Peroxidase-Like Activity of Ethylene Diamine Tetraacetic Acid and Its Application for Ultrasensitive Detection of Tumor Biomarkers and Circular Tumor Cells

Article abstract of DOI:10.1021/acs.analchem.6b02966

Ethylene diamine tetraacetic acid (EDTA) is such a powerful chelating agent that it may form stable complexes with most metal ions, which has wide applications in industry, agriculture, environment, and pharmaceutical technology. Recently, EDTA was found

Full text of DOI:10.1021/acs.analchem.6b02966

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Article
Peroxidase-like activity of EDTA and its application for ultrasensitive
detection of tumor biomarkers and circular tumor cells
Haowen Huang, Lanfang Liu, Lingyang Zhang, Qian Zhao,
Yuan Zhou, Shishan Yuan, Zilong Tang, and Xuanyong Liu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02966 • Publication Date (Web): 05 Dec 2016
Downloaded from http://pubs.acs.org on December 6, 2016
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Peroxidase-like activity of EDTA and its application for ultrasensitive detection
of tumor biomarkers and circular tumor cells
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†
Haowen Huang,* Lanfang Liu,† Lingyang Zhang,† Qian Zhao,† Yuan Zhou,†
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§
Shishan Yuan,‡ Zilong Tang,† Xuanyong Liu*
†
Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry
of Education, Hunan Provincial University Key Laboratory of QSAR/QSPR, School of
Chemistry and Chemical Engineering, Hunan University of Science and Technology,
Xiangtan, China.
‡School of Medicine, Hunan Normal University, Changsha, China
§State Key Laboratory of High Performance Ceramics and Superfine Microstructure,
Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050,
China
Corresponding authors:
Haowen Huang: hhwn09@163.com
Xuanyong Liu：xyliu@mail.sic.ac.cn
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ABSTRACT: Ethylene diamine tetraacetic acid (EDTA) is such a powerful chelating
agent that it may form stable complexes with most metal ions, which has wide
applications in industry, agriculture, environment and pharmaceutical technology.
Recently, EDTA was found to enhance the photocatalytic property of some materials.
Inspired by this fact of EDTA in the photocatalytic role, we further investigated the
photocatalytic property of EDTA and found much the same as that of natural
horseradish peroxidase (HRP). This significant discovery of peroxidaseꢀlike property
may extend the applications of conventional EDTA in life science. A novel and
colorimetric sensor based on the peroxidaseꢀlike EDTA and unique gold nanorods
(GNRs) was designed. Under light irradiation, EDTA may catalyze decomposition of
hydrogen peroxide and in situ regulate the longitudinal plasmon wavelength (LPW) of
GNRs, displaying various color solution as a readꢀout means. This colorimetric
nanosensor has a great potential to develop into a platform to quantitatively determine
analytes as long as the specific antibodies against them were available. Biomarkers of
different diseases, such breast cancer and prostate cancer, were detected with high
accuracy. Moreover, combined with immunomagnetic separation of circulating tumor
cells (CTCs) from blood, a visual readꢀout for detection of CTCs was established,
which has promising applications in clinical diagnosis, environmental monitoring, and
food quality control only using naked eyes.
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INTRODUCTION
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EDTA is such a powerful chelating agent that it may form stable complexes with
most metal ions. As an additive, EDTA can preserve color, flavor and prevent
deteriorative in the food because it deactivates the oxidation reactions catalyzed by
metal ions.1ꢀ5 EDTA is also recognized to directly inhibit the growth of bacteria in
food through disruption of the integrity of bacterial membrane by its chelation with
metal ions.6,7 In addition, EDTA facilitates to generate hydroxyl radical in the Fenton
reaction.8 The specific structure of EDTA favors the activation of H2O2 and the
generation of hydroxyl radicals. It has widely industrial applications such as pulp,
paper, metal, textile production and pharmaceutical technology.9ꢀ14 Recently, EDTA
was introduced to semiconductor materials to attend the photocatalytic process,
leading to significantly enhancing degradation rate of these particles to some organic
dyes and organic pollutions.15ꢀ20 There are six loneꢀpair electrons attributed to six
atoms of EDTA not only facilitate to coordinate with most of metal ions but also
serve as hole scavenger in the photocatalytic process. Inspired by the fact of EDTA in
the photocatalytic role, we further investigated the photocatalytic property of EDTA
with the special structure. Interestingly, our exploration demonstrated that the
conventional EDTA exhibits a fascinating photocatalytic property under light
irradiation, much the same as that of natural horseradish peroxidase (HRP). The
significant discovery of peroxidaseꢀlike EDTA suggests a costꢀeffective and easy
storage mimic enzyme with small molecule will provide promising applications in life
science.
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Enzymeꢀlinked immunosorbent assay (ELISA) is often extensively used in clinical
diagnosis, environmental monitoring and food quality control.21ꢀ24 ELISA works
merely on the natural enzymetic reactions, however, natural protein enzyme usually
subjects to environmental conditions and the stability is poor due to denaturation. As
alternative to the natural enzyme, the artificial enzyme mimicking such as various
types of materials have made a progress in recent years.25ꢀ29 Compared to the natural
enzyme, the enzyme mimetic nanoparticles have advantageous of easy preparation,
purification, stable and storage. Whereas, the nanoparticlesꢀbased biosensor remains
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uncertain factors because the size of nanoparticle is close to or larger than that of
conjugated proteins, in which the conformation of the conjugated protein may be
affected, resulting in variation of protein function. On the contrary, the small
molecular EDTA not only features stability, low cost and easy storage, but also hardly
affects the conjugated protein functions. Unquestionably, peroxidaseꢀlike EDTA is
excellent candidate to provide a good platform for the development of novel sensors.
In addition, conventional ELISA also meets challenge for detecting ultralow
concentration of species. Developing more sensitively ELISAꢀlike assay aroused the
interest of researchers.30ꢀ32
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In this paper, we firstly investigated the peroxidaseꢀlike activity of EDTA. Then,
a novel and colorimetric sensor based on the peroxidaseꢀlike EDTA and gold
nanorods (GNRs) was designed, allowing nakedꢀeye readout and determination of
ultraꢀtrace disease biomarkers. Moreover, combined with immunomagnetic separation
of circulating tumor cells (CTCs) from blood, a visual readꢀout for detection of CTCs
was established, which has potential implications in clinical diagnosis, environmental
monitoring, and food quality control only using naked eyes.
EXPERIMENTAL SECTION
Materials and Reagents. Hydrogen peroxide (H2O2), chloroauric acid
(HAuCl4·3H2O),
3,3,5,5ꢀtetramethylbenzidine (TMB), Nꢀhydroxysuccinimide
(NHS), ethylcarbodiimide hydrochloride (EDC), 2ꢀ (Nꢀmorpholino) ethanesulphonic
acid (MES), bovine serum albumin (BSA) were purchased from Aladdin Industrial
Corporation (Shanghai, China). Breast cancer antigen and rabbit polyclonal breast
cancer antibody were received from Solarbio Science and Technology Co., Ltd
(Beijing, China). Prostate specific antigen (PSA) and antiꢀPSA was received from
Shanghai Ji Ning Industrial Co., Ltd (Shanghai, China). Magnetic microbeads (CSMN
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Beadsꢀ1000, 1000 ꢀm) were obtained from Shanghai Suofei Biological
Pharmaceutical Co., Ltd. (Shanghai, China). All of the chemicals, unless mentioned
otherwise, were of analytical reagent grade and obtained from the commercial source.
The solutions of breast cancer antigen, breast cancer antibody, PSA, antiꢀPSA and
BSA were prepared using 0.01mol/L phosphate buffered saline (PBS) solution.
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Preparation of gold nanorods. GNRs were synthesized using a seedꢀmediated
silverꢀassisted growth method. Briefly, 100 ꢀL of ice cold 0.01M NaBH4 was added
to 100 ꢀL of CTAB solution containing 0.02 M HAuCl4, resulting in the formation of
a brownish yellow solution. Vigorous stirring was continued for 2 min and then the
seed solution was kept at room temperature (25°C) and used at least 2 h after
preparation. To prepare the GNRs, 1.5 mL of 0.02 M HAuCl4 and 1.0 mL of 0.01 M
AgNO3 were added to 30 mL of 0.1 M CTAB, followed by addition of 0.8 mL of 0.08
M ascorbic acid. Finally, 70 ꢀL of the seed solution was added and the color of the
solution gradually changed within 15 min.
Modification of protein with EDTA and magnetic microbeads. EDTA is
covalently attached to protein, such as BioꢀgoatꢀIgG, CA15ꢀ3 antibody, PSA antibody,
antiꢀEpCAM, via the –NH2 of amino acid residues on protein surface. Modification of
the antiꢀbodies was achieved by following process: EDTA mixed with 75 mM EDC
and 15 mM NHS and incubated for 20 min at room temperature. Then 1 mL of 0.01
mg/mL antibody solution was added to 10 mL of activated EDTA 1 h. After that, the
mixture solution was dialysis for 24 h and transferred the solution into a flask.
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The antiꢀEpCAMꢀconjugated beads were created by first mixing carboxyl
groupꢀcoated magnetic beads with 75 mM EDC and 15 mM NHS and incubated for
20 min at room temperature. Then 500 ꢀL of 0.02 mg/mL antiꢀEpCAM was added to
4 mL of the activated magnetic beads solution and incubated for 1 h. Subsequently,
500 ꢀL of 0.5 mg/mL BSA was added to the mixture solution to block the nonspecific
sites on the bead surface. Excess unbound protein was removed by magnetic
separation.
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Detection of avidin, CA15-3, and PSA. 96ꢀwell polystyrene plates were modified
with 350 ꢁL, 0.004 mg/mL antibody (BioꢀgoatꢀIgG or CA15ꢀ3 antibody or PSA
antibody) solution at 4℃ overnight. The plates were blocked with blocking buffer
(350ꢁL of 1 mg/mL BSA solution) for 1 h at room temperature after washing the
plates three times with wash buffer (0.01 mol/L PBS buffer solution). Subsequently,
the plates were washed three times with wash buffer, and 300 ꢁL analyte was added.
After 3 h, the plates were washed three times with wash buffer, 300 ꢁL of CA15ꢀ3
antibody labeled with EDTA was added for 1 h at room temperature.
After the all the plates were then washed three times, 250 ꢁL of hydrogen peroxide
(500 ꢁmol/L) and 100 ꢁL of GNRs were added to each well of the plate, and then the
mixture was irradiated by UV light for 30 min before recording absorption spectra.
There was also parallel test which was without the analyte in the well and carried out
at the same time with the detection of CA15ꢀ3. The photographs were taken after 30
min and the LPW of the resultant GNRs was recorded with an UVꢀVis
spectrophotometer.
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Application to human serum samples analysis. The human serum samples were
collected by Hunan Provincial Tumor Hospital supplied and stored at 4 °C until use.
All experimental procedures were performed in compliance with the relevant laws
and institutional guidelines. The proposed system was applied to the determination of
CA15ꢀ3 and PSA in human serum samples. The breast cancer serum samples were
diluted 1:1010 in PBS buffer solution. The PSA samples were diluted 105 times using
PBS buffer solution. 300ꢁL diluted sample solutions were added to wells in the
procedure of experiments.
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Cell Culture. The cancer cell line human breast cancer cells (MCFꢀ7) were
obtained from Cell Bank of Chinese Academy of Science, and cultured in the
RPMIꢀ1640 medium (Gibco, Life Technologies) supplemented with 10% fetal calf
serum (FCS) (Exell Biology, Inc, South America), and 1% antimicrobial of penicillin
and streptomycin (HyClone Laboratories, Inc, Utah, USA). The cells were cultured in
75 cm2 flasks (Thermo Fisher Scientific, USA) at 37 ℃ in humidified atmosphere
with 5% CO2. Based on the cell condition, cells were passaged at ratio of 1:2ꢀ1:3
every 2ꢀ4 days.
CTC isolation. To isolate CTCs from cellꢀspiked blood, a preꢀdetermined number
of cells were input into the healthy human blood. 10 mL of blood samples were
collected from healthy volunteers. Cells from cancer cell lines were trypsinised and
resuspended in media. Cells were counted with an improved Neubauer
haemocytometer (Hawksley, UK) and were diluted twice by 1 in 10. The appropriate
volume of cells containing 2,000 cells was added to 10 mL blood samples to give 200
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malignant cells/ml of whole blood. 10 ꢁL of magnetic microbead solution was used
for each test.
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RESULTS AND DISCUSSION
The peroxidase-like activity of EDTA. The peroxidaseꢀlike activity of EDTA was
tested with the peroxidaseꢀrequired substrate of 3,3,5,5ꢀtetramethylbenzidine (TMB)
in the presence of H2O2. Figure 1a shows that EDTA catalyzed the oxidation of TMB
by H2O2 to produce oxidized TMB (oxꢀTMB), along with the occurrence of blue color.
As control experiments, color variation was negligible in the absence of either EDTA
or H2O2, which indicates that both of them are indispensable for the reactions. It is
worthwhile to note that the colorimetric experiment must be carried out under light
irradiation, otherwise, no variation of color will be observed. This means the light
irradiation is necessary to trigger the photocatalytic activity of EDTA. Figure 1b
shows the schematic illustration of photocatlytic mechanism of EDTA. Under light
irradiation, EDTA accepts the photo energy to produce the photoꢀgenerated electrons,
which are trapped by H2O2 to produce OH• radicals. Subsequently, the generated OH•
radical quickly reacts with TMB to form oxꢀTMB, showing maximum absorbance at
652 nm. The resultant absorption intensity is proportional to the H2O2 concentration,
as shown in Figure S1. This suggests EDTA is similar to HRP that can catalyze H2O2
to oxidize TMB to form OxꢀTMB, and the color tonality was dependent on the H2O2
concentration.
To validate the mechanism illustrated in Figure 1b, the OH• group was
quantitatively assessed by using the fluorescent technique. The organic molecule of
terephthalic acid (TA) can effectively capture OH• radical and then generate highly
fluorescent 2ꢀhydroxyterephthalic acid which can be easily detected in fluorescence
spectrometer.33 Figure 1c shows the fluorescence spectra of the terephthalic acid in
the presence/absence of EDTA and H2O2 under light irradiation, which confirms that
OH• was produced in the presence of EDTA and H2O2. The fluorescence intensity
originated from the formed fluorescent compounds is proportional to the amount of
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generated hydroxyl radicals. Figure 1d depicts that fluorescence intensity varied from
different irradiation time at certain amount of EDTA. Similarly, the produced
2ꢀhydroxyterephthalic acid is proportional to the amount of EDTA under the same
irradiation time. On the contrary, the control experiment under no irradiation of light
(in the dark) demonstrated that no detectable fluorescence was found.
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To our surprise, in the absence of H2O2, EDTA can also transfer terephthalic acid to
fluorescent 2ꢀhydroxyterephthalic acid under sunlight irradiation, as shown in Figure
2a. This implies that EDTA is capability of producing photoꢀelectrons under sunlight
irradiation, photoꢀgenerated electrons could react with O2 dissolved in the aqueous to
ꢀ
produce superoxide radical (O2 ·) and then oxidize the terephthalic acid. Furthermore,
the photocatalytic activity of EDTA was evaluated by degradation of organic dye in
water. The photoꢀdegradation of rhodamine B (RhB) was tested as a model reaction to
assess photocatalytic performance of EDTA under UV, visible light irradiation.
Figure 2b displays the photoꢀdegradation of RhB under UV light irradiation in the
presence of EDTA, in which the intensity of the dye gradually decreased along with
increasing irradiation time; finally, the orange color of RhB was almost disappeared.
Similar experiment carried out under the sunlight irradiation indicates the color of
RhB in the presence of EDTA was almost completely gone, although the color of
RhB itself faded in some degree under the sunlight irradiation (Figure 2d). The above
discussion elucidates that EDTA plays a critical role in the photoꢀdegradation. In
particular, under room light irradiation, EDTA exhibits much the same as that of
natural HRP enables it to be an excellent candidate to develop more effective assay.
Fabrication of plasmonic nanosensor based on peroxidase-like EDTA. As
peroxidaseꢀlike molecule, although EDTA may replace the natural enzyme in the
conventional ELISA to fabricate more costꢀeffective assay, it is challenge to improve
the sensitivity and shortening detection time. Herein, the unique GNRs were
employed as indicator of colorimetric assay to develop ultraꢀsensitive and rapid assay.
As anisotropic nanoparticles, GNRs possess two plasmon bands: one is transverse
plasmon wavelength (TPW) and another is longitudinal plasmon wavelength (LPW)
appearing in visible or NIR range.34,35 Unlike TPW, LPW depends on the aspect ratio
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of GNRs. Thus, the LPW may be fineꢀtuned accompanied by adjusting the aspect
ratio of nanorods. The hydroxyl radicals generated from Fenton reaction has been
demonstrated to readily etch GNRs,36 leading to the decrease of aspect ratio of GNRs
and blueꢀshift of LPW. The degree of change of LPW directly depends on the
concentration of H2O2. This means the GNRs may be an excellent indicator to detect
H2O2 and relative targets associated with H2O2.
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Figure 3a shows the color tonality of the GNRs resulted from the asꢀprepared
GNRs reacting with various amounts of H2O2. The H2O2ꢀmediated color variation of
GNRs from purple to red and the corresponding change of LPW (Figure 3b) are a
very useful source for the development of colorimetric assay. As described above,
both GNRs and EDTA may be connected with H2O2. If combined with
immuneꢀreaction by attaching the EDTA onto an antibody toward an antigen (analyte),
the competitive reactions simultaneously imposed on H2O2 will facilitate to develop
EDTAꢀregulated plasmonic sensor with visual readout.
A colorimetric assay by combining peroxidaseꢀlike EDTA and GNRs was
accordingly designed, as shown in Scheme 1. An analyte is first captured by its
antibody immobilized on a disposable substrate, followed by reacting with another
analyte (antigen) recognized by antibody labeled with EDTA. Subsequently, GNRs
and H2O2 are added into the sandwich structure to display color for visual readout. In
this process, part of the H2O2 can be decomposed by EDTA and the rest of H2O2
reacting with GNRs. The competitive reactions simultaneously imposed on H2O2 will
modulate aspect ratio of GNRs and in turn their color variation dependent on the
captured amount of the analyte, displaying different color for naked eye readout. On
the other hand, if the analyte is of absence, the sandwich structure cannot be formed
and there will be no EDTA involved the competitive reactions. This strategy is not
only able to sense the analyte based on the color indicator of GNRs but also capable
of quantitatively determining it from the change of LPW.
In the designed colorimetric EDTAꢀbased plasmonic sensor, GNRs play a role
serving as a visual readꢀout means to detect target analyte (Figure 3c). This is actually
an in situ strategy and adopted in the design of plasmonic sensor. To make full use of
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H2O2ꢀbased reactions and the color variation of GNRs as a readout means, the optimal
conditions including the concentrations of H2O2 and aspect ratio of asꢀprepared GNRs
were explored. As a result, the LPW of GNRs in the range of 740 ~780 nm and the
concentration of H2O2 range from 200 to 650 ꢁM H2O2 are ideal parameters in this
colorimetric assay.
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In fact, the reaction between H2O2 and GNRs is so slow that the blue shift of LPW
of GNRs induced by H2O2 will not completely finish after 2 hours, which enables the
competitive reactions not to be suitable for fast detection in practical application. The
previous study revealed the reaction rate of H2O2 and GNRs might be quickly
accelerated by the introduction of Fenton reaction.36 However, our experiment
manifested iron ions associated with Fenton reaction would interfere with the color
variation and shift of LPW of GNRs. Here, a simply method of increasing reaction
rate was introduced only by UV irradiation, without introduction of any other
reactions. UV light was demonstrated to photocatalyze the decomposition of H2O2 to
generate strong oxidizing hydroxyl radical, which might significantly shorten the
reaction time due to OH• quickly reacting with GNRs.37 The reaction time may be cut
down to 30 min, significantly less than the conventional ELISA reaction time. Further
experiment illustrated the blueꢀshift of GNRs might be neglected when asꢀprepared
GNRs irradiated under the same irradiation. Thus, the UV irradiation was adopted in
the following study.
To evaluate the feasibility of this sensor, determination of protein avidin was
performed. The sensing avidin is based on the biotinꢀavidinꢀbiotin sandwich structure.
Biotin conjugated goat IgG (Bioꢀgoat IgG) was first absorbed on wells of a 96ꢀwell
polystyrene plate. After blocked by bovine serum albumin (BSA), various
concentrations of avidin were added to the wells. Afterwards, EDTAꢀconjugated
Bioꢀgoat IgG was added to form sandwiched structure, followed by addition of
aqueous GNRs and H2O2 to display color change. Figure 4a displays a distinguishable
color variation among the sample wells. In contrast, the lack of avidin presents
blueꢀcolored dispersion of GNRs. When the concentration of avidin was 1.0×10ꢀ20 M,
the color was close to the control wells. Thus, the limit of detection (LOD) of the
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colorimetric detection was 1.0×10ꢀ20 M by naked eyes. This plasmonic immunoassay
is highlighted by its naked eye readout with ultraꢀsensitvity. Furthermore, it is even
simple in fabrication, manipulation, storage and costꢀsaving compared to ELISA.
Detection of CA15-3 in human serum samples. Breast cancer is common
malignant tumor for female disease. CA15ꢀ3 is one of the most important breast
tumor markers. The level of CA15ꢀ3 could provide direct information to monitor the
patients’ post operative status and it can also predict recurrence and metastasis of
breast cancer.38,39 Analogous to the detection of avidin, the antibody of CA15ꢀ3 was
adsorbed on 96ꢀwell polystyrene plates and blocked with BSA. Figure 4b presents
that the color of the GNRs is dependent on the amount of CA15ꢀ3, and the LOD was
7.52×10ꢀ15 U/mL by naked eyes. The ultraꢀsensitive nanosensor was further applied to
analyze practical human serum samples. 10 breast cancer serum samples were used to
validate the practical application for determination of CA15ꢀ3. Owing to the
ultraꢀsensitivity of this sensor, all the human serum samples were diluted by PBS
buffer in the experiments, and only 100 ꢀL of the diluted sample was added to the
plates. Compared with the reference, the differentiated color of GNRs were
expectedly observed, which indicate that target molecules were detected in these sera,
as shown in Figure 4c.
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Although the colorimetric assay seems perfectly suitable for detecting analytes with
fewer resources, it is inherently inaccurate for quantifying the concentration of the
target molecule merely by naked eyes. As a matter of fact, LPW of the resulting
GNRs directly related to the change of H2O2. Clearly, the difference of LPW between
samples and reference may be used to accurately detect the biomarker. Linear
relationship was found from the difference of LPW of GNRs (ꢁλ=λ1ꢀλ0) between
reference and samples with various concentrations. At the range of 0.5 ~ 10×10ꢀ12
U/mL, the equation is ꢁλ=0.1875+ 0.037c and the linear regression coefficient is
0.9924. Where, λ1 represents the LPW of the sample and λ0 is the LPW of reference.
All of the samples were analyzed by the proposed approach and the CA15ꢀ3
concentrations were displayed in Figure 4d.
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Five of these samples were firstly measured in the local hospital were in agreement
with the results given by this assay. This confirms that this sensing system is
applicable to detection of CA15ꢀ3 in breast cancer serum with high accuracy.
Furthermore, prostate specific antigen (PSA) of prostate cancer patients was measured
using this assay. The PSA level of 10 human serum samples was successfully detected
illustrated in Figure S2. The determination of these biomarkers indicates the
EDTAꢀbased assay is of ultraꢀsensitivity. Consistency of the results determined by the
proposed assay and found by hospital displays this ultraꢀsensitive and accurate assay
can be extended to detect other disease markers.
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The effect of complex components on EDTA-based colorimetric assay. The
above determination of CAꢀ153 and PSA in human serum samples elucidates the
EDTAꢀbased sensor exhibit a good selectivity. However, in any real biological
samples, there will be many different potential sources of nonꢀspecific false signals
which could reduce the accuracy significantly. Similarly, it is possible that the color
of control measurement may be affected by complex components. To investigate the
effect of possibly nonꢀspecific adsorption on this assay, human serum sample without
the target analyte (PSA) was employed to verify the selectivity and liability of the
control. A simple and rapid method for separation of PSA was to functionalize
magnetic particles with antiꢀPSA that selectively captured the PSA in the human
serum, and then pulled the magnetic particles out of the mixture upon applying a
magnetic field. The resulted PSAꢀfree human serum is very complex due to
containing a great number of proteins and other components, which served as
background in the process of detection of PSA. Three kinds of species, including
reference, PSAꢀfree human serum with varied concentrations and original human
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serum containing PSA, were tested as shown in Figure 5a. From the photograph,
reference (lane 1) and PSAꢀfree human serum (lane 2) almost have the same color.
Meanwhile, the microwells of the lane 2 almost exhibit the same color. The
phenomenon indicates other complex components of human serum have little effect
on the detection of PSA in this study. On the other hand, the original human serum
sample displays orange color owing to the presence of PSA. Meanwhile, a series of
PSA were added to the PSAꢀfree human serum, and then detect the PSA again using
EDTAꢀbased colorimetric assay. Figure 5b shows a gradually colored change with the
increasing PSA concentration, and a good linear relationship was found between the
difference of LPW of GNRs (ꢁλ) and PSA concentration. In addition, replicate
experiments in measurements of SPA shown in Figure S3 illustrate a good
reproducibility of this method.
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Determination of breast cancer cells by EDTA-based colorimetric assay.
Further application of the EDTAꢀbased colorimetric assay toward breast cancer cells
was performed. Epithelial cell adhesion molecule (EpCAM) is a membraneꢀbound
glycoprotein with oncogenic properties, which often correlates with more aggressive
tumor behavior. Highꢀlevel EpCAM expression was found in premalignant lesions
and carcinomas of various origins, such as prostate, colorectal, ovarian, lung and
breast cancer.40ꢀ44 EpCAM expression in MCFꢀ7 cell lines is relatively high. Thereby,
MCFꢀ7 cells were tested to assess possibility of detection of cancer cells by this
colorimetric assay. The antibody antiꢀEpCAM adsorbed on polystyrene plates was
employed to capture breast cancer cells by specific reacting with the EpCAM on the
surface of cancer cells. After the captured breast cancer cells specifically react with
the second antiꢀEpCAM conjugated with EDTA, the GNRs and H2O2 were added into
the polystyrene plates. Other row of microꢀwells without samples was used as control
experiment. That is to say, the same GNRs and H2O2 were added in this row. Figure
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6a shows a distinguishable color among the two groups and the LPWs of the resulting
GNRs also were obtained by multiꢀmode microplate reader, indicating the feasibility
of this assay for visual detection of cancer cells.
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With the progress of tumor, CTCs will appear in blood because rare tumor cells
disseminated from primary tumors or metastatic sites enter the bloodstream. CTCs are
believed to play a critical role in the spread of disease throughout the body. They are a
noninvasive and repeatedly accessible source of tumor material, providing clinically
feasible diagnostic and prognostic markers of cancer. The analysis of CTCs is more
readily accomplished than conventional biopsy approaches. Therefore, there has been
considerable interest in analyzing CTCs as a potential source of clinical information
related to patient diseases.45,46
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MCFꢀ7 cell lines were used as CTC model in the colorimetric assay. These cell
lines have suitable characteristics for effective isolation based on size and density, and
they are relatively large compared with other blood cells. Thus, these model CTCs
maximize the combination of large cells and many magnetic beads bound on cells.
Using antiꢀEpCAM–coated magnetic beads, CTCs can be extracted from blood, and
be further conjugated on subtract, followed by visual read out, as schematic
illustration shown in Figure 6b. In this process, two other rows of microꢀwells without
samples were used as control experiment. Namely, the same GNRs and H2O2 were
added in one row, and GNRs and H2O was added to another row. Figure 6c displays
the visual readꢀout of CTCs in polystyrene plate. Obviously, the color and LPWs of
the GNRs attributed to samples ranging between the two kinds of references, which
elucidate the breast cancer cells were successfully isolated by antiꢀEpCAM–coated
magnetic beads. Interestingly, there are two wells in the sample row exhibiting similar
color to the references. It is possible that no MCFꢀ7 cells were captured in the two
wells, and this was confirmed by the fact that no MCFꢀ7 cells were found in the two
wells under the observation of microscopy. As a result, combined with
immunomagnetic separation, the colorimetric assay is successful for the visual
readꢀout CTCs.
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In this study, we discovered and investigated the peroxidaseꢀlike activity of
conventional EDTA. A straightforward plasmonic nanosensor based on EDTA was
developed to enable nakedꢀeye observation and determination of ultraꢀtrace disease
biomarkers and cancer cells. Under light irradiation, EDTA may catalyze
decomposition of hydrogen peroxide and in situ regulate the LPW of GNRs in the
presence of H2O2, displaying various color solution as a readꢀout means. This
colorimetric nanosensor has a great potential to develop into a platform to
quantitatively determine analytes as long as the specific antibodies against them were
available. The detection of CA15ꢀ3 and SPA of cancer serum samples indicate this
assay was applied to detect different biomarkers of diseases with high accuracy. The
colorimetric EDTAꢀbased nanosensor can not only detect target molecules but also
visually determine cancer cells. Moreover, combined with immunomagnetic
separation of CTCs from blood, a visual readꢀout for determination of CTCs was
established, providing a potential and promising strategy for diagnostic and
prognostic information related to patient diseases. The EDTAꢀbased colorimetric
assay takes the advantage of simple in fabrication, manipulation, storage,
costꢀeffective and fast detection, which has potential important implications in clinical
diagnosis, environmental monitoring, and food quality control only using naked eyes.
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ACKNOWLEDGMENTS
This work was supported by National Natural Science Foundation of China
(21375036, 21675049), National Science Foundation for Distinguished Young
Scholars of China (51525207).
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Competing financial interests
The authors declare no competing financial interests.
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Scheme 1. Schematic illustration of EDTAꢀbased plasmonic nanosenor. In sandwich
structure the target molecule is anchored to the substrate by capture antibodies and
recognized by other antibodies labeled with EDTA.
Figure 1. a: Photographs for H2O2 and TMB reaction in the absence (left) and
presence (right) of EDTA under room light irradiation. b: Schematic illustration of
photocatlytic mechansim of EDTA. c: Fluorescence spectra of the terephthalic acid in
the presence/absence of EDTA and H2O2 under light irradiation. d: Fluorescence
intensity of 2ꢀhydroxyterephthalic acid originated from terephthalic acid in the
presence of EDTA at various irradiation time.
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Figure 2. a: Fluorescence spectra of the terephthalic acid in the presence/absence of
EDTA under sunlight irradiation. b: Images of RhB irradiated by sunlight in the
presence (i) /absence (ii) of EDTA, the left represents initial RhB and the right is after
the irradiation. c: Absorption spectra of RhB under the UV irradiation, i represents
original RhB before irradiation, ii is the RhB after UV irradiation, iii represents the
RhB and EDTA after the UV irradiation. d: Absorption spectra of RhB irradiated by
sunlight, i represents original RhB before irradiation, ii is the RhB after sunlight
irradiation, iii represents the RhB and EDTA after the sunlight irradiation.
Figure 3. a: Photograph obtained by the GNRs reacting with various concentration of
H2O2 and the corresponding LPW of the resulting GNRs (b). c: Photograph displayed
in bottom row is obtained by the GNRs reacting with H2O2 in different concentration
of EDTA, the up row is the GNRs as reference.
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Figure 4. Colorimetric determination by EDTAꢀbased nanosensor. a: Detection of
avidin, the up row is the reference obtained by GNRs reacting with H2O2 and the
bottom row is the various concentrations of avidin in the presence of GNRs and H2O2.
b: Detection of CA15ꢀ3. c: Detection of CA15ꢀ3 of breast cancer serum samples, the
middle row is the reference and other represent the practical samples. d: The levels of
CA15ꢀ3 in sera of breast cancer by different methods.
Figure 5. Investigation of liability on EDTAꢀbased assay. a: Comparison of reference,
PSAꢀfree human serum and human serum containing PSA: lane 1 is the reference,
lane 2 represents the reference solution added various amounts of PSAꢀfree human
serum (from left to right, the added human serum is diluted 0, 2, 4, 8, 16, 32, 64, 128
times), lane 3 is the original human serum containing PSA. b: Colorimetric
determination of various concentrations of PSA added into the PSAꢀfree human
serum. c: Calibration curve of the difference of LPW of GNRs vs. concentration of
PSA corresponding to (b), and the equation is ꢁλ=2.7077+ 109c (R2=0.9937). Error
bars indicate the standard deviation of three independent measurements.
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Figure 6. Determination of breast cancer cells by EDTAꢀbased colorimetric
plasmonic nanosensor. a: Photograph of the detection of MCFꢀ7 cell lines by
colorimetric detection, and the LPW (nm) of GNRs were obtained by MultiꢀMode
Microplate Reader. b: A schematic illustration of visual readꢀout for determination of
CTCs by combined EDTAꢀbased sensor with immunomagnetic separation of CTCs
from blood. c: Photograph of the detection of CTCs in human blood, and the LPWs of
references are average.
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Analytical Chemistry
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