Label-free fluorimetric detection of CEA using carbon dots derived from tomato juice
Introduction
Tumor markers, appearing as molecules in blood or tissue, have been proved to be associated with carcinogenesis, and their related identification or measurement exhibited great importance for patient diagnosis or clinical management (Miyake et al., 2010; Eppler et al., 2002). The determination of tumor markers plays an important role in the early diagnosis of cancer, differentiating malignant from benign conditions, evaluating the extent of disease, monitoring tumor response to therapy, and predicting recurrence (Asawatreratanakul and Thavarungkul, 2006; Fakhraddin and Padide, 2006). Carcinoembryonic antigen (CEA), a tumor-associated antigen, is expressed in many malignancies such as lung cancer, ovarian carcinoma, breast cancer, and cystadenocarcinoma (Chen et al., 2009, Bisceglie et al., 2003). Accordingly, quantitative and selective detection of CEA is critical for clinical purposes. However, amounts of CEA in colon tissue of adults are reported as a low level of 2.5–5.0 ng mL−1 (Withofs et al., 2000; Zamcheck and Martin, 1981), which makes it quite a challenge to detect CEA level using available approaches. Currently, immunoassays and related techniques including enzyme immunoassay, chemiluminescence immunoassay, Raman spectroscopy, surface plasmon resonance and electrochemical immunoassay have been considered as major analytical methods towards detecting CEA (Gomez-Hens et al., 2009, Hui et al., 2009, Wang et al., 2009, Wang et al., 2009, Bi et al., 2009, Zhong et al., 2010). Howbeit, most of these methods show drawbacks, such as requiring radiation hazards, time-consuming, poor precision and sophisticated instrumentation, leading to a strong desire for the development of alternative approaches.
Aptamers, as artificial oligonucleotides (DNA or RNA) isolated through a selection process in vitro or systematic evolution of ligands by exponential enrichment (SELEX) (Osborne and Ellington, 1997; Wilson and Szostak, 1999), can bind to a wide variety of entities with high selectivity, specificity and affinity (Ellington and Szostak, 1990, Tuerk, 1990). Compared with other natural receptors such as antibodies and enzymes, aptamers show various unapproachable advantages including easy production by chemical synthesis, convenient acquisition from commercial sources and satisfactory chemical stability (Kwame et al., 2009, Nutiu and Li, 2005). In addition, aptamers generally exhibit high specificity and affinity for their targets, and their structural flexibility allows for adaptation of undergoing significant conformational changes once aptamers and their targets specifically bind with each other (Blind and Blank, 2015, Nielsen et al., 2010). Hence, these attractive superiorities potentiate them as ideal biosensing molecules (Billinge et al., 2014, Kim et al., 2015, Ara et al., 2014, Song et al., 2014), and aptamer have been exploited in biorecognition applications of proteins, cancer cells and cancer medicine (Li et al., 2014, Xiang et al., 2015, Ghasemi et al., 2015, Kim and Gu, 2014).
Currently, various types of nanomaterials have been employed to provide fluorescence response and construct ultrasensitive biosensors with the development of nanoscience and nanotechnology (Min et al., 2011, Zhang, 2012). Fluorescent nanomaterials, especially quantum dots (QDs) and noble metal nanoclusters (NCs), have been appearing as powerful and sensitive probes and attracted numerous attentions (Yang and Lian, 2014, Wen et al., 2014). Likewise, carbon dots (CDs) appear as a new class of ‘zero-dimensional’ fluorescent nanomaterials in the carbon family (Baker and Baker, 2010), and exhibit enormous prospects across the scientific disciplines owing to their ease of synthesis, abundance of raw material in nature, excellent chemical and colloidal stability, low photobleaching, scarcely optical blinking, favorable biocompatibility and superior water dispersibility (Hola et al., 2014, Song et al., 2014, Yang et al., 2013, Dong et al., 2012, Song et al., 2012). Consequently, CDs are attracting more attentions, and becoming a promising class of nanosensors for multifunctional purposes (Guo et al., 2013, Cheng et al., 2014, Gogoi et al., 2015, Cui et al., 2015). Also, CDs have been successfully applied in fields of clinical diagnostics, food industry and environmental safety (Miranda et al., 2011). Surprisingly, the fluorescent property of carbon dots has not been exploited to develop biosensors for detecting clinically important proteins.
Herein, we report a green synthesis of carbon dots by one-step hydrothermal treatment of tomato juice under comparative conditions of low temperature (150 °C) and short time (120 min) (Fig. 1(A)). The mechanism for the formation of CDs involved polymerization, dehydration and carbonization of the constituents of tomato juice such as saccharides, citric acid, and ascorbic acid (Hsu and Chang, 2012, Sun and Li, 2004). Again, both the precursor of CDs and the synthesis procedure are substantially environment-friendly, facilitating their biocompatibility and more extensive applications. More importantly, we developed a label-free fluorimetric assay with high sensitivity and selectivity for monitoring CEA level, while CDs served as the fluorescent probe and CEA-aptamer as the intermediate (Fig. 1(B)). Specifically, the fluorescence of CDs was obviously quenched once CEA-aptamer was introduced owing to the adsorption of CEA-aptamer (ssDNA) to the surface of CDs and the subsequent formation of CDs-aptamer complex. Upon the addition of CEA, the stronger binding affinity between CEA and their corresponding aptamers induced unwinding of CEA-aptamer from the CDs surface, leading to the recovery of quenched fluorescence. In the light of the adsorption and desorption of aptamers from the surface of CDs through a competitive mechanism, this proposed strategy showed satisfactory sensitivity and selectivity for detecting CEA in practical samples in a continuous and recyclable way. Besides, the CDs were used for cell imaging, potentiating them towards diverse purposes.
Section snippets
Chemicals
Tomatoes were bought from Yonghui Supermarket (Chongqing, China). CEA, AFP, CA125, CA15-3 were purchased from Boson Biotech Co., Ltd. (Beijing, China). The DNA were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China), and their corresponding sequences are as follows: 5′-ATACCAGCTTATTCAATT-3′ (CEA-aptamer). Metal ions (Fe3+, Ca2+, K+, Na+), thrombin, tyrosinase, dopamine, glucose oxidase, heparin sodium, bovine serum albumin (BSA) glucose, folic acid, norepinephrine (NE), bile
Characterization of the fluorescent CDs
To get an insight into the PL mechanism of the CDs, a series of characterizations had been successively performed. Accordingly, the CDs exhibited distinctly blue fluorescence with a peak maximum at 440 nm upon 367 nm excitation (Fig. 2(A)). Other fluorescent properties of the CDs were subsequently investigated in detail. The aqueous solution of CDs emitted obvious blue fluorescence (Fig. 2(A), photographⅡ) under UV light (365 nm), while appeared as transparent yellow under daylight (Fig. 2(A),
Conclusions
In summary, an innovative strategy to synthesize the blue-fluorescent carbon dots with tomato juice has been proposed for the first time. The whole synthesis procedure was simple, facile and environmental-friendly. Importantly, these CDs, rich in carboxyl groups on the surface, were employed for the quantification of CEA. Initially, CEA-aptamer adsorbed to the surface of CDs, depending on the π-π stacking interactions between CEA-aptamer and π-rich CDs, facilitated the fluorescence quenching of
Acknowledgements
We gratefully acknowledge financial support by Fundamental Research Funds for the Central Universities (XDJK2015A005 and XDJK2016D033), Innovative Research Project for Postgraduate Students of Chongqing (CYS14049).
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