ARTICLE IN PRESS

Journal of Magnetism and Magnetic Materials 311 (2007) 421424

www.elsevier.com/locate/jmmm

Biotinavidin amplied magnetic immunoassay for hepatitis B surface

antigen detection using GoldMag nanoparticles
An Yua, Tingting Genga, Qiang Fua, Chao Chena,b,, Yali Cuia,b,
a
Biochip Research and Development Center, Northwest University (National Engineering Research Center for Miniaturized Detection Systems),
Xian 710069, PR China
b
Shaanxi Lifegen Ltd. Co., Xian 710069, PR China
Available online 8 December 2006

Abstract

Using GoldMag Fe3 O4 =Au nanoparticles as a carrier, a biotinavidin amplied ELISA was developed to detect hepatitis B surface
antigen (HBsAg). A specic antibody was labeled with biotin and then used to detect the antigen with an antibody coated on GoldMag
nanoparticles by a sandwich ELISA assay. The results showed that 5 mol of biotin were surface bound per mole of antibody. The
biotinavidin amplied ELISA assay has a higher sensitivity than that of the direct ELISA assay. There is 5-fold difference between
HBsAg positive and negative serum even at dilution of 1:10000, and the relative standard deviation of the parallel positive serum at
dilution of 1:4000 is 5.98% n 11.
r 2006 Elsevier B.V. All rights reserved.

Keywords: GoldMag nanoparticles; Biotinavidin amplication; Sandwich; Immunoassay; HBsAg

GoldMag Fe3 O4 =Au nanoparticles are a new type of
composite particles, which have a typical core/shell
structure with Fe oxide as the core and a layer of Au
deposited on the core surface as the shell [13]. The
nanoparticles have the advantages of convenient binding to
biomolecules and easy separation using a magnetic eld.
We have reported that GoldMag nanoparticles have a
higher immobilization capacity for antibody (Ab) than
Dynabeadss [4]. Hepatitis B surface antigen (HBsAg) is
the primary diagnostic marker used for screening blood in
hospitals and health-care facilities [5]. Herein, using
biotinavidin as an amplifying system and GoldMag
nanoparticles with average diameter of 50 nm as the carrier
(TEM imageFig. 1), a sensitive sandwich method to
detect the HBsAg was established.
Corresponding author. Biochip Research and Development Center,
Northwest University (National Engineering Research Center for Miniatur-
ized Detection Systems), Xian 710069, PR China. Tel.: +86 29 8830 2427;
fax: +86 29 8830 3551.
Also corresponding author.
E-mail addresses: chaochen@nwu.edu.cn (C. Chen), yali_cui@mail.
com (Y. Cui).
Biotin can be conjugated with protein via primary
amines. Avidin is a glycoprotein that contains four
identical binding sites for biotin with extraordinarily high
afnity 1:3  1015 M1 [6]. The biotinavidin system is
widely used in specic targeting and assay design because
of the amplication effect. 4-hydroxyazobenzene-2-car-
boxylic acid (HABA) dye has a characteristic absorption
peak at 350 nm in solution and exhibits a characteristic
absorption band at 500 nm after binding with avidin. The
afnity of avidin and HABA 6  106 M1 is much less
than that of avidin and biotin, the addition of biotin or
biotinylated biomolecules results in displacement of HABA
from the binding sites. Therefore, the change of absorbance
at 500 nm can be used to measure the degree of
biotinylation of biomolecules when the biotinylated bio-
molecules is added to the avidinHABA complex [7]. The
biotinylated Ab was prepared as follows: NHS-biotin was
dissolved in dimethyl sulphoxide (DMSO) at a concentra-
tion of 1 mg/mL as stock solution. The Ab was dialyzed
against NaHCO3 buffer (0.1 M, pH 8.3) for 18 h at 4  C[8]
in order to remove sodium azide or other substances with
free amino groups. Then a given volume of NHS-biotin
solution was added to the Ab solution and the mixture

0304-8853/$ - see front matter r 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jmmm.2006.11.175
 ARTICLE IN PRESS
422 A. Yu et al. / Journal of Magnetism and Magnetic Materials 311 (2007) 421424
Fig. 1. Transmission electron microscopy of the GoldMag nanoparticles.
2.0
1.9
Absorbance @ 450 nm (O.D.)
1.8
1.7
1.6
1.5
1.4
1.3
10 12 14 16 18
NHS-Biotin (g)
Fig. 2. The relation between activity of the biotinylated Ab and the added
biotin.
shaken for 4 h at 37  C. After reaction, the biotinylated Ab
was dialyzed against PBS buffer (pH 7.4) for another 18 h
at 4  C to remove excess NHS-biotin and the obtained
biotinylated Ab was stored at 4  C for late use. The most
important step for this biotinylation process is to determine
the optimal amount of biotin in the reaction system.
Decient NHS-biotin leads to low efciency of biotinyla-
tion and excess NHS-biotin blocks out the antigen binding
sites on the antibody surface [9]. 10, 12, 14, 16, 18, and
20 mg of NHS-biotin was added to 100 mg of Ab solution,
respectively, and reacted for 4 h, the different biotinylated
Ab was dialyzed against PBS and the activity of the
samples was tested by the sandwich ELISA using GoldMag
nanoparticles (200 ng coating Ab, 500 ng biotinylated Ab,
horseradish peraxidase labeled avidin diluted 1:300). The
0.75
Absorbance @ 500 nm (O.D.)
0.70
0.65
0.60
0.55
0.50
0.0 0.5 1.0 1.5 2.0
D-biotin(g/L)
Fig. 3. Standard curve for the determination of biotin.
results in Fig. 2 indicate that 16 mg of NHS-biotin was the
optimum amount in biotinylation of 100 mg Ab.
The extent of biotinylation of Ab was quantitatively
analyzed by HABA dye assay. Avidin solution was prepared
in 0.05 M sodium phosphate buffer containing 0.15 M NaCl
(pH 6) at a concentration of 1 mg/mL, HABA was
dissolved in 10 mM NaOH at a concentration of 0.1 mg/
mL and 0.1, 0.4, 0.8, 1.2, 1.6 and 2:0 mg=mL of D-biotin
standard solution were prepared. 10 mL each of D-biotin
solution were added to the HABAavidin complex (400 mL
avidin solution plus 60 mL HABA solution), mixed well, and
the absorbance measured at 500 nm. The absorbance
decreases with increasing amount of biotin, as shown in
the standard curve in Fig. 3 R2 0:9991. This curve was
then used to determine the amount of biotin on the surface of
Ab after adding 10 mL of the biotinylated Ab to the
HABAavidin complex and measuring the absorbance at
500 nm. The relative standard deviation (RSD) for four
20 repeats was 2.3%. The difference between the absorbance of
avidinHABA and the average value of sample was 0.0238
A500 avidin2HABA  A500 biotin sample reaction mixture , this value
was used to calculate the degree of biotinylation of Ab using
the following equation:
Moles Biotin/Mole Biotinylated Ab
A500 avidin2HABA  A500 biotin sample reaction mixture  A0 =k
A0 0:0138 is the absorbance of background caused by
PBS; k is the slope of the standard curve (herein is 0:124).
The calculation result indicate that 5 mol biotin were
bound per mole of Ab surface.
Another specic Ab was immobilized on GoldMag nano
particle surface and used as carrier to determine the
HBsAg. 10 mg Ab dissolved in 600 mL Tris-HCl buffer
(0.02 M, pH 7.4) was added to 1 mg of GoldMag
nanoparticles. The mixture was incubated at 37  C for
30 min. The GoldMag nanoparticles coupled with Ab were
magnetically separated from the free Ab and rinsed 3 times
with PBST. The Ab coupled GoldMag nanoparticles were
 ARTICLE IN PRESS
A. Yu et al. / Journal of Magnetism and Magnetic Materials 311 (2007) 421424
0.5
0.4
1 Pre-coupled IgG
2 Post-coupled IgG
0.3
Absorbance
3 Washing
0.2 1 1.2
0.1 2 0.8
3 0.6
0.0
240 260 280 300 320 340 360 380 400
Wavelength (nm)
Fig. 4. UV spectra of pre- and post-coupled Ab solutions and the washing
solution.
blocked by bovine albumin at 37  C for 30 min and were
washed 5 times with PBST. Finally, the Ab coupled
GoldMag nanoparticles were re-suspended in 560 mL PBS
and ready to use. The absorbance of the pre- and post-
coupled Ab solution at 280 nm was measured by spectro-
photometer (Fig. 4) and the result indicates that there is a
signicant change of absorbance of Ab solution before and
after coupling. The amount of the Ab coated onto GoldMag
nanoparticles was calculated using the following equation:
OD280 pre  D1  OD280 post  D2
 100%  adding Abmg,
OD280 pre  D1
where D1 and D2 are the dilution factors of pre- and post-
coupling antibody solutions, respectively. The result
showed that 10 mg Ab had been almost coated on 1 mg of
GoldMag nanoparticles for the next sandwich ELISA.
In the sandwich ELISA method, the combination of two
antibodies recognizing different epitopes of the antigen
(Ag) increases both the sensitivity and the specicity of the
assay [10]. For this biotinavidin amplied magnetic
ELISA system, a specic Ab labeled with biotin and
another Ab coated on GoldMag nanoparticles were used to
capture HBsAg to form a AbAgAbbiotin complex and
to determine HBsAg. 10 mL of GoldMag nanoparticles
coupled with 200 ng Ab in each tube were used in the
sandwich ELISA. Serum from patients with HBV infection
was diluted with calf serum and added to each tube. The
mixture was incubated for 30 min at 37  C. After the rst
incubation, the GoldMag nanoparticles were washed 3
times by PBST. 50 mL of biotinylated Ab was added to each
tube and the mixture was incubated for 20 min at 37  C.
After the second incubation, the GoldMag nanoparticles
were washed 3 times by PBST. 50 mL of HRP labeled avidin
(diluted to 1:300) were then added to each tube and
incubated for 5 min at room temperature. After that, the
GoldMag nanoparticles were washed 2 times with PBST.
Finally the TMB substrate was added to each tube. The
423
2.4 2
2.2
Absorbance @ 450 nm (O.D.)
2.0
1. DIRECT ELISA
1.8 2. BA-ELISA
1.6
1.4 1
1.0
0.4
0.2
1:1000 1:2000 1:4000 1:6000 1:8000 1:10000
Dilution of serum
Fig. 5. Comparison of the biotinavidin amplied magnetic ELISA
system and the direct ELISA system for the detection of hepatitis B
surface antigen using GoldMag nanoparticles.
mixture was incubated and stopped with 2 M H2 SO4
solution. A pronounced yellow color developed at room
temperature after 5 min. The absorbance of each tube was
measured at 450 nm. Herein, six dilutions of the serum
from patients with HBV infection1:1000, 1:2000, 1:4000,
1:6000; 1:8000, 1:10 000 were chosen to react in the
sandwich ELISA, respectively, and the direct ELISA
without biotinavidin amplication was also performed
for comparison (Fig. 5).
The data in Fig. 5 show that the biotinavidin amplied
magnetic ELISA system is more sensitive than direct
ELISA in six dilutions of the serum. At dilution of 1:10000,
the value of the positive serum (0.72) also differed from the
negative serum (0.13). However, the positive serum at
1:10000 is nearly the same as the negative serum in the
direct ELISA assay. The serum (diluted 1:4000) was also
detected 11 times by the biotinavidin amplied magnetic
ELISA. The average value of the positive serum is 0.95 and
the average value of the negative serum is 0.09. The relative
standard deviation (RSD) of the positive serum is 5.98%.
In conclusion, biotinavidin amplied magnetic ELISA
system using GoldMag nanoparticles was developed and
used to detect HBsAg. The detection system is more
sensitive than without biotinavidin amplication effect.
The high sensitivity of this method reveals great potential
for clinical diagnosis.
This work was supported by the National Natural
Science Foundation of China (No. 20273050) and the
National High Technology Research and Development
Program of China (2005AA205220).
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