Journal of Immunological Methods xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Journal of Immunological Methods

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

Research paper

A copper based enzyme-free fluorescence ELISA for HER2 detection

Shiyu Tiana, Ke Zenga, Aijun Yangb, Qin Wangb, Minghui Yanga,⁎
a
College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
b
Center for Reproductive medicine, Affiliated Hospital of Jining Medical University, Jining, Shandong 272029, China

A R T I C L E I N F O A B S T R A C T

Keywords: We reported an enzyme-free ELISA to detect breast cancer biomarker human epidermal growth factor receptor 2
Fluorescence (HER2) in human serum samples. Instead of enzymes (such as horseradish peroxidase) used in traditional ELISA,
Breast cancer CuO nanoparticles were utilized as the signal probe. Compared to traditional enzymes, CuO nanoparticles have
ELISA the advantages of low cost and good stability. After dissolving CuO nanoparticles with acid, the Cu (II) ions
CuO nanoparticle
generated catalyzed the reaction of o-phenylenediamine with ascorbic acid to produce fluorescent quinoxaline
Serum
derivative molecules. The immunoassay displays high sensitivity and good selectivity towards HER2 with de-
tection limit as low as 9.65 pg·mL− 1. The assay was successfully applied to the analysis of HER2 in serum of
breast cancer patients. The analysis results demonstrated the HER2 level in the serum samples determined by our
assay were in good agreement with those determined by commercial HER2 ELISA kit. This enzyme-free ELISA
assay can be easily adapted to the detection of other analytes. With these merits, the simple, sensitive and cost
effective fluorescence immunoassay shows great potential for clinical applications.

1. Introduction
The enzyme-linked immunosorbent assay (ELISA), a well developed
testing method for detecting antibodies/antigens or different other in-
dicators, is considered to be the most popular analysis tool in hospital
diagnosis, medicinal and food industry (Zeng et al., 2017; Satija et al.,
2016; Li et al., 2013; Yang et al., 2014). ELISA has advantages of good
specificity, low cost and high-throughput compared to other methods,
such as electrochemistry, surface-enhanced Raman spectroscopy (SERS)
and surface plasmon resonance (SPR) (Kiyota et al., 2017; Pappert
et al., 2010). In traditional ELISA, enzymes, such as horseradish per-
oxidase and alkaline phosphatase are often employed to generate am-
plified detection signals (He et al., 2017; Shen et al., 2016; Guo et al.,
2017). However, the enzymes are usually expensive and the catalytic
activity of enzymes is sensitive to the environmental changes, such as
temperature and acidity. Enzyme denaturation caused by these reasons
will make ELISA results inaccurate and lack of reproducibility.
Enzyme-free ELISA is then developed to solve the above issues. In
enzyme-free ELISA, inorganic or small organic substances are used in-
stead of enzymes to be linked onto binding molecules to catalyze spe-
cific reaction and generate amplified detection signals. These kinds of
substance are usually much more stable and cost effective than en-
zymes. Such substances include DNA and different nanomaterials (Hu
et al., 2015; Li et al., 2009; Si et al., 2014).
Breast cancer is the highest incidence of women malignant
carcinoma and the second leading cause of cancer death in women in
United States. Human epidermal growth factor receptor 2 (HER2), en-
coded by the ErbB2 gene, is one of the human epidermal growth factor
receptor family members (Hu et al., 2017; Ngamcherdtrakul et al.,
2016; Elster et al., 2015). It is found that the fluctuation of HER2
content is closely related to the occurrence of breast cancer. According
to the statistical data, up to 30% of breast cancer patients are accom-
panied with the over-expression of the HER2 (Yang et al., 2017; Ross
and Fletcher, 1998). Therefore, it is important to develop methods for
precise detection of HER2 protein for early breast cancer diagnosis.
Two FDA-approved detective methods are applied to clinical testing of
HER2, immunohistochemistry (IHC) and fluorescence in situ hy-
bridization (FISH). These two methods focus on the cellular HER2
analysis that require medical infrastructure, pathology, and cytology
expertise which is not accessible to some region with few medical
equipment especially in developing countries. Recent studies have
shown that extracellular HER2 value also reflects the status of breast
cancer. The serum HER2 concentration was elevated in 20%–50% of
patients with primary breast cancer and 50%–62% for metastatic
cancer (Mitri et al., 2012). Normal individuals have a HER2 con-
centration between 2 and 15 ng·mL− 1 in the blood, and breast cancer
patients have blood HER2 levels from 15 to 75 ng·mL− 1. Our group
recently developed an approach for detecting extracellular HER2 by
using electrochemical aptasensor based on DNA generated electro-
chemical current (Hu et al., 2017). However, efforts are still needed to

⁎
Corresponding author.
E-mail address: yangminghui@csu.edu.cn (M. Yang).

http://dx.doi.org/10.1016/j.jim.2017.09.002
Received 30 July 2017; Received in revised form 22 August 2017; Accepted 11 September 2017
0022-1759/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Tian, S., Journal of Immunological Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.09.002
S. Tian et al.
Fig. 1. Schematic illustration of the copper ion based ELISA.
develop simple and easy operating methods for HER2 analysis in serum
samples.
In this work, we reported an enzyme-free ELISA for HER2 detection
utilizing copper oxide nanoparticles (CuO NPs) as signal amplification
probe. After dissolving CuO NPs with acid, the generated Cu (II) ions
catalyzed the reaction of o-phenylenediamine (OPDA) with ascorbic
acid (AA) to produce fluorescent quinoxaline derivative molecules. The
new ELISA is easy to operate, highly sensitive and selective to HER2
contents in serum samples of breast cancer patients, which means the
assay has wide potential clinical applications.
2. Experimental
2.1. Regents and equipment
The polyclonal rabbit anti-HER2 antibody was purchased from
Sangon Biotech Company (Shanghai, China). Peptide with sequence of
CKLRLEWNR was synthesized from GL Biochem Co. Ltd. (Shanghai,
China). HER2 proteins and L-ascorbic acid (AA) were obtained from
Abcam (Cambridge, UK). CuO nanoparticles were purchased from
Sigma–Aldrich. All other reagents used were of analytical grade.
Double-deionized water was used throughout the experiments. A
0.01 M phosphate buffer (pH 7.4) containing 0.1% Tween 20 was used
as the washing buffer.
Transmission electron microscope (TEM) images of CuO were ob-
tained from Tecnai G2 60-300 (FEI. Co. Ltd). UV–Vis experiments were
performed on a Shimadzu UV2450 spectrophotometer (Shimadzu,
Japan). Size distribution data were obtained using Zetasizer ZEN 3600
(Malvern Instruments Ltd). The fluorescence spectra were recorded on a
fluorescence spectrophotometer (Hitachi, F-7000). The electrospray
ionization mass spectrometry (ESI-MS) data were measured by Bruker
compact mass spectrometry. The 1H NMR was tested by Bruker ascend
400 NMR spectrometer.
2.2. Preparation of CuO–peptide conjugates
The CuO–peptide conjugates were prepared according to a previous
report (Qu et al., 2011). Briefly, about 0.75 mg CuO NPs were added
into 7.5 mL phosphate buffer followed by ultrasonication for about
30 min. Then, 750 μg·mL− 1 peptide solution (0.5 mL) was added into
2
Journal of Immunological Methods xxx (xxxx) xxx–xxx
the CuO NPs solution and then vortex-shaken for about 3 h at 37 °C to
conjugate peptide onto the CuO. Finally, the solution was centrifuged
for 10 min at 12,000 rpm to remove the excess peptide and the pre-
cipitate was re-dispersed in 7.5 mL phosphate buffer. Then, 200 μL of
25 mM 6-mercapto-1-hexanol (MCH) solution was added into 800 μL
peptide modified CuO NPs solution, and after 4.5 h stirring at room
temperature, unbounded MCH were removed by centrifuge. Finally, the
obtained nanoparticles were re-dispersed in 800 μL of pH 7.4 buffer and
stored at 4 °C when not in use.
2.3. ELISA measurements
The 5 μg·mL− 1 anti-HER2 antibody (100 μL) was added into the
wells of the 96-well plate and incubated overnight. The plate was wa-
shed with phosphate buffer containing 0.1% Tween 20 three times and
the wells were then blocked with 1 mg·mL− 1 BSA for 1.5 h at 37 °C.
After washing again, 100 μL of various concentrations of HER2 stan-
dard solutions were added into the plate and incubated for 3 h at 37 °C.
Similarly, the plate was washed and then, 100 μL peptide modified CuO
NPs were added and incubated for 2 h at 37 °C. After another round of
washing, 50 μL HCl solution (pH = 2) was added into each well and
reacted for 30 min at room temperature under shaking to dissolve the
CuO nanoparticles. The resulting acid solution containing the dissolved
Cu2 + ions was then reacted with 200 μL solution containing AA
(0.5 mM) and OPDA (6.5 mM). After another 30 min shaking at room
temperature, the fluorescence emission spectra were collected at the
excitation wavelength of 347 nm.
3. Results and discussion
3.1. Feasibility study
The schematic representation of the fluorescence enzyme-free ELISA
immunoassay is shown in Fig. 1. We occasionally found acid solution
containing OPDA and AA can produce strong fluorescence emission at
around 430 nm (excited at 347 nm) in the presence of either Cu (II) or
CuO nanoparticles. However, the fluorescence intensity generated due
to the same amount of Cu (II) was about two times higher than that due
to CuO nanoparticles. So the catalytic behavior of Cu (II) towards OPDA
and AA was investigated. To study this phenomenon, UV–Vis spectra
S. Tian et al.
0.6
A OPDA+AA
0.4
Absorbance (a.u.)
0.2
0.0
300
8000
B OPDA
Fluorecence intensity (a.u.)
6000
4000
2000
0
400
Wavelength (nm)
Fig. 2. UV–Vis spectra (A) and fluorescence spectra (B) of acid solution (pH = 2) con-
taining different compounds.
and fluorescence spectra of the mixing solutions were recorded. As
shown in Fig. 2A, for UV–Vis spectrum, a broad peak is appeared at
350 nm for acid solution (pH = 2) containing 100 μM Cu (II), 6.5 mM
OPDA and 0.5 mM AA. No absorbance were observed in the absent of
Cu (II), which means the reaction between OPDA and AA did not occur
without Cu (II). The fluorescence data was also in consistent with the
above results (Fig. 2B). The fluorescence intensity of the mixture so-
lution was increased either with the increase of reaction time or with
the increase of Cu (II) concentrations (Supporting information, Fig. S1).
These data indicated that the three components, Cu (II), OPDA as well
as AA are all indispensable for the generation of fluorescence, and Cu
(II) was acted as catalyst to catalyze the reaction between OPDA and
AA. The generated fluorescence compound was believed to be qui-
noxaline derivative.
To prove the formed fluorescence molecules were quinoxaline de-
rivative, the formed molecules were characterized by electrospray io-
nization mass spectrometry (ESI-MS) and nuclear magnetic resonance
spectroscopy (1H NMR). OPDA was commonly employed for the de-
tection of AA in the presence of oxidants such as ascorbate oxidase,
tempol and hydroxide (Vislisel et al., 2007; Sánchez-Mata et al., 2000;
Wu et al., 2003). In this assay, the oxidant was substituted by Cu (II).
We suppose in this system, Cu (II) was first reacted with AA to form
dehydro-ascorbic acid (DHAA), and then the condensation reaction of
OPDA with DHAA occurred to produce 3-(dihydroxyethyl)furo[3,4-b]
quinoxaline-1-one (abbreviated as quinoxaline-one, Fig. 3). The ESI-MS
data was obtained using the mixed solution containing OPDA/AA/
Cu2 + without further separation. [Quinoxaline-one + H]+ was found
Journal of Immunological Methods xxx (xxxx) xxx–xxx
at a characteristic ion peak of m/z 247.07. For NMR characterization,
AA
OPDA the characteristic peaks of quinoxaline protons were observed as a
doublet peak at 6.09 ppm of protons 1 and a multiple peak at 8.09 ppm
OPDA+Cu (II) of protons 2–5(Supporting information, Fig. S2). Combining the 1H
AA+Cu (II)
NMR with ESI-MS results, we confirm the formed fluorescence molecule
OPDA+AA+ Cu (II)
is quinoxaline-one.
In order to optimize the performance of the assay, different para-
meters that affect the fluorescence of the system were optimized, such
as the molar ratio of OPDA versus AA and the pH of solution for Cu (II)
catalyzed reaction. More details are shown in supporting information
(Supporting information, Figs. S3–S4). The optimal pH value for the
reaction was found to be 2 and the ratio of OPDA/AA was 13:1.
Based on the above results, CuO nanoparticles were utilized to carry
out the ELISA assy. The dissolving of CuO nanoparticles by acid will
350 400 produce Cu (II) ions. The CuO NPs utilized were characterized by SEM
Wavelength (nm) and TEM, which indicated the spherical morphology of the CuO NPs
(Fig. 4). The average size of CuO NPs was 104 nm calculated by dy-
namic light scattering (DLS) (Supporting information, Fig. S5). To
perform the ELISA assay, peptides with the sequence of CKLRLEWNR
that can bind specifically with HER2 molecules were conjugated onto
AA
CuO to turn the CuO NPs into signal probe (Hu et al., 2017). The thiol
moiety on the peptide can form SeCu bond with Cu atom on the surface
OPDA+AA
OPDA+Cu (II) of CuO and the MCH immobilized onto the CuO surface can prevent
non-specific adsorption between the nanoparticles and molecules in
AA+Cu (II)
OPDA+AA+Cu (II) detection solution. With the immobilization of anti-HER2 antibodies
into the wells of the 96 plate, the following specific capture of HER2
molecules and then CuO probe, the Cu (II) generated by dissolving CuO
probe will then catalyze the reaction of OPDA and AA to generate
fluorescence. The resulted fluorescence intensity is proportional to the
HER2 concentration in a specific range.
3.2. The performance of the assay
440 480 520 Under the optimized experimental conditions, different concentra-
tions of HER2 standard solutions were analyzed to test the linear range
of the assay towards HER2. As can be seen from Fig. 5, the fluorescence
intensity increased with the increase of HER2 concentrations and a
good linear relationship for HER2 was obtained range from 25 pg·mL− 1
to 5 ng·mL− 1 with R square value of 0.996 (inset of Fig. 5). The limit of
detection of the assay for HER2 was calculated to be about
9.65 pg·mL− 1 based on S/N of 3. The American Medical Association
(AMA) has approved a serum HER-2 oncoprotein test intended to
quantitatively measure HER-2 protein in serum with reference range
(s) < 12.8 ng·mL− 1. The sensitivity of this ELISA means it can be
applied for clinical testing of HER2 in diluted samples.
The analytical performances of our assay were compared with lit-
erature reported assays for HER2 detection. From Table 1, it can be seen
the performance of our assay were comparable or even better than
literature reports. In addition, this enzyme-free ELISA has advantages of
low cost and high-throughput.
In addition of sensitivity, good selectivity is of great importance for
precise detection of target analytes in complex samples. Different pro-
teins and molecules that present in the serum samples, such as the β-site
amyloid precursor protein cleaving enzyme 1 (BACE1), human IgG,
mucin 1 (MUC1), carcinoembryonic antigen (CEA) and P53 were uti-
lized as potential interferents to study the selectivity of the assay. The
concentration of HER2 tested was 100 pg·mL− 1 while the concentration
of the above potential interferents studied was 10 times higher than
HER2. The fluorescence intensity occurred by the above potential in-
terferents were negligible when compared to HER2 (Fig. 6), indicating
good selectivity of the assay.
3.3. Real sample analysis
To further prove the potential of the assay in clinical applications,
eight serum samples from four different breast cancer patients were
3
S. Tian et al. Journal of Immunological Methods xxx (xxxx) xxx–xxx

Fig. 3. The possible mechanism of the reaction process use Cu2 + as catalyst.

obtained. For each patient, two samples were collected, one collected
before tumor removal surgery, and one collected 5 days after tumor
removal surgery. The HER2 contents in the 8 samples were determined
by commercial HER2 ELISA kit and our assay. To test the samples by
our assay, the serum samples were diluted 500 times, which can lower
sample consumption (< 1 μL of serum samples is needed, lower than
that needed for commercial assay) and minimize sample matrix effect.
The HER2 concentrations acquired by our assay were in good consistent
with the value obtained from the commercial kits (as shown in Table 2).
In addition, the testing results demonstrated the HER2 levels of the four
patients are all dropped after surgery. These results demonstrated the
assay has wide potential clinical application in diagnosis and in breast
cancer therapy.

Fig. 4. SEM and TEM (inset) image of the CuO NPs.

4. Conclusion

In summary, we reported an enzyme-free ELISA strategy for HER2

2500 5 ng/mL 2000
Fluorescence intensity

detection. The enzyme used in conventional ELISA is replaced with

1600
inorganic CuO nanoparticles. The assay shows low detection of limits of
Fluorencence intensity (a.u.)

2000 1200 9.65 pg·mL− 1 and a wide linear relationship for detecting HER2 from
0 pg/mL 800 25 pg·mL− 1 to 5 ng·mL− 1. Compared with traditional ELISA im-
1500 400 munoassay, our strategy of using CuO nanoparticle as signal probe
lowered the cost and improved the stability of the assay. By replace the
10 100 1000 10000
Concentration of HER2 (pg/mL) binding ligands, this assay can be extended to detect other kinds of
1000
biomarkers and find wide clinical applications. However, further efforts
are still needed to simplify the detection procedures, for example, using
500 nanoparticles itself to trigger the generation of fluorescence.

0
350 400 450 500 550 Acknowledgments
Wavelength (nm)
The authors thank the support of this work by the National Key
Fig. 5. The fluorescence response of the enzyme-free ELISA towards different con- Basic Research Program of China (2014CB744502), the National
centrations of HER2. The inset is the calibration curve of the assay towards HER2. Error
Natural Science Foundation of China (No. 21575165), the Natural
bar = RSD (n = 3).
Science Foundation of Shandong Province (NO: ZR2012HL002 and NO:
ZR2015HL022) and the Open Foundation of State Key Laboratory of
Chemo/Biosensing and Chemometrics of Hunan University (2016008).

4
S. Tian et al. Journal of Immunological Methods xxx (xxxx) xxx–xxx

Table 1
Compare the performance of the assay towards HER2 with literature reports.

Detection method Signal tag Linear range (ng·mL− 1) Detection limit (ng·mL− 1) Reference

Fluorescence CdSe/ZnS nanocrystal \ 8 (Qiu et al., 2016)

PEMS None 0.05–2 0.0253 (Loo et al., 2011)
PEMS None 6–60 5 (Capobianco et al., 2011)
Fluorescence Silica nanospheres 1–10,000 1 (Rossi et al., 2006)
Fluorescence Photonic crystals 3–200 0.3 (Sinibaldi et al., 2017)
Electrochemical Magnetic beads 0–15 6 (Mitri et al., 2012)
Optical Resonator None 13–100 11 (Gohring et al., 2010)
Fluorescence CuO nano 0.025–5 0.00965 This work

PEMS = piezoelectric microcantilever.

1000 the OptoFluidic ring resonator biosensor. Sensors Actuators B Chem. 146, 226–230.
Guo, L., Chen, D., Yang, M., 2017. DNA-templated silver nanoclusters for fluorometric
determination of the activity and inhibition of alkaline phosphatase. Microchim. Acta
184, 2165–2170.
He, S., Li, X., Gao, J., Tong, P., Chen, H., 2017. Development of sandwich ELISA for
FLuorescence intensity

750 testing bovine beta-lactoglobulin allergenic residues by specific polyclonal antibody

against human IgE binding epitopes. Food Chem. 227, 33–40.
Hu, R., Liu, T., Zhang, X.-B., Yang, Y., Chen, T., Wu, C., et al., 2015. DLISA: a DNAzyme-
based ELISA for protein enzyme-free immunoassay of multiple analytes. Anal. Chem.
87, 7746–7753.
500 Hu, L., Hu, S., Guo, L., Shen, C., Yang, M., Rasooly, A., 2017. DNA generated electric
current biosensor. Anal. Chem. 89, 2547–2552.
Kiyota, K., Kawatsu, K., Sakata, J., Yoshimitsu, M., Akutsu, K., Satsuki-Murakami, T.,
et al., 2017. Development of monoclonal antibody-based ELISA for the quantification
of orange allergen Cit s 2 in fresh and processed oranges. Food Chem. 232, 43–48.
250 Li, J., Zhang, T., Ge, J., Yin, Y., Zhong, W., 2009. Fluorescence signal amplification by
cation exchange in ionic nanocrystals. Angew. Chem. Int. Ed. 48, 1588–1591.
Li, D., Ying, Y., Wu, J., Niessner, R., Knopp, D., 2013. Comparison of monomeric and
polymeric horseradish peroxidase as labels in competitive ELISA for small molecule
detection. Microchim. Acta 180, 711–717.
0 Loo, L.N., Capobianco, J.A., Wei, W., Gao, X., Wan, Y.S., Shih, W.H., et al., 2011. Highly
BACE1 IgG MUC1 CEA P53 HER2 sensitive detection of HER2 extracellular domain in the serum of breast cancer pa-
tients by piezoelectric microcantilevers. Anal. Chem. 83, 3392.
Different samples Mitri, Z., Constantine, T., O'Regan, R., 2012. The HER2 receptor in breast cancer: pa-
thophysiology, clinical use, and new advances in therapy. Chemother. Res. Pract.
Fig. 6. Selectivity of the enzyme-free ELISA for HER2 over other potential interferents. 2012, 743193.
Error bar = RSD (n = 3). Ngamcherdtrakul, W., Castro, D.J., Gu, S., Morry, J., Reda, M., Gray, J.W., et al., 2016.
Current development of targeted oligonucleotide-based cancer therapies: perspective
on HER2-positive breast cancer treatment. Cancer Treat. Rev. 45, 19–29.
Table 2 Pappert, G., Rieger, M., Niessner, R., Seidel, M., 2010. Immunomagnetic nanoparticle-
Determination of HER2 in breast cancer patients before and 5 days after surgery by the based sandwich chemiluminescence-ELISA for the enrichment and quantification of
E. coli. Microchim. Acta 168, 1–8.
enzyme-free assay and commercial HER2 kit.
Qiu, X., Wegner, K.D., Wu, Y., Henegouwen, P.M.P.V.B.E., Jennings, T.L., Hildebrandt,
N., 2016. Nanobodies and antibodies for duplexed EGFR/HER2 immunoassays using
Sample Status Commercial ELISA assay Our assay (ng·mL− 1) Terbium-to-Quantum Dot FRET. Chem. Mater. 28, 8256–8267.
(ng·mL− 1) Qu, W., Liu, Y., Liu, D., Wang, Z., Jiang, X., 2011. Copper-mediated amplification allows
readout of immunoassays by the naked eye. Angew. Chem. Int. Ed. 50, 3442–3445.
1 Before surgery 42.00 ± 4.04 40.70 ± 3.14 Ross, J.S., Fletcher, J.A., 1998. The HER-2/neu oncogene in breast cancer: prognostic
5 days after 38.45 ± 3.38 38.17 ± 4.25 factor, predictive factor, and target for therapy. Stem Cells 16, 413–428.
surgery Rossi, L.M., Shi, L., Rosenzweig, N., Rosenzweig, Z., 2006. Fluorescent silica nanospheres
for digital counting bioassay of the breast cancer marker HER2/nue. Biosens.
2 Before surgery 39.53 ± 4.70 36.2 ± 3.74
Bioelectron. 21, 1900–1906.
5 days after 31.13 ± 2.47 27.04 ± 2.75 Sánchez-Mata, M.C., Cámara-Hurtado, M., Díez-Marqués, C., Torija-Isasa, M.E., 2000.
surgery Comparison of high-performance liquid chromatography and spectrofluorimetry for
3 Before surgery 70.67 ± 9.06 59.78 ± 3.06 vitamin C analysis of green beans (Phaseolus vulgaris L.). Eur. Food Res. Technol. 210,
5 days after 54.52 ± 8.40 43.83 ± 4.05 220–225.
surgery Satija, J., Punjabi, N., Mishra, D., Mukherji, S., 2016. Plasmonic-ELISA: expanding hor-
4 Before surgery 34.83 ± 1.73 37.22 ± 2.88 izons. RSC Adv. 6, 85440–85456.
5 days after 33.52 ± 2.55 32.00 ± 3.23 Shen, C., Li, X., Rasooly, A., Guo, L., Zhang, K., Yang, M., 2016. A single electrochemical
biosensor for detecting the activity and inhibition of both protein kinase and alkaline
surgery
phosphatase based on phosphate ions induced deposition of redox precipitates.
Biosens. Bioelectron. 85, 220–225.
Results are mean ± S.D. (n = 5). Si, Z., Li, Y., Quan, H., Qi, H., Li, T., Yang, M., 2014. ELISA type fluorescence turn-on
immunoassay based on Fe3 + induced fluorescence enhancement. Sensors Actuators
B Chem. 202, 663–666.
Appendix A. Supplementary data Sinibaldi, A., Sampaoli, C., Danz, N., Munzert, P., Sibilio, L., Sonntag, F., et al., 2017.
Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/
fluorescence platform based on Bloch surface waves. Biosens. Bioelectron. 92,
Supplementary data to this article can be found online at http://dx.
125–130.
doi.org/10.1016/j.jim.2017.09.002. Vislisel, J.M., Schafer, F.Q., Buettner, G.R., 2007. A simple and sensitive assay for as-
corbate using a plate reader. Anal. Biochem. 365, 31–39.
Wu, X., Diao, Y., Sun, C., Yang, J., Wang, Y., Sun, S., 2003. Fluorimetric determination of
References ascorbic acid with o-phenylenediamine. Talanta 59, 95–99.
Yang, M., Yi, X., Wang, J., Zhou, F., 2014. Electroanalytical and surface plasmon re-
Capobianco, J., Shih, W.Y., Adams, G.P., Shih, W., 2011. Label-free Her2 detection and sonance sensors for detection of breast cancer and Alzheimer's disease biomarkers in
dissociation constant assessment in diluted human serum using a longitudinal ex- cells and body fluids. Analyst 139, 1814.
tension mode of a piezoelectric microcantilever sensor. Sensors Actuators B Chem. Yang, H.-Y., Ma, D., Liu, Y.-R., Hu, X., Zhang, J., Wang, Z.-H., et al., 2017. Impact of
160, 349–356. hormone receptor status and distant recurrence-free interval on survival benefits
Elster, N., Collins, D.M., Toomey, S., Crown, J., Eustace, A.J., Hennessy, B.T., 2015. from trastuzumab in HER2-positive metastatic breast cancer. Sci. Report. 7.
HER2-family signalling mechanisms, clinical implications and targeting in breast Zeng, K., Tian, S., Wang, Z., Shen, C., Luo, J., Yang, M., et al., 2017. An ELISA for the
cancer. Breast Cancer Res. Treat. 149, 5–15. determination of human IgG based on the formation of a colored iron(II) complex and
Gohring, J.T., Dale, P.S., Fan, X., 2010. Detection of HER2 breast cancer biomarker using photometric or visual read-out. Microchim. Acta 184, 2791–2796.