNPs can be employed in an automated rapid andsensitive CLEIA.

3. Conclusions

Therma-Max MNPs with a diameter of less than 100 nmdisperse well in an aqueous solution and can be magneticallycollected by adding a small amount of flocculants at a constanttemperature. The MNPs were used to develop an automatedsandwich CLEIA for measurement of TSH. The MNPs proved to begood magnetic carriers for CLEIA in terms of high sensitivity,specificity, and reproducibility. Due to their good dispersibility inan aqueous solution, an accurate liquid handling allows the MNPsto be easily used in automated devices.

Acknowledgements

We thank Dr. Hiromasa Nagao (GeneWold Ltd.,) for histechnical advice and Mr. Koji Kikumiya (Tecan Japan Co., Ltd.,)for his help with the automation program.

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