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Highly sensitive and specific colorimetric detection of cancer cells via dual-aptamer target binding strategy
Biosensors and Bioelectronics 73 (2015) 1–6
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Highly sensitive and specific colorimetric detection of cancer cells via dual-aptamer target binding strategy Kun Wang a,c,d, Daoqing Fan a,c, Yaqing Liu a,b,c, Erkang Wang a,c,n a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China School of Food Engineering and Biological Technology, Tianjin University of Science and Technology, Tianjin 300222, China c University of Chinese Academy of Sciences, Beijing, 100039, China d Department of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 March 2015 Received in revised form 4 May 2015 Accepted 21 May 2015 Available online 22 May 2015
Simple, rapid, sensitive and specific detection of cancer cells is of great importance for early and accurate cancer diagnostics and therapy. By coupling nanotechnology and dual-aptamer target binding strategies, we developed a colorimetric assay for visually detecting cancer cells with high sensitivity and specificity. The nanotechnology including high catalytic activity of PtAuNP and magnetic separation & concentration plays a vital role on the signal amplification and improvement of detection sensitivity. The color change caused by small amount of target cancer cells (10 cells/mL) can be clearly distinguished by naked eyes. The dual-aptamer target binding strategy guarantees the detection specificity that large amount of noncancer cells and different cancer cells (104 cells/mL) cannot cause obvious color change. A detection limit as low as 10 cells/mL with detection linear range from 10 to 105 cells/mL was reached according to the experimental detections in phosphate buffer solution as well as serum sample. The developed enzymefree and cost effective colorimetric assay is simple and no need of instrument while still provides excellent sensitivity, specificity and repeatability, having potential application on point-of-care cancer diagnosis. & 2015 Published by Elsevier B.V.
Keywords: Colorimetric detection Cytosensor Dual-aptamer Nanomaterial Breast cancer cells
1. Introduction On the world cancer day of 2014, the International Agency for Research on Cancer reported that it was about 8.2 million deaths from cancer in 2012 and cancer has been the biggest cause of mortality worldwide. Early and accurate diagnosis is absolutely critical for saving patients' life (Irish et al., 2006; Uhr, 2007). With the current techniques, however, cancers usually cannot be detected until the tumor has grown fairly large especially in the developing countries. It is already too late for curing. What's more, the diagnostic techniques are also time consuming, high-cost and usually require expensive instrumentation. For example, breast cancer is diagnosed by combination of computed tomography and magnetic resonance imaging. Some diagnostic approaches may even couple with radioactive risk (Brindle, 2008; Li et al., 2012; Mosmann, 1983). It has reached on a consensus that developing rapid, simple and low-cost detection approaches for sensitive and specific detection of cancer cells would make a huge impact on n Corresponding authors at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (E. Wang).
http://dx.doi.org/10.1016/j.bios.2015.05.044 0956-5663/& 2015 Published by Elsevier B.V.
improvement in survival rate, reduction in mortality and decrease in therapy cost (Sidransky, 1997; Tothill, 2009). To address the challenge various strategies have been provided for high sensitive detection of cancer cells. A fluorescence imaging assay developed by Tan's group reaches a detection limit of 250 cells (Smith et al., 2007). By taking rolling circle amplification strategy, 163 Ramos cells were successfully detected through chemiluminescence imaging array (Bi et al., 2013). Detection limit as low as 10 cells/mL was reached according to the designed sensitive electrochemical approach (Li et al., 2012; Liu et al., 2013; Xu et al., 2013). The above developed assays significantly improve the detection sensitivity of cancer cell. However, all the above detections depend on instrument measurement, limiting their potential application in point-of-care disease diagnosis. Nanomaterial-based colorimetric assay provides a simple, costeffective and convenient readout way for non-instrument detection and has drawn an increasing interest for point-of-care cancer diagnosis (Elghanian et al., 1997). Gold nanoparticle (AuNP) has been confirmed to be excellent candidate for colorimetric sensors due to its distance-dependent surface plasmon resonance (SPR) adsorption property (Liu et al., 2009; Medley et al., 2008; Valentini et al., 2013; Xu et al., 2009). While, the distance-dependence intrinsic property of AuNP limits its applications in early cancer
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diagnosis in that the color change of AuNP would be hardly monitored in case of a relatively low density of biomarkers on the cancer cell membrane (Lu et al., 2010; Shlyahovsky et al., 2007; Xie et al., 2012; Zhang et al., 2014b). To improve detection sensitivity, signal amplification strategies have been developed. In very recently, a detection limit of 40 cells/mL for colorimetric detection was reached by combining cyclic signal amplification assay under the help of nicking endonuclease (Zhang et al., 2014b). By making use of catalytic activity of platinum nanoparticle on colorimetric reaction of chromogenic substrates, a detection limit of 30 cells was obtained according to the calculation of three times the standard deviation (3s) (Zhang et al., 2014a). The previous works provide valuable instruction for further developing sensitive cytosensors. Herein, we developed one enzyme-free strategy for rapid, sensitive and specific detection of cancer cells by coupling the high specificity of aptamer to targets and high catalytic activity of PtAu nanoparticle. The developed assay can differentiate target cancer cells from non-cancer cells and different cancer cell types. A detection limit of 10 cells/mL is reached according to practical experiment results while not calculation of 3s.
2. Experimental section 2.1. Materials DNA oligonucleotides were synthesized by Shanghai Sangon Biotechnology Co (Shanghai, China). VEGF165 binding aptamer 5′GGGCC CGTCC GTATG GTGGG TGTGC TGGCT TTTAA AAAAA AAA -3′. MUC1 binding aptamer 5′- GCAGT TGATC CTTTG GATAC CCTGG TTTTTA AAA DNA oligonucleotide solutions were prepared by dissolving DNAs in ultrapure water and quantified by measuring UV– visible absorption spectroscopy at 260 nm. (3-aminopropyl) triethoxysilane (APTES), 2-[N-Morpholino] ethanesulfonic acid (MES), sodium acrylate and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (St. Louis, MO). The other chemicals were purchased from Aladin (Shanghai, China) and used as received without further purification. All other chemicals not mentioned here were of analytical reagent grade and used as received. Milli-Q ultrapure water (18.2 MΩ) was used throughout. 2.2. Apparatus Absorbance measurements were performed on a Cary 500 Scan UV/Vis/NIR Spectrophotometer (Varian, USA). Transmission electron microscopy (TEM) measurements were conducted on a HITACHI H-8100 EM (Hitachi, Tokyo, Japan) with an accelerating voltage of 200 kV. 2.3. Cell culture The cells were cultured in DMEM medium (GIBCO) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The cell density was determined by a Petroff Hausser cell counter (USA). 2.4. Preparation of aptamer modified PtAuNP The PtAuNP was synthesized with citrate reduction according to previously reported method (Tseng et al., 2012). 100 mL aqueous solution of HAuCl4 (0.9 mM) and H2PtCl6 (0.1 mM) was stirred under heating in a round-bottom flask. Then trisodium citrate solution (40 mM, 10 mL) was quickly added. When the color of solution changed from pale yellow to deep red, the solution was cooled down to room temperature after heating for an additional
15 min under stirring. The aptamers of Human mucin-1 (AptMUC1) were immobilized on the PtAuNP by taking advantage of the preferential adsorption of adenine nucleotides (dA) on gold (Opdahl et al., 2007; Pei et al., 2012). The AptMUC1 mixed with PtAuNP at ratios of 200:1. 500 mM citrateHCl buffer (pH 3.0) was then added to the mixture (Zhang et al., 2012). The concentration of citrateHCl in the final buffer was 10 mM. Afterward, the excess AptMUC1 was removed by washing the conjugates in 10 mM sodium phosphate buffer (pH 7.4) with 0.1 M NaCl via centrifugation (10,000 rpm, 30 min, 4 °C). The synthesized nanomaterials were short named as AptMUC1-PtAuNP. 2.5. Preparation of aptamer modified magnetic beads (AptVEGF-MB) First, the monodispersed Fe3O4 nanoparticle was synthesized according to solvothermal method (Xuan et al., 2009). FeCl3 6H2O (0.54 g), C3H3O2Na (1.5 g) and NaOAc (1.5 g) were dissolved in ethylene glycol (20 mL) under magnetic stirring to form a clear solution. The mixture was then transferred into a teflonlined stainless-steel autoclave, keeping 12 h at 200 °C. After cooling down to room temperature, SiO2 was used to enwrap the Fe3O4 nanoparticle in order to avoid the catalytic interference of Fe3O4 nanoparticle. The obtained nanoparticle was then washed several times with ultrapure water and ethanol and then dispersed in mixture of ethanol (80 mL) and ultrapure water (20 mL) followed by the addition of ammonia (1 mL) and ethylsilicate (200 μL) to form Fe3O4/SiO2 nanoparticles. After being separated, the resulted product mixed with APTES (100 μL) and ammonia (2 mL), the resultant amino-functionalized Fe3O4/SiO2 nanoparticles were washed with water for several times. Through electrostatic interaction, the AuNP (13- nm, synthesized with citrate reduction as previous report (Hill and Mirkin, 2006)) was adsorbed on the amino-functionalized magnetic beads and short named as MB. Finally, the MB was dispersed in ultrapure water (10 mL) before use. The diameter of MB was about 200 nm evaluated by TEM measurements (See Fig. S1 in supporting information (SI)). To prepare aptamer functionalized MB, 100 μL of MB suspension was first dispersed in 4 mL PBS followed by addition of aptamer of vascular endothelial growth factor 165 (AptVEGF) (10 μM, 20 μL) under continuous shaking for 16 hours. The AptVEGF was then immobilized on AuNP through the interaction of poly A with Au (Opdahl et al., 2007; Pei et al., 2012). The unbounded AptVEGF was removed by washing with PBS several times. Finally, the conjugates were dispersed in PBS (100 μL) for further use, which was short named as AptVEGF-MB. 2.6. Cell detecion 20 μL of AptVEGF-MB and 20 μL of AptMUC1-PtAuNP were added into 960 μL of PBS buffer containing human breast adenocarcinoma cells (MCF-7 cells) at a certain concentration and incubated at 37 °C for 30 min. Then complexes of (AptVEGF-MB)/MCF-7/(AptMUC1-PtAuNP) was formed, which can be efficiently separated by magnetic field (See Fig. S2 in S1 separation the complexes of (AptVEGF-MB)/ MCF-7/(AptMUC1-PtAuNP) were dispersed into 500 μL of MESAc buffer (25 mM, contain 20 mM KAc, pH 4.5). 10 mL TMB solution (20 mM in dimethyl sulfoxide) and 5 mL H2O2 (30% w/v) were then sequentially added to the above mixture. After 2 min the reaction was stopped by adding equal volume of 2 mM H2SO4. The absorbance was measured by using UV–vis spectrophotometer. 2.7. Human Serum Samples The human serum samples were diluted with PBS buffer with ratio of 1:4. The human serum sample, containing human breast
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adenocarcinoma cells (MCF-7 cells) at a certain concentration, was analyzed following the same procedure as that for cancer cell detection.
3. Result and discussion 3.1. Principle of colorimetric detection cancerous cell based on the dual-aptamer target binding strategy Breast cancer is the most common type of cancer affecting women, causing approximately 45,000 deaths per year (Han et al., 2006; Kerr, 2002). MCF-7 cells represent one of the most widely used experimental models for in vitro studies on breast carcinoma (Xie et al., 2012) and were selected as model cell to validate the developed analytical approach. Two biomarkers, MUC1 and VEGF165 over-expressed on the membrane of MCF-7 cells, were selected as target sites (Mukhopadhyay et al., 2011; Zhao et al. 2012). The aptamers of MUC1 and VEGF (AptMUC1 and AptVEGF) present high affinity to the corresponding proteins (Borbas et al., 2007; Guo et al., 2003; Ikebukuro et al., 2007; Rahn et al., 2001; Wei et al., 2012) and were used as recognition agents to improve the detection specificity according to dual-aptamer target binding. The colorimetric cancer cell detection is based on the color change of TMB in the presence of H2O2. PtAu nanoparticle exhibits excellent catalytic activity and was selected as catalyst for the colorimetric reaction. Scheme 1 outlines the detection principle which is based on the new developed bio-bar-code nanoprobe strategy (Nam et al., 2003). In briefly, the aptamer of MUC1 modified on PtAu nanoparticle (AptMUC1-PtAuNP) acts as sensing probe to specifically recognize MUC1 and also as bar code to enhance signal generation. The aptamer of VEGF modified on magnetic bead (AptVEGF-MB) acts as capture probe to specifically recognize VEGF. Here, the mixture of AptVEGF-MB and AptMUC1-PtAuNP functions as the “detection kit”. After incubating MCF-7 cells in the detection kit, the capture probe of AptVEGF-MB and signal probe of AptMUC1-PtAuNP were trapped on the membrane of cancer cells, forming bio-bar-code configuration complex. The signal was then monitored after separating the bio-bar-code complex from the excess of AptMUC1-PtAuNP with the help of magnetic field. The color change depends on the amount of target cancer cell and also the incubation time of the cell in the detection kit. Moreover, the catalytic activity of PtAuNP plays a critical role on the detection sensitivity in our provided strategy. Thus, the catalytic activity of PtAuNP and the incubation time were optimized in the following experiments.
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3.2. Optimization of experimental conditions The bimetallic PtAu nanoparticle (PtAuNP) has been confirmed to present excellent peroxidase property on catalyzing fluorogenic substrate (Tseng et al., 2012). In our investigation, PtAuNP was synthesized according to the reported method and was used to catalyze the colorimetric reaction of TMB in the presence of H2O2. The catalytic efficiency could be modulated by varying the ratios of Au to Pt. Learned from Fig. 1, AuNP presents lowest peroxidase activity. With increasing the ratio of Pt and Au from Pt0.5AuNP to Pt1AuNP, the peroxidase activity of bimetallic PtAuNP increases correspondingly. With further increasing the ratio of Pt, however, the peroxidase activity of PtAuNP significantly decreases, which is ascribed to the decreasing of active sites on the particle surface (Tseng et al., 2012). So the Pt1AuNP was selected as catalyst to the signal generator. The TEM image, Fig. 1B, indicates that the asprepared Pt1AuNP has an average diameter of 13 nm. The X-ray photoelectron spectra of the Pt1AuNP, Fig. 1C, present the featured peaks at 84.0 eV and 87.7 eV for Au(0) 4f7/2 and Au(0) 4f5/2 and 71.0 eV and 74.4 eV for Pt(0) 4f7/2 and Pt(0) 4f5/2, respectively. To endow reorganization function, aptamers are modified on the Pt1AuNP surface. After removing the excess aptamer, the functionalized nanoparticle is characterized by measuring the UV absorbance spectra, Fig. 2. Except that the feature peaks of nanoparticle are monitored at 540 nm from Fig. 2 for both PtAuNP (black curve) and AptMUC1-PtAuNP (red curve), a new peak at 260 nm is found for AptMUC1-PtAuNP, Fig. 2 (red curve), indicating the successful anchoring of aptamer on the nanoparticle surface. The optimal incubation time of cancer cell in the detection kit is explored by monitoring the absorbance spectra change of TMB with incubation time (See Fig. S3A in SI). The absorbance peak of TMB at 452 nm significantly increases with increasing incubation time to 30 min and then reaches a plateau (See Fig. S3B in SI). Thus, 30 min is selected as the incubation time. 3.3. Sensitive and specific colorimetric detection of MCF-7 cells Sensitive and specific diagnostic approaches are of great importance for early and accurate detection. The sensitivity of our provided strategy is investigated by monitoring the color and absorbance change of TMB in the presence of different numbers of cancer cell, MCF-7, Fig. 3. In the absence of the target cancer cell (MCF-7), the AptMUC1-PtAuNP could not be linked together with AptVEGF-MB and would be removed from the detection system through magnetic separation. In the presence of MCF-7 cell, more and more AptMUC1-PtAuNP/MCF-7/AptVEGF-MB complexes are generated with increasing the target cell (from 10 to 105 cells/mL), leading an obvious colorimetric reading from light yellow to dark
Scheme 1. Schematic diagram of colorimetric detection of cancer cells on the basis of dual-aptamer target binding and bio-bar-code strategy.
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Fig. 1. A) Catalytic ability of PtAuNP with different ratio of Pt to Au on the colorimetric reaction of TMB in the presence of H2O2. B) TEM imaging of the as-prepared Pt1/AuNP. C) XPS of Au (red curve) and Pt (black curve) obtained from the as-prepared Pt1/AuNP. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. The UV–vis absorption spectra of PtAuNP (black curve) and AptMUC1-PtAuNP (red curve). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
yellow, Fig. 3A. Due to the excellent catalytic efficiency of PtAuNP, the color change caused by 10 cells/mL could be clearly distinguished with the naked eyes. The colorimetric signal as a function of cell number is further monitored by UV–vis spectrometry, Fig. 3B. The absorbance of TMB at 452 nm linearly depends on the logarithm number of target cells ranging from 10 cells/mL to 105 cells/mL with a regression coefficient of 0.999, Fig. 3C. Thus, a detect limit as low as 10 cells/mL with a wide detection range is obtained according to the practical measurement while not the calculation of three times standard deviation of the blank measurement. To the best of our knowledge, 10 cells/mL is the lowest detection limit for colorimetric detection of cancer cell. In previous
reports, the detection limit of colorimetric cancer cell detection is reduced from 800 cells (Liu et al., 2009) to 125 cells (Zhang et al., 2014a) due to the intrinsic property of nanomaterials. Recently, a detection limit of 40 cells for visual cancer cell detection is reached according to cell-triggered cyclic enzymatic signal amplification strategy (Zhang et al., 2014b). With our proposed strategy, the detection sensitivity is significantly improved and 10 cells/mL can be clearly distinguished by naked eyes on the basis of the synergistic action of high catalytic activity of PtAuNP and magnetic separation and concentration. The results demonstrate that the proposed assay presents a more sensitive sensing platform for cancer cells detection. Distinguishing target cancer cell from non-cancer cell and different cancer cell types is significant important while a major hurdle for cancer diagnosis and therapy (Liu et al., 2014). To address the challenge, we make use of the unique signatures of different cell types to specifically detect target cancer cells. According to our provided assay, the dual aptamer-based cytosensor can differentiate MCF-7 cancer cell from non-cancer cells, human embryonic kidney 293 cells (HEK 293) and human hepatocyte cells (HL7702), and also other cancer cells, human liver hepatocellular carcinoma cells (HeGp2). In the presence of large amount of HEK 293, HL7702 and HeGp2 cells (104 cells/mL) the respective solution color shown in Fig. 4A is similar as that in the absence of target cancer cell MCF-7, Fig. 3A. In the presence of small amount of MCF-7 (10 cells/mL), however, a remarkable color change can be easily monitored by naked eyes. The corresponding absorbance signal detection at 452 nm further confirms the colorimetric result and the excellent selectivity of the developed strategy, Fig. 3B. The different colorimetric results depend on the cell type and the
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Fig. 3. A) Photographs and B) UV–vis absorption spectra of the TMB–H2O2 against different concentrations of MCF-7 cells: 0, 10, 102, 103, 104, 105 cells/mL. C) Linear relationship between the absorbance value at 452 nm and the logarithm of MCF-7 concentration. The error bar was obtained according to five independent experimental results.
adopted dual-aptamer target binding assay. The used biomarkers, VEGF and MUC1, are limited expressed on membrane of the used control cells (Carpini et al., 2010; Guo et al., 2003). Due to the high specific affinity of the used aptamers to the targets the HEK 293 and HL 7702 cannot be trapped by AptVEGF-MB and AptMUC1-PtAuNP to form bio-bar-code configuration, causing no obvious color change. The cancer cells of HeGp2 are MUC1-negative human hepatic cancer cell line. Though the HeGp2 cells could be trapped by the AptVEGF-MB, they could not be trapped by AptMUC1-PtAuNP, leading no obvious color change. It is similar as the colorimetric response of the TMB–H2O2 system if incubate MCF-7 in the mixture of AptMUC1-PtAuNP and MB without modifying AptVEGF (See Fig. S4 in SI). The experimental results confirm that the combination of dual-aptamer target-binding with nanotechnology strategy in our
assay can efficiently reduce cross reaction and improve selectivity. Comparing with reported colorimetric methods for cancer cells detection (Table S1 in SI), the strategy developed by us presents higher sensitivity. The dual-aptamer target binding strategy makes it possible to distinguish cancer cells not only from non-cancer cells but also from different cancer cell types. To explore the feasibility of the developed strategy in biological medium, the as-prepared cytosensor was used to detect MCF-7 cells in human serum. Learned from curve (a) in Fig. 5, the absorbance intensity of TMB at 452 nm linearly increases with logarithm of cancer cell concentration from 0 to 105 cells/mL. The slope of curve (a) is very close to that of the cancer cell detection in buffer solution, curve (b), which is repotted from Fig. 3C for convenient comparison. The results indicate the excellent anti-
Fig. 4. A) Photograph and absorbance of the sensing system to different cells, HEK 293 (104 cells/mL), HL 7702 (104 cells/mL), HeGp2 (104 cells/mL) cells, and MCF-7 cell (10 cells/mL).
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the online version at http://dx.doi.org/10.1016/j.bios.2015.05.044.
References
Fig. 5. Linear relationship between the absorbance value at 452 nm in serum (a) and PBS (b) against the logarithm of MCF-7 concentration. The error bar was obtained according to five independent experimental results.
interference ability and reproducibility of the developed strategy.
4. Conclusions In summary, we have provided a simple, efficient, enzyme-free and convenient colorimetric assay for high sensitive and specific detection of MCF-7 cancer cells with acceptable reproducibility. On the membrane of MCF-7 cancer cell VEGF and MUC1 are overexpressed. The target cells act as bridge to link AptVEGF-MB and AptMUC1-PtAuNP together. The high catalytic activity of PtAuNP makes it possible for sensitive detection 10 cells/mL in phosphate buffer solution and also in serum by naked eyes without need of instrument help, which is more sensitive than previously reported colorimetric assays. The dual-aptamer target binding strategy with two different biomarkers as recognition elements improves the detection specificity and provides a more accurate way for cancer cell detection. The developed assay can be used to distinguish cancer cells not only from non-cancer cells but also from different cancer cell types. Comparing with conventional methods, the developed colorimetric strategy provides a sensitive, specific, simplicity, enzyme-free and cost-effective assay and has potential application on point-of-care cancer diagnosis.
Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 21105095 and 211900040), State Key Instrument Developing Special Project of Ministry of Science and Technology of China (2012YQ170003), the Instrument Developing Project of the Chinese Academy of Sciences (YZ201203) and the Natural Science Foundation of Jilin Province, China (No. 20130101117JC).
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Highly sensitive and specific colorimetric detection of cancer cells via dual-aptamer target binding strategy Kun Wang a,c,d, Daoqing Fan a,c, Yaqing Liu a,b,c, Erkang Wang a,c,n a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China School of Food Engineering and Biological Technology, Tianjin University of Science and Technology, Tianjin 300222, China c University of Chinese Academy of Sciences, Beijing, 100039, China d Department of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 March 2015 Received in revised form 4 May 2015 Accepted 21 May 2015 Available online 22 May 2015
Simple, rapid, sensitive and specific detection of cancer cells is of great importance for early and accurate cancer diagnostics and therapy. By coupling nanotechnology and dual-aptamer target binding strategies, we developed a colorimetric assay for visually detecting cancer cells with high sensitivity and specificity. The nanotechnology including high catalytic activity of PtAuNP and magnetic separation & concentration plays a vital role on the signal amplification and improvement of detection sensitivity. The color change caused by small amount of target cancer cells (10 cells/mL) can be clearly distinguished by naked eyes. The dual-aptamer target binding strategy guarantees the detection specificity that large amount of noncancer cells and different cancer cells (104 cells/mL) cannot cause obvious color change. A detection limit as low as 10 cells/mL with detection linear range from 10 to 105 cells/mL was reached according to the experimental detections in phosphate buffer solution as well as serum sample. The developed enzymefree and cost effective colorimetric assay is simple and no need of instrument while still provides excellent sensitivity, specificity and repeatability, having potential application on point-of-care cancer diagnosis. & 2015 Published by Elsevier B.V.
Keywords: Colorimetric detection Cytosensor Dual-aptamer Nanomaterial Breast cancer cells
1. Introduction On the world cancer day of 2014, the International Agency for Research on Cancer reported that it was about 8.2 million deaths from cancer in 2012 and cancer has been the biggest cause of mortality worldwide. Early and accurate diagnosis is absolutely critical for saving patients' life (Irish et al., 2006; Uhr, 2007). With the current techniques, however, cancers usually cannot be detected until the tumor has grown fairly large especially in the developing countries. It is already too late for curing. What's more, the diagnostic techniques are also time consuming, high-cost and usually require expensive instrumentation. For example, breast cancer is diagnosed by combination of computed tomography and magnetic resonance imaging. Some diagnostic approaches may even couple with radioactive risk (Brindle, 2008; Li et al., 2012; Mosmann, 1983). It has reached on a consensus that developing rapid, simple and low-cost detection approaches for sensitive and specific detection of cancer cells would make a huge impact on n Corresponding authors at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (E. Wang).
http://dx.doi.org/10.1016/j.bios.2015.05.044 0956-5663/& 2015 Published by Elsevier B.V.
improvement in survival rate, reduction in mortality and decrease in therapy cost (Sidransky, 1997; Tothill, 2009). To address the challenge various strategies have been provided for high sensitive detection of cancer cells. A fluorescence imaging assay developed by Tan's group reaches a detection limit of 250 cells (Smith et al., 2007). By taking rolling circle amplification strategy, 163 Ramos cells were successfully detected through chemiluminescence imaging array (Bi et al., 2013). Detection limit as low as 10 cells/mL was reached according to the designed sensitive electrochemical approach (Li et al., 2012; Liu et al., 2013; Xu et al., 2013). The above developed assays significantly improve the detection sensitivity of cancer cell. However, all the above detections depend on instrument measurement, limiting their potential application in point-of-care disease diagnosis. Nanomaterial-based colorimetric assay provides a simple, costeffective and convenient readout way for non-instrument detection and has drawn an increasing interest for point-of-care cancer diagnosis (Elghanian et al., 1997). Gold nanoparticle (AuNP) has been confirmed to be excellent candidate for colorimetric sensors due to its distance-dependent surface plasmon resonance (SPR) adsorption property (Liu et al., 2009; Medley et al., 2008; Valentini et al., 2013; Xu et al., 2009). While, the distance-dependence intrinsic property of AuNP limits its applications in early cancer
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diagnosis in that the color change of AuNP would be hardly monitored in case of a relatively low density of biomarkers on the cancer cell membrane (Lu et al., 2010; Shlyahovsky et al., 2007; Xie et al., 2012; Zhang et al., 2014b). To improve detection sensitivity, signal amplification strategies have been developed. In very recently, a detection limit of 40 cells/mL for colorimetric detection was reached by combining cyclic signal amplification assay under the help of nicking endonuclease (Zhang et al., 2014b). By making use of catalytic activity of platinum nanoparticle on colorimetric reaction of chromogenic substrates, a detection limit of 30 cells was obtained according to the calculation of three times the standard deviation (3s) (Zhang et al., 2014a). The previous works provide valuable instruction for further developing sensitive cytosensors. Herein, we developed one enzyme-free strategy for rapid, sensitive and specific detection of cancer cells by coupling the high specificity of aptamer to targets and high catalytic activity of PtAu nanoparticle. The developed assay can differentiate target cancer cells from non-cancer cells and different cancer cell types. A detection limit of 10 cells/mL is reached according to practical experiment results while not calculation of 3s.
2. Experimental section 2.1. Materials DNA oligonucleotides were synthesized by Shanghai Sangon Biotechnology Co (Shanghai, China). VEGF165 binding aptamer 5′GGGCC CGTCC GTATG GTGGG TGTGC TGGCT TTTAA AAAAA AAA -3′. MUC1 binding aptamer 5′- GCAGT TGATC CTTTG GATAC CCTGG TTTTTA AAA DNA oligonucleotide solutions were prepared by dissolving DNAs in ultrapure water and quantified by measuring UV– visible absorption spectroscopy at 260 nm. (3-aminopropyl) triethoxysilane (APTES), 2-[N-Morpholino] ethanesulfonic acid (MES), sodium acrylate and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (St. Louis, MO). The other chemicals were purchased from Aladin (Shanghai, China) and used as received without further purification. All other chemicals not mentioned here were of analytical reagent grade and used as received. Milli-Q ultrapure water (18.2 MΩ) was used throughout. 2.2. Apparatus Absorbance measurements were performed on a Cary 500 Scan UV/Vis/NIR Spectrophotometer (Varian, USA). Transmission electron microscopy (TEM) measurements were conducted on a HITACHI H-8100 EM (Hitachi, Tokyo, Japan) with an accelerating voltage of 200 kV. 2.3. Cell culture The cells were cultured in DMEM medium (GIBCO) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The cell density was determined by a Petroff Hausser cell counter (USA). 2.4. Preparation of aptamer modified PtAuNP The PtAuNP was synthesized with citrate reduction according to previously reported method (Tseng et al., 2012). 100 mL aqueous solution of HAuCl4 (0.9 mM) and H2PtCl6 (0.1 mM) was stirred under heating in a round-bottom flask. Then trisodium citrate solution (40 mM, 10 mL) was quickly added. When the color of solution changed from pale yellow to deep red, the solution was cooled down to room temperature after heating for an additional
15 min under stirring. The aptamers of Human mucin-1 (AptMUC1) were immobilized on the PtAuNP by taking advantage of the preferential adsorption of adenine nucleotides (dA) on gold (Opdahl et al., 2007; Pei et al., 2012). The AptMUC1 mixed with PtAuNP at ratios of 200:1. 500 mM citrateHCl buffer (pH 3.0) was then added to the mixture (Zhang et al., 2012). The concentration of citrateHCl in the final buffer was 10 mM. Afterward, the excess AptMUC1 was removed by washing the conjugates in 10 mM sodium phosphate buffer (pH 7.4) with 0.1 M NaCl via centrifugation (10,000 rpm, 30 min, 4 °C). The synthesized nanomaterials were short named as AptMUC1-PtAuNP. 2.5. Preparation of aptamer modified magnetic beads (AptVEGF-MB) First, the monodispersed Fe3O4 nanoparticle was synthesized according to solvothermal method (Xuan et al., 2009). FeCl3 6H2O (0.54 g), C3H3O2Na (1.5 g) and NaOAc (1.5 g) were dissolved in ethylene glycol (20 mL) under magnetic stirring to form a clear solution. The mixture was then transferred into a teflonlined stainless-steel autoclave, keeping 12 h at 200 °C. After cooling down to room temperature, SiO2 was used to enwrap the Fe3O4 nanoparticle in order to avoid the catalytic interference of Fe3O4 nanoparticle. The obtained nanoparticle was then washed several times with ultrapure water and ethanol and then dispersed in mixture of ethanol (80 mL) and ultrapure water (20 mL) followed by the addition of ammonia (1 mL) and ethylsilicate (200 μL) to form Fe3O4/SiO2 nanoparticles. After being separated, the resulted product mixed with APTES (100 μL) and ammonia (2 mL), the resultant amino-functionalized Fe3O4/SiO2 nanoparticles were washed with water for several times. Through electrostatic interaction, the AuNP (13- nm, synthesized with citrate reduction as previous report (Hill and Mirkin, 2006)) was adsorbed on the amino-functionalized magnetic beads and short named as MB. Finally, the MB was dispersed in ultrapure water (10 mL) before use. The diameter of MB was about 200 nm evaluated by TEM measurements (See Fig. S1 in supporting information (SI)). To prepare aptamer functionalized MB, 100 μL of MB suspension was first dispersed in 4 mL PBS followed by addition of aptamer of vascular endothelial growth factor 165 (AptVEGF) (10 μM, 20 μL) under continuous shaking for 16 hours. The AptVEGF was then immobilized on AuNP through the interaction of poly A with Au (Opdahl et al., 2007; Pei et al., 2012). The unbounded AptVEGF was removed by washing with PBS several times. Finally, the conjugates were dispersed in PBS (100 μL) for further use, which was short named as AptVEGF-MB. 2.6. Cell detecion 20 μL of AptVEGF-MB and 20 μL of AptMUC1-PtAuNP were added into 960 μL of PBS buffer containing human breast adenocarcinoma cells (MCF-7 cells) at a certain concentration and incubated at 37 °C for 30 min. Then complexes of (AptVEGF-MB)/MCF-7/(AptMUC1-PtAuNP) was formed, which can be efficiently separated by magnetic field (See Fig. S2 in S1 separation the complexes of (AptVEGF-MB)/ MCF-7/(AptMUC1-PtAuNP) were dispersed into 500 μL of MESAc buffer (25 mM, contain 20 mM KAc, pH 4.5). 10 mL TMB solution (20 mM in dimethyl sulfoxide) and 5 mL H2O2 (30% w/v) were then sequentially added to the above mixture. After 2 min the reaction was stopped by adding equal volume of 2 mM H2SO4. The absorbance was measured by using UV–vis spectrophotometer. 2.7. Human Serum Samples The human serum samples were diluted with PBS buffer with ratio of 1:4. The human serum sample, containing human breast
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adenocarcinoma cells (MCF-7 cells) at a certain concentration, was analyzed following the same procedure as that for cancer cell detection.
3. Result and discussion 3.1. Principle of colorimetric detection cancerous cell based on the dual-aptamer target binding strategy Breast cancer is the most common type of cancer affecting women, causing approximately 45,000 deaths per year (Han et al., 2006; Kerr, 2002). MCF-7 cells represent one of the most widely used experimental models for in vitro studies on breast carcinoma (Xie et al., 2012) and were selected as model cell to validate the developed analytical approach. Two biomarkers, MUC1 and VEGF165 over-expressed on the membrane of MCF-7 cells, were selected as target sites (Mukhopadhyay et al., 2011; Zhao et al. 2012). The aptamers of MUC1 and VEGF (AptMUC1 and AptVEGF) present high affinity to the corresponding proteins (Borbas et al., 2007; Guo et al., 2003; Ikebukuro et al., 2007; Rahn et al., 2001; Wei et al., 2012) and were used as recognition agents to improve the detection specificity according to dual-aptamer target binding. The colorimetric cancer cell detection is based on the color change of TMB in the presence of H2O2. PtAu nanoparticle exhibits excellent catalytic activity and was selected as catalyst for the colorimetric reaction. Scheme 1 outlines the detection principle which is based on the new developed bio-bar-code nanoprobe strategy (Nam et al., 2003). In briefly, the aptamer of MUC1 modified on PtAu nanoparticle (AptMUC1-PtAuNP) acts as sensing probe to specifically recognize MUC1 and also as bar code to enhance signal generation. The aptamer of VEGF modified on magnetic bead (AptVEGF-MB) acts as capture probe to specifically recognize VEGF. Here, the mixture of AptVEGF-MB and AptMUC1-PtAuNP functions as the “detection kit”. After incubating MCF-7 cells in the detection kit, the capture probe of AptVEGF-MB and signal probe of AptMUC1-PtAuNP were trapped on the membrane of cancer cells, forming bio-bar-code configuration complex. The signal was then monitored after separating the bio-bar-code complex from the excess of AptMUC1-PtAuNP with the help of magnetic field. The color change depends on the amount of target cancer cell and also the incubation time of the cell in the detection kit. Moreover, the catalytic activity of PtAuNP plays a critical role on the detection sensitivity in our provided strategy. Thus, the catalytic activity of PtAuNP and the incubation time were optimized in the following experiments.
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3.2. Optimization of experimental conditions The bimetallic PtAu nanoparticle (PtAuNP) has been confirmed to present excellent peroxidase property on catalyzing fluorogenic substrate (Tseng et al., 2012). In our investigation, PtAuNP was synthesized according to the reported method and was used to catalyze the colorimetric reaction of TMB in the presence of H2O2. The catalytic efficiency could be modulated by varying the ratios of Au to Pt. Learned from Fig. 1, AuNP presents lowest peroxidase activity. With increasing the ratio of Pt and Au from Pt0.5AuNP to Pt1AuNP, the peroxidase activity of bimetallic PtAuNP increases correspondingly. With further increasing the ratio of Pt, however, the peroxidase activity of PtAuNP significantly decreases, which is ascribed to the decreasing of active sites on the particle surface (Tseng et al., 2012). So the Pt1AuNP was selected as catalyst to the signal generator. The TEM image, Fig. 1B, indicates that the asprepared Pt1AuNP has an average diameter of 13 nm. The X-ray photoelectron spectra of the Pt1AuNP, Fig. 1C, present the featured peaks at 84.0 eV and 87.7 eV for Au(0) 4f7/2 and Au(0) 4f5/2 and 71.0 eV and 74.4 eV for Pt(0) 4f7/2 and Pt(0) 4f5/2, respectively. To endow reorganization function, aptamers are modified on the Pt1AuNP surface. After removing the excess aptamer, the functionalized nanoparticle is characterized by measuring the UV absorbance spectra, Fig. 2. Except that the feature peaks of nanoparticle are monitored at 540 nm from Fig. 2 for both PtAuNP (black curve) and AptMUC1-PtAuNP (red curve), a new peak at 260 nm is found for AptMUC1-PtAuNP, Fig. 2 (red curve), indicating the successful anchoring of aptamer on the nanoparticle surface. The optimal incubation time of cancer cell in the detection kit is explored by monitoring the absorbance spectra change of TMB with incubation time (See Fig. S3A in SI). The absorbance peak of TMB at 452 nm significantly increases with increasing incubation time to 30 min and then reaches a plateau (See Fig. S3B in SI). Thus, 30 min is selected as the incubation time. 3.3. Sensitive and specific colorimetric detection of MCF-7 cells Sensitive and specific diagnostic approaches are of great importance for early and accurate detection. The sensitivity of our provided strategy is investigated by monitoring the color and absorbance change of TMB in the presence of different numbers of cancer cell, MCF-7, Fig. 3. In the absence of the target cancer cell (MCF-7), the AptMUC1-PtAuNP could not be linked together with AptVEGF-MB and would be removed from the detection system through magnetic separation. In the presence of MCF-7 cell, more and more AptMUC1-PtAuNP/MCF-7/AptVEGF-MB complexes are generated with increasing the target cell (from 10 to 105 cells/mL), leading an obvious colorimetric reading from light yellow to dark
Scheme 1. Schematic diagram of colorimetric detection of cancer cells on the basis of dual-aptamer target binding and bio-bar-code strategy.
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Fig. 1. A) Catalytic ability of PtAuNP with different ratio of Pt to Au on the colorimetric reaction of TMB in the presence of H2O2. B) TEM imaging of the as-prepared Pt1/AuNP. C) XPS of Au (red curve) and Pt (black curve) obtained from the as-prepared Pt1/AuNP. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. The UV–vis absorption spectra of PtAuNP (black curve) and AptMUC1-PtAuNP (red curve). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
yellow, Fig. 3A. Due to the excellent catalytic efficiency of PtAuNP, the color change caused by 10 cells/mL could be clearly distinguished with the naked eyes. The colorimetric signal as a function of cell number is further monitored by UV–vis spectrometry, Fig. 3B. The absorbance of TMB at 452 nm linearly depends on the logarithm number of target cells ranging from 10 cells/mL to 105 cells/mL with a regression coefficient of 0.999, Fig. 3C. Thus, a detect limit as low as 10 cells/mL with a wide detection range is obtained according to the practical measurement while not the calculation of three times standard deviation of the blank measurement. To the best of our knowledge, 10 cells/mL is the lowest detection limit for colorimetric detection of cancer cell. In previous
reports, the detection limit of colorimetric cancer cell detection is reduced from 800 cells (Liu et al., 2009) to 125 cells (Zhang et al., 2014a) due to the intrinsic property of nanomaterials. Recently, a detection limit of 40 cells for visual cancer cell detection is reached according to cell-triggered cyclic enzymatic signal amplification strategy (Zhang et al., 2014b). With our proposed strategy, the detection sensitivity is significantly improved and 10 cells/mL can be clearly distinguished by naked eyes on the basis of the synergistic action of high catalytic activity of PtAuNP and magnetic separation and concentration. The results demonstrate that the proposed assay presents a more sensitive sensing platform for cancer cells detection. Distinguishing target cancer cell from non-cancer cell and different cancer cell types is significant important while a major hurdle for cancer diagnosis and therapy (Liu et al., 2014). To address the challenge, we make use of the unique signatures of different cell types to specifically detect target cancer cells. According to our provided assay, the dual aptamer-based cytosensor can differentiate MCF-7 cancer cell from non-cancer cells, human embryonic kidney 293 cells (HEK 293) and human hepatocyte cells (HL7702), and also other cancer cells, human liver hepatocellular carcinoma cells (HeGp2). In the presence of large amount of HEK 293, HL7702 and HeGp2 cells (104 cells/mL) the respective solution color shown in Fig. 4A is similar as that in the absence of target cancer cell MCF-7, Fig. 3A. In the presence of small amount of MCF-7 (10 cells/mL), however, a remarkable color change can be easily monitored by naked eyes. The corresponding absorbance signal detection at 452 nm further confirms the colorimetric result and the excellent selectivity of the developed strategy, Fig. 3B. The different colorimetric results depend on the cell type and the
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Fig. 3. A) Photographs and B) UV–vis absorption spectra of the TMB–H2O2 against different concentrations of MCF-7 cells: 0, 10, 102, 103, 104, 105 cells/mL. C) Linear relationship between the absorbance value at 452 nm and the logarithm of MCF-7 concentration. The error bar was obtained according to five independent experimental results.
adopted dual-aptamer target binding assay. The used biomarkers, VEGF and MUC1, are limited expressed on membrane of the used control cells (Carpini et al., 2010; Guo et al., 2003). Due to the high specific affinity of the used aptamers to the targets the HEK 293 and HL 7702 cannot be trapped by AptVEGF-MB and AptMUC1-PtAuNP to form bio-bar-code configuration, causing no obvious color change. The cancer cells of HeGp2 are MUC1-negative human hepatic cancer cell line. Though the HeGp2 cells could be trapped by the AptVEGF-MB, they could not be trapped by AptMUC1-PtAuNP, leading no obvious color change. It is similar as the colorimetric response of the TMB–H2O2 system if incubate MCF-7 in the mixture of AptMUC1-PtAuNP and MB without modifying AptVEGF (See Fig. S4 in SI). The experimental results confirm that the combination of dual-aptamer target-binding with nanotechnology strategy in our
assay can efficiently reduce cross reaction and improve selectivity. Comparing with reported colorimetric methods for cancer cells detection (Table S1 in SI), the strategy developed by us presents higher sensitivity. The dual-aptamer target binding strategy makes it possible to distinguish cancer cells not only from non-cancer cells but also from different cancer cell types. To explore the feasibility of the developed strategy in biological medium, the as-prepared cytosensor was used to detect MCF-7 cells in human serum. Learned from curve (a) in Fig. 5, the absorbance intensity of TMB at 452 nm linearly increases with logarithm of cancer cell concentration from 0 to 105 cells/mL. The slope of curve (a) is very close to that of the cancer cell detection in buffer solution, curve (b), which is repotted from Fig. 3C for convenient comparison. The results indicate the excellent anti-
Fig. 4. A) Photograph and absorbance of the sensing system to different cells, HEK 293 (104 cells/mL), HL 7702 (104 cells/mL), HeGp2 (104 cells/mL) cells, and MCF-7 cell (10 cells/mL).
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the online version at http://dx.doi.org/10.1016/j.bios.2015.05.044.
References
Fig. 5. Linear relationship between the absorbance value at 452 nm in serum (a) and PBS (b) against the logarithm of MCF-7 concentration. The error bar was obtained according to five independent experimental results.
interference ability and reproducibility of the developed strategy.
4. Conclusions In summary, we have provided a simple, efficient, enzyme-free and convenient colorimetric assay for high sensitive and specific detection of MCF-7 cancer cells with acceptable reproducibility. On the membrane of MCF-7 cancer cell VEGF and MUC1 are overexpressed. The target cells act as bridge to link AptVEGF-MB and AptMUC1-PtAuNP together. The high catalytic activity of PtAuNP makes it possible for sensitive detection 10 cells/mL in phosphate buffer solution and also in serum by naked eyes without need of instrument help, which is more sensitive than previously reported colorimetric assays. The dual-aptamer target binding strategy with two different biomarkers as recognition elements improves the detection specificity and provides a more accurate way for cancer cell detection. The developed assay can be used to distinguish cancer cells not only from non-cancer cells but also from different cancer cell types. Comparing with conventional methods, the developed colorimetric strategy provides a sensitive, specific, simplicity, enzyme-free and cost-effective assay and has potential application on point-of-care cancer diagnosis.
Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 21105095 and 211900040), State Key Instrument Developing Special Project of Ministry of Science and Technology of China (2012YQ170003), the Instrument Developing Project of the Chinese Academy of Sciences (YZ201203) and the Natural Science Foundation of Jilin Province, China (No. 20130101117JC).
Appendix A. Supplementary material Supplementary data associated with this article can be found in
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