Peroxidase-Like Activity of Ethylene Diamine Tetraacetic Acid and Its

Subscriber access provided by NEW YORK UNIV

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

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, 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: [email protected] Xuanyong Liu：[email protected]

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 peroxidase-like property may extend the applications of conventional EDTA in life science. A novel and colorimetric sensor based on the peroxidase-like 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 read-out 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 read-out for detection of CTCs was established, which has promising applications in clinical diagnosis, environmental monitoring, and food quality control only using naked eyes.


Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION 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.1-5 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.9-14 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.15-20 There are six lone-pair 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 peroxidase-like EDTA suggests a cost-effective and easy storage mimic enzyme with small molecule will provide promising applications in life science. Enzyme-linked immunosorbent assay (ELISA) is often extensively used in clinical diagnosis, environmental monitoring and food quality control.21-24 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.25-29 Compared to the natural enzyme, the enzyme mimetic nanoparticles have advantageous of easy preparation, purification, stable and storage. Whereas, the nanoparticles-based biosensor remains



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 24

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, peroxidase-like 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 ELISA-like assay aroused the interest of researchers.30-32 In this paper, we firstly investigated the peroxidase-like activity of EDTA. Then, a novel and colorimetric sensor based on the peroxidase-like EDTA and gold nanorods (GNRs) was designed, allowing naked-eye readout and determination of ultra-trace disease biomarkers. Moreover, combined with immunomagnetic separation of circulating tumor cells (CTCs) from blood, a visual read-out 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

(HAuCl4·3H2O),

Reagents.

Hydrogen

peroxide

3,3,5,5-tetramethylbenzidine

(H2O2),

(TMB),

chloroauric

acid

N-hydroxysuccinimide

(NHS), ethylcarbodiimide hydrochloride (EDC), 2- (N-morpholino) 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 anti-PSA was received from Shanghai Ji Ning Industrial Co., Ltd (Shanghai, China). Magnetic microbeads (CSMN


Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Beads-1000,

1000

µm)

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, anti-PSA and BSA were prepared using 0.01mol/L phosphate buffered saline (PBS) solution. Preparation of gold nanorods. silver-assisted growth method.

GNRs were synthesized using a seed-mediated

Briefly, 100 µL of ice cold 0.01M NaBH4 was added

to 100 µL 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 µL 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 Bio-goat-IgG, CA15-3 antibody, PSA antibody, anti-EpCAM, via the –NH2 of amino acid residues on protein surface. Modification of the anti-bodies 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.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The anti-EpCAM-conjugated beads were created by first mixing carboxyl group-coated magnetic beads with 75 mM EDC and 15 mM NHS and incubated for 20 min at room temperature. Then 500 µL of 0.02 mg/mL anti-EpCAM was added to 4 mL of the activated magnetic beads solution and incubated for 1 h. Subsequently, 500 µL 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. Detection of avidin, CA15-3, and PSA. 96-well polystyrene plates were modified with 350 µL, 0.004 mg/mL antibody (Bio-goat-IgG or CA15-3 antibody or PSA antibody) solution at 4℃ overnight. The plates were blocked with blocking buffer (350µL 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 µL analyte was added. After 3 h, the plates were washed three times with wash buffer, 300 µL of CA15-3 antibody labeled with EDTA was added for 1 h at room temperature. After the all the plates were then washed three times, 250 µL of hydrogen peroxide (500 µmol/L) and 100 µL 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 CA15-3. The photographs were taken after 30 min and the LPW of the resultant GNRs was recorded with an UV-Vis spectrophotometer.


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 CA15-3 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. 300µL diluted sample solutions were added to wells in the procedure of experiments. Cell Culture. The cancer cell line human breast cancer cells (MCF-7) were obtained from Cell Bank of Chinese Academy of Science, and cultured in the RPMI-1640 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 1:2-1:3 every 2-4 days. CTC isolation. To isolate CTCs from cell-spiked blood, a pre-determined 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

malignant cells/ml of whole blood. 10 µL of magnetic microbead solution was used for each test.

RESULTS AND DISCUSSION The peroxidase-like activity of EDTA. The peroxidase-like activity of EDTA was tested with the peroxidase-required substrate of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2. Figure 1a shows that EDTA catalyzed the oxidation of TMB by H2O2 to produce oxidized TMB (ox-TMB), 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 photo-generated electrons, which are trapped by H2O2 to produce OH• radicals. Subsequently, the generated OH• radical quickly reacts with TMB to form ox-TMB, 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 Ox-TMB, 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 2-hydroxyterephthalic 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


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


generated hydroxyl radicals. Figure 1d depicts that fluorescence intensity varied from different irradiation time at certain amount of EDTA. Similarly, the produced 2-hydroxyterephthalic 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. To our surprise, in the absence of H2O2, EDTA can also transfer terephthalic acid to fluorescent 2-hydroxyterephthalic acid under sunlight irradiation, as shown in Figure 2a. This implies that EDTA is capability of producing photo-electrons under sunlight irradiation, photo-generated 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 photo-degradation 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 photo-degradation 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 photo-degradation. 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 peroxidase-like molecule, although EDTA may replace the natural enzyme in the conventional ELISA to fabricate more cost-effective 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 ultra-sensitive 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of GNRs. Thus, the LPW may be fine-tuned 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 blue-shift 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. Figure 3a shows the color tonality of the GNRs resulted from the as-prepared GNRs reacting with various amounts of H2O2. The H2O2-mediated 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 immune-reaction by attaching the EDTA onto an antibody toward an antigen (analyte), the competitive reactions simultaneously imposed on H2O2 will facilitate to develop EDTA-regulated plasmonic sensor with visual readout. A colorimetric assay by combining peroxidase-like 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 EDTA-based plasmonic sensor, GNRs play a role serving as a visual read-out 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


Page 10 of 24

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H2O2-based reactions and the color variation of GNRs as a readout means, the optimal conditions including the concentrations of H2O2 and aspect ratio of as-prepared 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 µM H2O2 are ideal parameters in this colorimetric assay. 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 blue-shift of GNRs might be neglected when as-prepared 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 biotin-avidin-biotin sandwich structure. Biotin conjugated goat IgG (Bio-goat IgG) was first absorbed on wells of a 96-well polystyrene plate. After blocked by bovine serum albumin (BSA), various concentrations of avidin were added to the wells. Afterwards, EDTA-conjugated Bio-goat 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 blue-colored dispersion of GNRs. When the concentration of avidin was 1.0×10-20 M, the color was close to the control wells. Thus, the limit of detection (LOD) of the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

colorimetric detection was 1.0×10-20 M by naked eyes. This plasmonic immunoassay is highlighted by its naked eye readout with ultra-sensitvity. Furthermore, it is even simple in fabrication, manipulation, storage and cost-saving compared to ELISA. Detection of CA15-3 in human serum samples. Breast cancer is common malignant tumor for female disease. CA15-3 is one of the most important breast tumor markers. The level of CA15-3 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 CA15-3 was adsorbed on 96-well polystyrene plates and blocked with BSA. Figure 4b presents that the color of the GNRs is dependent on the amount of CA15-3, and the LOD was 7.52×10-15 U/mL by naked eyes. The ultra-sensitive 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 CA15-3. Owing to the ultra-sensitivity of this sensor, all the human serum samples were diluted by PBS buffer in the experiments, and only 100 µL 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. 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=λ1-λ0) between reference and samples with various concentrations. At the range of 0.5 ~ 10×10-12 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 CA15-3 concentrations were displayed in Figure 4d.


Page 12 of 24

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 CA15-3 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 EDTA-based assay is of ultra-sensitivity. Consistency of the results determined by the proposed assay and found by hospital displays this ultra-sensitive and accurate assay can be extended to detect other disease markers. The effect of complex components on EDTA-based colorimetric assay. The above determination of CA-153 and PSA in human serum samples elucidates the EDTA-based sensor exhibit a good selectivity. However, in any real biological samples, there will be many different potential sources of non-specific 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 non-specific 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 anti-PSA 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 PSA-free 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, PSA-free human serum with varied concentrations and original human



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

serum containing PSA, were tested as shown in Figure 5a. From the photograph, reference (lane 1) and PSA-free 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 PSA-free human serum, and then detect the PSA again using EDTA-based 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. Determination of breast cancer cells by EDTA-based colorimetric assay. Further application of the EDTA-based colorimetric assay toward breast cancer cells was performed. Epithelial cell adhesion molecule (EpCAM) is a membrane-bound glycoprotein with oncogenic properties, which often correlates with more aggressive tumor behavior. High-level EpCAM expression was found in premalignant lesions and carcinomas of various origins, such as prostate, colorectal, ovarian, lung and breast cancer.40-44 EpCAM expression in MCF-7 cell lines is relatively high. Thereby, MCF-7 cells were tested to assess possibility of detection of cancer cells by this colorimetric assay. The antibody anti-EpCAM 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 anti-EpCAM conjugated with EDTA, the GNRs and H2O2 were added into the polystyrene plates. Other row of micro-wells without samples was used as control experiment. That is to say, the same GNRs and H2O2 were added in this row. Figure


Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6a shows a distinguishable color among the two groups and the LPWs of the resulting GNRs also were obtained by multi-mode microplate reader, indicating the feasibility of this assay for visual detection of cancer cells. 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 MCF-7 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 anti-EpCAM–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 micro-wells 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 read-out 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 anti-EpCAM–coated magnetic beads. Interestingly, there are two wells in the sample row exhibiting similar color to the references. It is possible that no MCF-7 cells were captured in the two wells, and this was confirmed by the fact that no MCF-7 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 read-out CTCs.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CONCLUSIONS In this study, we discovered and investigated the peroxidase-like activity of conventional EDTA. A straightforward plasmonic nanosensor based on EDTA was developed to enable naked-eye observation and determination of ultra-trace 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 read-out 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 CA15-3 and SPA of cancer serum samples indicate this assay was applied to detect different biomarkers of diseases with high accuracy. The colorimetric EDTA-based nanosensor can not only detect target molecules but also visually determine cancer cells. Moreover, combined with immunomagnetic separation of CTCs from blood, a visual read-out for determination of CTCs was established, providing a potential and promising strategy for diagnostic and prognostic information related to patient diseases. The EDTA-based colorimetric assay takes the advantage of simple in fabrication, manipulation, storage, cost-effective and fast detection, which has potential important implications in clinical diagnosis, environmental monitoring, and food quality control only using naked eyes.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21375036, 21675049), National Science Foundation for Distinguished Young Scholars of China (51525207).

REFERENCES 1. Polavarapu, S.; Oliver, C. M.; Ajlouni, S.; Augustin, M. A. J. Agric. Food Chem. 2012, 60, 444–450. 2. Hajeb, P.; Jinap, S. J. Agric. Food Chem. 2012, 60, 6069–6076.


Page 16 of 24

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3. Habiba, U.; Ali, S.; Farid, M.; Shakoor, M. B.; Rizwan, M.; Ibrahim, M.; Abbasi, G. H.; Hayat, T.; Ali, B. Environ Sci Pollut Res. 2015, 22, 1534–1544. 4. Hurrell, R. F.; Reddy, M. B.; Burri, J.; Cook, J. D. Brit. J. Nutr. 2000, 84, 903-910. 5. Davidsson, L.; Ziegler, E.; Zeder, C.; Walczyk, T.; Hurrell, R. Am. J. Clin. Nutr. 2005, 81, 104-109. 6. Kubo, I.; Lee, S. H.; Ha, T. J. J. Agric. Food Chem. 2005, 53, 1818−1822. 7. Zhang, R.; Chen, M.; Lu, Y.; Guo, X.; Qiao F.; Wu, L. Scientific Reports 2015, 5, 12944. 8. Imlay, J. A.; Chin, S. M.; Line, S. Science 1998, 240, 640-642. 9. Yin, K.; Giannis, A.; Wong, A. S. Y.; Wang, J. Y. Water Air Soil. Pollut. 2014, 225, 2024. 10. Plante, B.; Benzaazoua, M.; Bussière, B.; Kandji, E. H. B.; Chopard, A.; Bouzahzah, H. Environ Sci. Pollut. Res. 2015, 22, 7882–7896. 11. Belal, F.; Aly, F. A.; Walash, M. I.; Kenawy, I. M.; Osman, A. M. Osman. II Farmaco 1998, 53, 365-368. 12. Krokidis, A. A.; Megoulas, N. C.; Koupparis, M. A. Anal. Chim. Acta 2005, 535, 57–63. 13. Rouhollah, H.; Mojtaba, S.; Nasim, N. AAPS Phamscitech 2013, 14, 764-769. 14. Heydari, R.; Shamsipur, M.; Naleini, N. AAPS PharmSciTech 2013, 14, 764-769. 15. Yu, Y.; Chen, G.; Wang, X.; Jia, D.; Tang, P.; Lv, C. RSC Adv. 2015, 5, 74174–74179. 16. Zhang, H.; Tong, T.; Cao, W. H.; Chen, J. G.; Jin, D. R.; Cheng, J. R. J. Sol-Gel Sci. Technol. 2015, 75, 481–485. 17. Su, E. C.; Huang, B. S.; Liu, C. C.; Wey, M. Y. Renew. Energ. 2015, 75, 266-271. 18. Lee, S. S.; Bai, H.; Liu, Z.; Sun, D. D. Environ. Sci. Technol. 2015, 49, 2541–2548. 19. Trinidad, C. S.; Cruz, A. M.; Cuéllar, E. L. Environ Sci Pollut Res. 2015, 22, 792–799. 20. Yang, B.; Zhang, J.; Zhang, Y.; Deng, S.; Yu, G.; Wu, J.; Zhang, H.; Liu, J. Chem. Eng. J. 2014, 250, 222–229.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

21. Lequin, R. M. Clin. Chem. 2005, 51, 2415-2418. 22. Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S.C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C. Nat. Biotechnol. 2010, 28, 595-599. 23. Huang, C. H.; Sedlak, D. L. Toxicol. Chem. 2001, 20, 133-139. 24. Asensio, L.; Gonzalez, I.; Garcia, T.; Martin, R. Food Control 2008, 19, 1-8.

25. Han, D.; Wu, C.; You, M.; Zhang, T.; Wan, S.; Chen, T.; Qiu, L.; Zheng, Z.; Liang, H.; Tan, W. Nat. Chem. 2015, 7, 835-841.

26. Wang, G. L.; Xu, X. F.; Qiu, L.; Dong, Y. M.; Li, Z. J.; Zhang, C. ACS Appl. Mater. Interfaces 2014, 6, 6434-6442. 27. Chen, Z.; Yin, J. J.; Zhou, Y. T.; Zhang, Y.; Song, L.; Song, M.; Hu, S.; Gu, N. 2012 6, 4001–4012. ACS Nano, 2012, 28. Kirkorian, K.; Ellis, A.; Twyman, L. J. Chem. Soc, Rev. 2012, 41, 6138-6159. 29. Vernekar, A. A.; Sinha, D.; Srivastava, S.; Paramasivam, P. U.; D’Silva, P.; Mugesh, G. Nat.Commun. 2014, 5, 5301. 30. de la Rica, R.; Stevens, M. M. Nat. Nanotechnol. 2012, 7, 821-824. 31. Rodríguez-Lorenzo, L.; de la Rica, R.; Álvarez-Puebla, R. A.; Liz-Marzán, L. M.; Stevens, M. M. Nat. Mater. 2012, 11, 604-607. 32. Zhao, Q.; Huang, H.; Zhang, L.; Wang, L.; Zeng, Y.; Xia, X.; Liu, F.; Chen, Y. Anal. Chem. 2016, 88, 1412-1418. 33. Sun, W. Z.; Pan, Y.; Zhao, L.; Zhou, X. G. Chem. Eng. Technol. 2008, 31, 1402-1409. 34. Chen, H.; Shao, L.; Li, Q.; Wang, J. F. Chem. Soc. Rev. 2013, 42, 2679-2724. 2014 136, 35. Takahata, R.; Yamazoe, S.; Koyasu, K.; Tsukuda, T. J. Am. Chem. Soc. 2014, 8489–8491. 36. Chen, S.; Zhao, Q.; Zhang, L.; Xia, X.; Huang, H. Anal. Method 2015, 7, 1018–1025.


Page 18 of 24

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37. Sun, W. Z.; Pan, Y.; Zhao, L.; Zhou, X. G. Chem. Eng. Technol. 2008, 31, 1402-1409. 38. Sandri, M. T.; Salvatici, M.; Botteri, E.; Passerini, R.; Zorzino, L.; Rotmensz, N.; Bottari, F. Breast Cancer Res. Tr. 2012, 132, 317-326. 39. Samy, N.; Ragab, H. M.; Maksoud, Abd, N.; Shaalan, M. Cancer Biomark 2010, 6, 63-72. 40. Cristofanilli, M.; Budd, T.; Ellis, M.J.; Stopeck, A.; Matera, J.; Miller, C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W.M.M.; Hayes, D. F. N. Engl. J. Med. 2004, 351, 781-791. 41. Cohen, S. J.; Punt, C. J.; Iannotti, N.; Saidman, B. H.; Sabbath, K. D.; Gabrail, N. Y.; Picus, J.; Morse, M.; Mitchell, E.; Miller, M. C.; Doyle, G. V.; Tissing H.; Terstappen L. W. M. M.; Meropol, N. J. J. Clin. Oncol. 2008, 26, 3213-3221. 42. de Bono, J. S.; Scher, H. I.; Montgomery, R. B.; Parker, C.; Miller, M. C.; Tissing, H.; Raghavan, D. Clin. Cancer Res. 2008, 14, 6302-6309. 43. Burges, A.; Wimberger, P.; Kümper, C.; Gorbounova, V.; Sommer, H.; Schmalfeldt, B.; Lahr, A. Clin. Cancer Res. 2007, 13, 3899-3905. 44. Chen, Q.; Ge, F.; Cui, W.; Wang, F.; Yang, Z.; Guo, Y.; Lin, P. P. Clin. Chem. Acta 2013, 419, 57-61. 45. Mostert, B.; Sleijfer, S.; Foekens, J. A.; Gratama, J. W. Cancer Treat. Rev. 2009, 35, 463-474. 46. Gradilone, A.; Naso, G.; Raimondi, C.; Cortesi, E.; Gandini, O.; Vincenzi, B.; Frati, L. Ann. Oncol. 2011, 22, 86-92.

Competing financial interests The authors declare no competing financial interests.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FIGURES AND CAPTIONS

Scheme 1. Schematic illustration of EDTA-based 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 2-hydroxyterephthalic acid originated from terephthalic acid in the presence of EDTA at various irradiation time.


Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Colorimetric determination by EDTA-based 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 CA15-3. c: Detection of CA15-3 of breast cancer serum samples, the middle row is the reference and other represent the practical samples. d: The levels of CA15-3 in sera of breast cancer by different methods.

Figure 5. Investigation of liability on EDTA-based assay. a: Comparison of reference, PSA-free human serum and human serum containing PSA: lane 1 is the reference, lane 2 represents the reference solution added various amounts of PSA-free 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 PSA-free 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.


Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Determination of breast cancer cells by EDTA-based colorimetric plasmonic nanosensor. a: Photograph of the detection of MCF-7 cell lines by colorimetric detection, and the LPW (nm) of GNRs were obtained by Multi-Mode Microplate Reader. b: A schematic illustration of visual read-out for determination of CTCs by combined EDTA-based 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.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC


Page 24 of 24

Peroxidase-Like Activity of Ethylene Diamine Tetraacetic Acid and Its

Recommend Documents