Copper-mediated amplification allows readout of immunoassays by the naked eye

Article abstract of DOI:10.1002/anie.201006025

An eye for color: Antibodies modified by CuO nanoparticles (NPs) are subjected to immunoreaction with the release of Cu^II, which can be detected by click chemistry. The Cu acts as a catalyst that induces aggregation of Au NPs functionalized with azide and alkyne groups, which can be seen as a color change (see picture). The detection of HIV in blood serum of infected patients is demonstrated. Copyright

Full text of DOI:10.1002/anie.201006025

Communications
DOI: 10.1002/anie.201006025
Click Chemistry
Copper-Mediated Amplification Allows Readout of Immunoassays by
the Naked Eye**
Weisi Qu, Yingyi Liu, Dingbin Liu, Zhuo Wang,* and Xingyu Jiang*
A new method of labeling antibodies for the colorimetric
detection of immunoassays without using advanced equip-
ment is described. Colorimetric detections are convenient and
effective in many applications because the readout requires
only human eyes. Gold nanoparticles (Au NPs) are useful in
colorimetric assays because the aggregation of solutions with
low concentrations of Au NPs displays a clear color change.
The aggregation of Au NPs in solution has yielded many
assays,[1] including those for ions,[2,3] small molecules,[4]
DNA,[5,6] proteins,[7] and cancerous cells.[8,9] Advances in
CuI-catalyzed tethering reactions, such as “click chemis-
try”[10,11] in aqueous solutions at room temperature, have
allowed for new types of chemistry for Au NP-based
biochemical analysis.
Our previous work shows that CuI-based click chemistry
Scheme 1. Immunoassay based on CuO-labeled antibody and click
chemistry.
allows high sensitivity and selectivity[2] for chemical analysis.
The fact that copper is used as a catalyst (a small amount of it
is required for the reaction) ensures the sensitivity, and the
fact that the reaction is orthogonal to most known chemical
reactions ensures the selectivity. In the presence of CuII with
sodium ascorbate as the reductant, Au NPs that have azide-
and alkyne-terminated groups undergo aggregation as the
result of CuI-catalyzed click chemistry. At ambient temper-
ature, this process can be monitored by the naked eye. This
method is highly specific even in the presence of high
concentrations of mixtures of other cations and interfering
molecules. We reasoned that if such assays can be extended to
detect species other than CuII, it might be generally useful for
many different types of highly sensitive and selective assays.
We now report a colorimetric immunoassay based on detect-
ing CuII released from copper monoxide nanoparticle (CuO
NP)-labeled antibodies as the secondary antibody, in place of
the fluorescent dye- or enzyme-labeled secondary antibodies
traditionally used in immunoassays (Scheme 1).
One of the bottlenecks for developing “point of care”
(POC) detection[12,13] is the readout method. With the rapid
development of nanoscience, many nanomaterials have been
widely applied in bioanalysis as the label of probe molecules,
to realize various signal output modes.[14] Because of the size
effects and optical properties of nanomaterials, many of these
methods showed good sensitivity, but many of them relied on
bulky and complex equipment. Generally applicable readout
methods by the naked eye alone for immunoassays have the
potential to revolutionize analytical sciences based on the lab-
on-a-chip format.[15] Naked-eye-based results simply need to
match those from instruments, such as fluorescence assays, to
be widely applicable.[16]
Herein, we provide a method of labeling antibodies for
the highly sensitive and selective colorimetric detection of
immunoassays without the use of advanced equipment.
Human immunodeficiency virus (HIV) is the cause of
acquired immune deficiency syndrome (AIDS), a worldwide
pandemic. Because there is no cure for AIDS or effective
vaccine against HIV, clinical assays for HIV are one of the
major tools for prevention. Amongst all the methods for
detecting HIV,[17] the detection of virus-elicited antibody (in
human blood serum) is most widely applied.[18,19] Because
AIDS affects many resource-poor regions, simple and sensi-
tive assays for the detection of infection by HIV are essential
for controlling this pandemic.[20]
[*] W. Qu,[+] Y. Liu,[+] D. Liu, Prof. Z. Wang, Prof. X. Jiang
CAS Key Lab for Biological Effects of Nanomaterials and Nano-
safety, National Center for Nanoscience and Technology
11 Beiyitiao, ZhongGuanCun, Beijing 100190 (P.R. China)
Fax: (+86)10-8254-5631
E-mail: wangz@nanoctr.cn
xingyujiang@nanoctr.cn
[+] These authors contributed equally to this work.
[**] We thank L. S. Song and Y. Zhang for useful discussions. Financial
support for this work was from the National Science Foundation of
China (90813032, 20890020, 21025520), the Ministry of Science and
Technology (2009CB930000, 2011CB933201), the Ministry of Health
(2008ZX10001-010), and the Chinese Academy of Sciences (KJCX2-
YW-M15).
The classical method for HIV detection is based on
enzyme-linked immunosorbent assay (ELISA) in microwells,
with the most current forms employing enzyme-labeled,
fluorescent or chemiluminescent antibodies for readout.
This type of ELISA needs instruments for detection, and
hence is costly and requires hours of work and skilled labor.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201006025.
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New chemical methods that allow sensitive detection of HIV
infection are therefore particularly urgently needed. In
developed economies, such tests are typically carried out in
centralized laboratories. Instrument-free detection for HIV
may help dramatically reduce soaring medical costs and
enable the general public to easily test for such diseases. Our
method can rapidly analyze HIV antibodies without using
advanced instruments, by detecting copper through a colori-
metric approach based on the aggregation of Au NPs and
click chemistry.
Our approach relies on antibodies modified by CuO NPs.
When CuII is released into the solution by HCl, it can be
assayed by the detection mixture (DM, comprising azide- and
alkyne-functionalized Au NPs and sodium ascorbate) as a
naked-eye-based readout (Scheme 1). It can be applied to
essentially any biochemical assay relying on immunoreactions
that take place on a solid/liquid interface. Instead of an
enzyme or a fluorophore, CuO NPs are the label that modifies
the secondary antibody. After immobilization of the anti-
bodies, we added acid to dissolve the CuO NPs to produce
copper ions, which in turn can be detected with high
sensitivity and specificity by click chemistry in which copper
acts as a catalyst, thereby inducing aggregation of Au NPs
functionalized with azide and alkyne groups (the components
of click chemistry[2]). Since the aggregation of Au NPs can be
monitored with the naked eye alone (dispersed Au NPs show
a red color while aggregated Au NPs result in a purple or blue
color), no instrument is needed for the readout. Because the
method can detect the antibody without equipment, we
applied it to the diagnosis of diseases relying on immuno-
assays, such as HIV.
To enhance the sensitivity of the DM and decrease the
time for the detection compared with our earlier work, we
optimized the conditions that would influence the limit of
detection (LOD) for the DM, such as the ratio between the
ligand (alkyne/azide-terminated thiol) and the stabilizing
agent (polyethylene glycol-terminated thiol[21]; for details of
optimization of the conditions, see the Supporting informa-
tion, Table S1). Based on our early work, we synthesized a
new alkyne ligand (compound 1 in Scheme 1; for the synthetic
protocol and characterization, see the Supporting Informa-
tion) to improve the reactivity of alkyne toward the azide. The
LOD for CuII is 1 mm and the assay takes about 10 minutes for
the color change of the DM to be observed by the naked eye.
To show that our method can be useful for ELISA-type
assays, we first investigated the detection of the antibody of a
model protein, ovalbumin (OVA). In this assay, the antigen
was OVA, the primary antibody was rabbit anti-OVA, and the
secondary antibody was goat anti-rabbit immunoglobulin G
(IgG) labeled with CuO NPs. We mainly tested whether
factors that would normally affect such experiments might
adversely influence the selectivity of our assay (Figure 1). We
incubated OVA in a 96-well plate, blocked the wells with 5%
fetal bovine serum (FBS), and incubated the primary anti-
body. CuO NP-labeled secondary antibody was used as the
probe. After immobilization of these molecules on the wells,
hydrochloric acid released copper ions from the immobilized
antibodies; we added the DM to every well and observed the
color change after 10 minutes (for the details of optimizing
Figure 1. Detection of a model antibody (the antibody of OVA). Photo-
graphs taken 10 min after adding the DM to microwells are shown for
each sample. The reagents added to each well are indicated below the
images.
the immunoassay conditions, see the Supporting Information,
Figures S5 and S6).
Only wells A1 and A2 showed color changes. Well A1 had
CuO NP-labeled secondary antibody immobilized on the well
by the immunoreaction. Well A2 only had secondary antibody
labeled with CuO NPs. The reason for the color change was
that this antibody was nonspecifically adsorbed on the well.
Well A3 had no OVA; instead it had bovine serum albumin
(BSA), where neither the primary antibody nor the secon-
dary-antibody-labeled CuO NPs could adsorb on the well.
Well A4 had no primary antibody, so the secondary-antibody-
labeled CuO NPs could not adsorb on the well. Well A5 was
negative because CuO NP-labeled antibody could not adsorb
on the well as a result of effective blocking that prevented
nonspecific adsorption after. Well A6 had only OVA to
exclude the influence of antigen. Well A7 had only FBS to
exclude the influence of the blocking reagent. Well A8 had
goat anti-rabbit IgG without CuO NPs to exclude the
influence of secondary antibody. Well A9 had only hydro-
chloric acid and well A10 had only the DM. Wells A1 and A2
showed positive results, while wells A3 to A10 showed
negative results, as expected. We carried out the experiment
to exclude the possible factors affecting the results, and
verified that CuO NPs were labeled with the antibody
successfully.
To apply our method of detection to a model HIV
diagnosis, we assayed rabbit serum with anti-gp41 IgG
(Figure 2). The HIV-1 gp41 antigen was incubated in the 96-
well plate and the wells were blocked with 5% FBS. Rabbit
serum containing rabbit anti-gp41 IgG of serially diluted
concentration (from 1/200 to 1/25600) was added to the wells.
The goat anti-rabbit IgG labeled with CuO NPs by non-
specific adsorption was added last. The control experiments
included a nonspecific rabbit IgG as the negative control,
blank control, and BSA as the unrelated protein control.
After incubation, HCl was used to produce copper ions and
the DM was added to every well. Some wells had a color
change from red to purple or blue. In Figure 2a, it can be seen
that the color of the liquids from wells B3 to B8 changed
10 minutes after adding the DM (the corresponding time is
shown in the Supporting Information, Figure S6, Table S2).
The higher the concentration of the primary antibody, the
faster the color change of the solution, so the rabbit anti-gp41
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Communications
highest detectable dilution in our assay is
6400 times by naked-eye readout). The
LOD for our method rivals that of
absorbance or fluorescence, two com-
monly used reporting systems for micro-
plate readers. Contrary to existing report-
ing systems, the new method requires no
equipment at all.
To test the utility of our approach for
real samples, and to explore the possible
interference of endogenous Cu present in
blood plasma, we used this method to
distinguish the serum of HIV-infected
patients from that of uninfected patients
(Figure 3). Wells C1 to C3 had serum
samples of different HIV-infected
patients diluted 40 times and the secon-
dary antibody was rabbit anti-human IgG
labeled with CuO NPs. The other con-
ditions were the same as those for the
HIV model experiment. Because differ-
ent patients have different concentrations
of anti-HIV antibody in the blood serum,
the wells showed different color changes,
while the color of control wells had no
change. We can distinguish the positive
blood serum from the negative by the
naked eye, because the serum sample of
the HIV-negative samples did not cause a
color change of the DM. This experiment
Figure 2. Model HIV assay. a) An immunoassay was performed on a 96-well plate that
detected anti-HIV IgG with serially diluted concentrations from wells B8 to B1. Well B9 had
nonspecific rabbit IgG as negative control, well B10 was the blank control, and well B11 had
BSA as the unrelated protein control. Photographs of the DM on microwells are shown for
each sample and the reagents added to every well are indicated below. b) The absorbance (A)
of each microwell was measured by a UV/Vis microplate reader (at wavelength l). Inset: plot
of A566/A530 against logCAb for rabbit IgG assay. c) Response curve of the fluorescence signals
(F) versus different concentrations of rabbit anti-gp41 IgG under the FITC-goat anti-rabbit IgG
as secondary antibody. Error bars indicate one standard deviation from the mean of three
assays. Photographs were taken 10 min after adding the DM.
IgG diluted 6400 times could be detected by the naked eye
and its concentration was about 150 ngmLÀ1.
We next compared the sensitivity of the method with UV/
Vis absorption (for detecting the aggregation of Au NPs) and
fluorophore-labeled secondary antibody (which is the current
industry standard for clinical immunoassays of HIV). We
measured the UV/Vis absorption in each well by a microplate
reader (Figure 2b). To our amazement, the sensitivity for
immunoassay readout by the naked eye was at the same level
as that measured by UV/Vis spectrometry.
In addition, as shown in Figure 2b, increasing the
concentration of primary antibody in the well also results in
a clear increase in the absorbance at 566 nm (A566) and a
decrease in the absorbance at 530 nm (A530). The ratio
between A566 and A530 was linear with the logarithm of
primary antibody concentration CAb (r= 0.991), which dem-
onstrated that the assay described here could be used for
visual detection of HIV at high sensitivity. When comparing
the analytical performance of our method with that of
immunoassays that measure the fluorescence of secondary
antibody labeled by fluorescein isothiocyanate (FITC, Fig-
ure 2c; we carried out the same immunoassay using FITC-
labeled secondary antibody instead of the CuO NP-labeled
secondary antibody), the LOD for the concentration of the
primary antibody, at a signal-to-noise ratio of 3, is approx-
imately at the primary antibody diluted 12800 times (the
Figure 3. Detection of real HIV-1 samples. Wells C1 to C3 had blood
serum from different HIV-1-infected patients. Well C4 had blood serum
from uninfected individuals. Well C5 had no blood serum as blank
control. Well C6 had BSA as the unrelated protein control. The
reagents added to every well are indicated below the photographs. P1,
P2, and P3 indicate different HIV-1-infected patients. Photographs
were taken 10 min after adding the DM.
indicates that endogenous Cu in the serum does not affect our
approach.
The high selectivity and sensitivity of the immunoassay
result from the highly selective and sensitive nature of the
copper-catalyzed reaction. This new immunoassay may there-
fore be useful for many applications, including clinical assays
in resource-poor settings. Since immunoassays comprise the
largest class of assays for proteins (and are becoming more
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our approach to have wide-ranging applications from ana-
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related assays. Such types of assays may pave the way for
highly sensitive and selective detection of molecules through
the indirect detection of metal ions.
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Received: September 26, 2010
Revised: December 28, 2010
Published online: March 8, 2011
Keywords: antibodies · click chemistry · colorimetric detection ·
.
nanoparticles · immunoassays
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