Download Application of the Biological Conjugate between Antibody and

Anal. Chem. 2002, 74, 96-99
Application of the Biological Conjugate between
Antibody and Colloid Au Nanoparticles as Analyte
to Inductively Coupled Plasma Mass Spectrometry
Chao Zhang,† Zhenyu Zhang,† Binbing Yu,‡ Jinjun Shi,† and Xinrong Zhang*,†
Department of Chemistry and Department of Biological Sciences and Biotechnology, Tsinghua University,
100084, Beijing, P.R. China
This paper describes the study of atomization of nanoparticles by inductively coupled plasma mass spectrometry (ICPMS) and developes a novel nonisotopic immunoassay by coupling sandwich-type immunoreaction to
ICPMS. The goat-anti-rabbit immunoglobulin G (IgG)
labeled with colloidal gold nanoparticles served as an
analyte in ICPMS for the indirect measurement of rabbitanti-human IgG. Matrix effect studies showed the gold
signal was not sensitive to the organic matrix. A relatively
good correlation (r2 ) 0.9528) between the proposed
method and enzyme-linked immunosorbent assay has
been obtained. The method may have significant potential
as an important ICPMS-based nonisotopic immnoassay
method for the simultaneous determination of biologic
analytes of interest by labeling different kinds of inorganic
nanoparticles.
Since its introduction in the early 1960s, radioimmunoassay
(RIA) has revolutionized the biological measurement due to its
extremely high sensitivity and specificity.1 Even today, radioimmunoassay still plays a major role in clinic diagnoses, medicine
and related areas.2 The special disposal of radioactive waste after
use and the potential radioactive hazards, however, has been the
big problem.
The sensitively nonisotopic methods may overcome the
drawbacks of radioactive detection and have great potential in
many research fields such as DNA sequencing, clinical diagnostics, and biological assays.3-6 Therefore, a plethora of nonisotopic
labels has been employed, such as chemiluminescent, fluorescent
labels, metal atoms, as the alternatives of RIA method.7-9
* Corresponding author: (tel) +86-10-62787678; (fax) +86-10-62770327; (email) [email protected].
† Department of Chemistry.
‡ Department of Biological Sciences and Biotechnology.
(1) Chard, T. An introduction to radioimmunoassay and related techniques, 2nd
ed.; Elsevier Biomedical Press: Amsterdam, 1982.
(2) Development in Radioimmunoassay and Related Procedures; IAEA: Vienna,
1991 (Proc. Symp. Vienna, 1991).
(3) Kricka, L., Ed. Nonisotopic probing, blotting, and sequencing; Academic
Press: NewYork, 1995,
(4) Knemeyer, J. P.; Marme, N.; Sauer, M. Anal. Chem. 2000, 72, 3717-3724.
(5) Gelmini, S.; Caldini, A.; Becherini, L.; Capaccioli, S.; Pazzagli, M.; Orlando,
C. Clin. Chem. 1998, 44, 2133-2138.
(6) Rossler, A. Clin. Chim. Acta 1998, 270, 101-114.
(7) Martin, C.; Bresnick, L.; Juo, R. R.; Voyta, J. C.; Bronstein, I. Biotechniques
1991, 11, 110-113.
(8) Gite, S.; Mamaev, S.; Olejnik, J.; Rothschild, K. Anal. Biochem. 2000, 279,
218-225.
96 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
The expanding availability of a variety of nanostructures with
unique properties at nanoscale dimensions has attracted widespread attention in their use in biotechnological systems. Besides,
since nanoparticles are similar in size range to many common
biomolecules, they are more suitable for integration with biological
systems.10-13 For instance, the use of highly luminescence
semiconductor nanocrystals (quantum dots) as fluorescent biological labels has been used in ultra-sensitive biological detection.14
Inductively coupled plasma mass spectrometry (ICPMS), as
an outstanding method for trace element determination, has
gained a very wide acceptance due to its extremely high sensitivity
and element specificity. However, the excellent performances of
ICPMS mainly focus on the field of inorganic element analysis. It
is well-known that apart from high sensitivity, ICPMS can offer
powerful ability for simultaneous determination of inorganic
elements in a few minutes. In principle, by selecting proper
nanoparticles of elements and labeling them to the biological
molecules, ICPMS-based nonisotopic immunoassay may open new
possibility to simultaneously detect biological analytes of interest.
At the present level of development, colloidal Au nanoparticlelabeled antibody has been chosen as the model protein and the
possibility of a sandwich-type immunoreaction coupled to ICPMS
was explored. Colloidal Au nanoparticles are ideal markers in
biotechnological systems for several reasons: first, they can be
readily prepared in a wide range of sizes, from about 2 nm to
above 100 nm; second, the specific activities of micromolecules
can be retained when coupling micromolecules to colloidal Au
nanoparticles; third, the gold particles can be easily visualized as
dense strictures within biological entities in the transmission
electron microscopy.15 As an excellent biological tag, colloidal Au
nanoparticles have been extensively employed to label a broad
range of biological receptors, such as protein A, immunoglobulin
G (IgG), and glucose oxidase, and applied to surface-enhanced
(9) Li. M.; Selvin, P. R. Bioconjugate Chem. 1997, 8, 127-132.
(10) Reichert, J.; Csaki, A.; Kohler, J. M,; Fritzsche, W. Anal. Chem. 2000, 72,
6025-6029.
(11) Taton, T. A.; Mirkin, C. A.; Letsinger, R. Science 2000, 289, 1757-1760.
(12) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V.
C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 1214212150.
(13) Sondi, I.; Siiman, O.; Koester, S.; Matijevic, E. Langmuir 2000, 16, 31073118.
(14) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018.
(15) Verkleij, A. J.; Leunissen, J. L. M. Immuno-gold-labeling in cell biology; CRC
Press: Boca Raton, FL, 1989.
10.1021/ac0103468 CCC: $22.00
© 2002 American Chemical Society
Published on Web 11/30/2001
Table 1. Operating Conditions of Elan 6000 ICPMS
rf power
coolant argon flow
auxiliary argon flow
nebulizer argon flow
operating frequency
sample uptake rate
detector mode
scanning mode
dwell time
sweeps per reading
readings per replicate
replicates
internal standard used
1100 W
15 L min-1
1.2 L min-1
1 L min-1
40 MHz
1.4 mL min-1
pulse mode
peak hopping
50 ms
3
60
1
In
Raman scattering (SERS), surface plasmon resonance (SPR), and
immunoblotting.16-18
In the proposed paper, the atomization of different kinds of
nanosized materials including TiO2, La2CuO4, Y2O3, and colloidal
Au nanoparticles in the ICP torch has been investigated. Also, a
sandwich-type immunoreaction using colloidal Au nanoparticlelabeled antibody coupled to ICPMS was demonstrated. Human
IgG was first immobilized to the solid phase. Afterward, the bound
antigen was allowed to capture rabbit-anti-human IgG antibody
specifically followed by detection of goat-anti-rabbit second
antibody labeled with colloidal Au nanoparticles with ICPMS.
Results indicated that the ICPMS-based nonisotopic immunoassay
may have potential for the determination of biological analytes of
interest.
EXPERIMENTAL SECTION
Instrumentation. A Elan 6000 ICPMS (PE- Sciex, Concord,
Canada) was used for this experiment. The instrumental optimal
operating parameters are summarized in Table 1. Prior to analysis,
the X, Y positions of the torch, rf power, nebulizer gas flow, and
lens were optimized using 10 µg/L Mg, Rh, and Pb in 2% nitric
acid. A multielement solution containing 10 µg/L Be, Co, In, and
Pb, respectively, was used to calibrate the lens autosettings and
to establish a linear relationship between lens voltage and mass.
The enzyme-linked immunosorbent assay (ELISA) results were
obtained by measuring the absorbance at 490 nm with the BioRad model 550 microplate reader.
Reagents and Immunoreaction Buffers. Deionized water
(18 MΩ cm) was used in all the experiment (Beijing ShaungFeng
purity water equipment factory). Polystyrene 96-well microtiter
plates (Nanc) were used to perform the immunoreaction. Human
IgG, goat IgG, rabbit-anti-human IgG antibody, and bovine serum
albumin (BSA) were purchased from Beijing Xin Jing Ke biotechnology Co. Ltd. (Beijing, China). TiO2, La2CuO4, Y2O3, colloidal
Au nanoparticles, and goat-anti-rabbit colloidal Au conjugate were
synthesized in our laboratory. The buffers used were as follows:
(A) coating buffer, 0.05 M carbonate/bicarbonate buffer solution,
pH 9.6; (B) assay buffer, 0.01 M sodium phosphate-buffered saline
(PBS) containing 1% BSA, pH 7.4; (C) washing buffer, buffer B
with 0.05% Tween 20
(16) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71,
4903-4908.
(17) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 51775183.
(18) Chevallet, M.; Procaccio, V.; Rabilloud, T. Anal. Biochem. 1997, 251, 6972.
Figure 1. TEM photograph of colloidal Au.
Preparation of Colloidal Gold Nanoparticles. Colloidal Au
was prepared according to the literature with slight modification.15,19 Briefly, after boiling 0.01% HAuCl4 with 0.05% trisodium
citrate in aqueous solution for 15-30 min, the resulting colloidal
suspension was cooled and filtered through a 0.45-µm Millipore
membrane. The diameter of particle was ∼15 nm (Figure 1), as
confirmed by Hitachi H-800 transmission electron microscopy.
Preparation of a Colloidal Gold-Antibody Conjugate.
Antibody-colloidal conjugates were prepared according to the
modification in the literature.15-17 The goat-anti-rabbit antibody
(10% more than the minimum amount, which was determined
using a flocculation test) was added to 1 mL of pH-adjusted
colloidal Au suspension followed by incubation at room temperature for 1 h. The conjugate was centrifuged at 45000g for 30-60
min, and the soft sediment was resuspended in 0.01mol L-1 Trisbuffered saline. Addition of glycerol to a final concentration of
50% allows storage of the colloidal Au goat-anti-rabbit conjugate
at -20 °C for several months.
Immunoassay Protocol. The immunoassay was conducted
by following the typical procedure for sandwich-type immunoreaction (Figure 2). Initially, a polystyrene 96-well microtiter plate
was coated using 200 µL of human IgG (diluted to 10 µg/well
with bicarbonate buffer, pH9.6) and incubated at 4 °C overnight.
The unbound antigen was washed away with PBS containing 0.05%
Tween-20 (PBS-T). After washing, the wells were incubated with
PBS containing 1% BSA for 1 h at 37 °C. Afterward, the plate was
washed three times with PBS-T; Series dilutions of rabbit-antihuman IgG antibodies with assay buffer were pipetted into the
wells and incubated for 2 h at 37 °C. Plates were washed six times
with PBS-T followed by addition of colloidal Au-labeled goat-anti(19) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22.
Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
97
Figure 2. Scheme of sandwich-type immunoreaction.
Table 2. Comparison of Atomization of Nanoparticle
Suspensions and Their Solutions
relative intensitya
TiO2
Y2O3
La2CuO4
Au
a
in solution
nanoparticle suspension
100 ( 3.3
100 ( 2.9
100 ( 2.1
100 ( 1.8
98.6 ( 3.2
100.5 ( 1.7
101.2 ( 3.4
100.8 ( 3.7 (colloidal Au)
99.1 ( 2.5 (colloidal Auantibody conjugate)
Five replicates; concentrations, 10 ng/mL
rabbit antibody (1:200 dilution with PBS containing 1% BSA) to
each well and incubation for 4 h at 37 °C followed by washing six
times with PBS-T.
External calibration was used for the quantitative determination
of rabbit-anti-human IgG. In was used as internal standard element
to correct the fluctuation of the instrument. A 200-µL aliquot of
1% HNO3 solution containing 1 ng/mL In was added to each well.
Samples to be analyzed were placed at room temperature for 3
min and introduced to the ICPMS by peristaltic pump.
RESULTS AND DISCUSSION
Atomization of Nanosized Meterials in ICP. For most
atomic spectroscopic methods, it is necessary to solubilize the
sample in a suitable solvent before it can be introduced into the
instrument. The atomization, therefore, is an important factor that
affects the sensitivity of the analyte of interest. In this paper, a
comparison of atomization of TiO2, La2CuO4, Y2O3, and Au
nanoparticle suspensions as well as their solutions was conducted
in ICP. La2CuO4 and Y2O3 nanoparticle suspensions were divided
into two groups (nanoparticle suspensions and their corresponding
solutions dissolved by 1% nitric acid) and introduced into the ICP
system. The same concentrations of Ti and Au standard solutions
were prepared in order to compare the results with that obtained
from TiO2, colloidal Au nanoparticles in ICP. The results in this
experiment shown in Table 2 suggested that the nanoparticles in
ICP have the same atomization efficency as that of ion solutions,
indicating the excellent atomic efficiency in the ICP torch.
Study of the Matrix Effect of Colloidal Au NanoparticleLabeled Antibody. Sample composition and interaction in plasma
commonly affect the result in ICPMS determinations when a
nebulizer chamber system is used. These interferences are
generally the result of either sample matrix effects that influence
98 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
Table 3. Nonspecific Binding and Specificity for the
Determination of Rabbit-Anti-Human IgG
capture
antibody
analyte
Relative Au
intensity (n)4)
human IgG
human IgG
human IgG
none
goat IgG
blank
goat-anti-human IgG
rabbit-anti-human IgG
rabbit-anti-human IgG
rabbit-anti-human IgG
3.5 ( 0.17
5.9 ( 0.31
100 ( 6.5
4.8 ( 0.19
4.1 ( 0.21
rabbit-anti-human IgG and goat-anti-human IgG concentration, 5 ng/
mL; human IgG and goat IgG concentration, 10 µg/mL.
aerosol formation or the formation of ions in the plasma. The
matrix effect of colloidal Au nanoparticle-labeled antibody was
investigated prior to coupling to the immunoreaction. The preformance of Au solution, colloidal Au nanoparticle solution, and
colloidal Au nanoparticle-labeled antibody solution in the ICP torch
were examined. Table 2 indicated no difference was observed by
comparing the results obtained from these solutions, indicating
that the Au signal was not sensitive to the organic matrix.
Immunoreaction. The immunoreaction was conducted by
using the procedure for the sandwich type. The scheme is shown
in Figure 2. Human IgG was immobilized on the solid phase
followed by using 1% BSA to block the nonspecific binding site.
After the blocking step, the rabbit-anti-human IgG and colloidal
Au-labeled goat-anti-rabbit antibody were added to form a sandwich complex of human IgG-rabbit-anti-human IgG-colloid goldlabeled goat-anti-rabbit IgG. Since the antigen-antibody complex
can be dissociated under some extreme physiochemical conditions
such as high temperature, low pH, and strong ionic strength, 1%
HNO3 was employed to get the measurable signals of Au eluted
from the solid surface after completion of the immunoassay in
this experiment.
Specificity. The specific recognition of antigenic species was
studied. Goat-anti-rabbit IgG labeled with colloidal Au nanoparticles was used for immune recognition. Human IgG immobilized
on the plate served as the capture antibody. The results in Table
3 showed that the Au signal only increased after immunoreaction
of rabbit-anti-human antibody with human IgG coated on the plate.
The Au signal obtained from goat-anti-human IgG, however, was
slightly higher than that from the blank, suggesting that only
rabbit-anti-human IgG was specifically captured by the substrate
as well as bound with colloidal Au goat-anti-rabbit IgG. Since
colloidal Au nanoparticles with extremely high particle density
Figure 3. Dependence of Au intensity on rabbit-anti-human IgG
concentration.
Table 4. Intra-assay and Interassay for
Rabbit-anti-human IgG Determination
rabbit-anti-human
IgG concn (ng/mL)
mean ( SD
RSD
(%)
2.5
10
50
Intra-assay (n ) 6)
2.34 ( 0.18
10.3 ( 0.58
48.5 ( 2.1
7.7
5.6
4.3
2.5
10
50
Interassay (n ) 5)
2.41 ( 0.16
10.5 ( 0.78
49.5 ( 3.6
6.6
7.4
7.3
have strong adsorption activity, it is necessary to determine the
extent of the nonspecific binding (NSB) of colloidal Au-labeled
antibody in the assay. The results in Table 3 showed that the Au
signal from NSB was less than 5% by adding colloidal gold-labeled
goat-anti-rabbit IgG to either the blank microtiter plate or the plate
coated by using goat IgG, indicating an acceptable NSB level.
Analytical Performance. The dilution test of colloidal Au goatanti-rabbit IgG showed that the detection limit of the colloidal Au
goat-anti-rabbit IgG was 0.008 ng/mL by series dilution. After
immunoreaction, the detection limit for the rabbit-anti-human IgG
was 0.4 ng/mL (3σ). The dependence of Au intensity on rabbitanti-human IgG concentration was shown in Figure 3. A good
linear relationship between Au intensity and rabbit-anti-human IgG
in the concentration range between 0.8 and 50 ng/mL was
obtained (r2 ) 0.9846). The departure from linearity was observed
when the concentration of rabbit-anti-human IgG was up to 100
ng/mL.
The reproducibility of an assay was expressed in term of values
for a within-batch (intra-assay) and a between-batch (interassay)
Figure 4. Correlation of ICPMS and ELISA for the determination
of rabbit-anti-human IgG.
relative standard deviation (RSD) in the presented paper. The
obtained mean values, standard deviation (SD), and RSD by
replicate analyses (n ) 6) in the intra-assay and in the interassay
(n ) 5) are reported in Table 4. The RSD values were all below
10%, indicating an acceptable level of precision.
Finally, a correlation of results for rabbit-anti-huamn IgG serum
by ICPMS and ELISA was investigated. The results of comparative
studies were shown in Figure 4. It can be seen that relatively good
correlation was obtained (r2 ) 0.9528) between these methods.
CONCLUSION
A novel nonisotopic method using immunoreaction coupled
to ICPMS has been developed. Studies have shown that the
immunoassays may be successful by detecting nanoparticlelabeled antibody with ICPMS. Compared with other nonisotopic
methods, the proposed detection has a wide choice of label and,
thus, may expand the range of application. The ICPMS-based
nonisotopic method demonstrated here may open up new possibilities for biological assays and clinical diagnoses. Furthermore,
it has potential to be applicable to simultaneous determination of
several biological or clinical analytes of interest by selecting proper
nanoparticles of inorganic elements and labeling them to the
biological molecules.
ACKNOWLEDGMENT
This work is supported by National Natural Science Foundation
of China (20075014)
Received for review March 23, 2001. Accepted September
25, 2001.
AC0103468
Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
99