Aptamer-based fluorescent and visual biosensor for multiplexed monitoring of cancer cells in microfluidic paper-based analytical devices
Introduction
Cancer, as a complex disease, greatly threatens human health, and until now, it remains the second leading cause of death worldwide [1]. Simultaneous monitoring of different types of cancer cells in a single assay plays a critically important role in the process of early cancer screening, cancer metastasis and cancer therapies [2], [3], [4]. Currently, various cyto-sensors have been applied in the field of cancer research including chemiluminescence [5], electrochemical impedance spectroscopy [6], electrochemistry [7] and fluorescence [8], and so on. These conventional methods possess high sensitivity and selectivity for nondestructive detection of intact cancer cells. However, there is still an urgent need for a rapid, facile, portable, disposable and low-cost detection methods for the earlier and sensitive detection of cancer cells, for clinical and point-of-care applications, especially in developing countries, resource-limited and remote regions. Recently, microfluidic paper-based analytical devices (μ-PADs) have been introduced into immunoassay as promising tool for point-of-care diagnosis due to their low-cost, ease-to-operate, portable platforms and disposability [9], [10]. To the best of our knowledge, in the past, the primary analytical technology on μ-PADs relied on colorimetric methods [11] and fluorescent techniques [12], [13]. So, we combined the fluorescent technology and visual detection on μ-PADs to achieve the quantitive determination of cells providing distinct advantages, such as visual detection, high detection sensitivity and specificity.
With the remarkable development of nanotechnology, nanomaterials with different chemical and physical properties have brought a variety of novel approaches to the diagnosis and therapy of diseases based on the use of nanoparticles (NPs) [14]. Quantum dots (QDs) have gained more and more attention because of their unique properties such as a large stokes shift, high fluorescence efficiency, broad excitation spectra [15], photobleaching resistance [16], narrow, symmetric, and tunable emission spectra and simultaneous excitation of multiple fluorescence colors [17], which are not possessed by traditional organic dyes. Meanwhile, the good biocompatibility has enabled QDs labeling for the detection of various biological analytes, such as DNA [18], protein [19] and cells [20]. Signal amplification strategies are generally necessary for the construction of a sensitive fluorescent probe, because inadequate target-to-background signal ratio and the effects of photobleaching can limit the sensitivity. More significantly, many efforts have been directed to mesoporous silica nanoparticles (MSNs) for a wide arrange of applications due to its unique structural features including large surface-to-volume ratios, uniform pore diameters and a great diversity in surface functionalization [21], which enables conjugation of more number biomolecules to improve the sensitivity of assay. There, MSNs were prepared to load more QDs for signal amplification.
Tumor cells as targets, specific DNA aptamers against tumour cells was used to distinguish cancer cells from normal cells or other particular tumor type among various strains [22], [23]. Recent research has also seen the development of cancer cell study using self-assembled DNA nanostructures [24] and DNA based extracellular matrix [25]. The combination of NPs and DNA enables a modular and signal-amplified sensing strategy. Therefore, it is possible that such functionalized DNA nanomaterials may become one of the most important tools in cancer study. Graphene oxide (GO) is a single-atom-thick and two-dimensional carbon material, containing large quantities of oxygen atoms in the form of epoxy, hydroxyl, and carboxyl groups [26]. Recently, GO has received rapidly increasing attention due to its high quenching efficiency, large surface area, good water dispersibility, biocompatibility and facile surface modification, and so on, making it a promising candidate for biosensors [27]. GO can act as an efficient fluorescence quencher based on the photoinduced electron transfer mechanism or energy transfer mechanism. GO interacts with single-stranded DNA through π–π stacking interaction with nucleobases and efficiently quenches fluorescence of QDs [28], [29]. Based on this principle, a range of fluorescent biosensors have been developed for the detection of DNA [30], metal ions [31], proteins [32] and small molecules [33].
In this work, we designed a cancer cell sensing platform composed of different colored MSNs/QDs-labeled aptamers and GO for visual, low-cost, sensitive, facile, disposable, real-time monitoring of multiple cancer cells on μ-PADs. Aptamers were selected from three different cancer cell lines due to their high specificity and affinity. The fluorescence of these MSNs/QDs–DNA bioconjugates was quenched by GO to produce a cancer cell capture probe. Once the probe was exposed to the target cells, the corresponding colored probe would be released and emit strong fluorescence, indicating the existence of the corresponding target cell. Our results indicated that these MSNs/QDs–DNA bioconjugates could be used as a highly sensitive and selective platform for sensing multiple cancer cells simultaneously. This method can not only determine cancer cells but also realize other bioanalysis.
Section snippets
Materials
All chemicals and solvents related to cell culture and assay used here were aseptic after heat sterilization treatment. Tetraethoxysilane (TEOS) and (1-hexadecyl)trimethylammonium bromide (CTAB) were purchased from Sigma (St. Louis, MO, USA). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Alfa Aesar China Ltd. (3-aminopropyl) triethoxysilane (APTES) was purchased from Chemical Reagent Co. (Tianjin, China). Graphene oxide
Characterization of QDs, MSNs/QDs and MSNs/QDs–DNA
Here, yellow colored QDs and its corresponding MSNs/QDs, MSNs/QDs–DNA were used as models for the following characterization.
This work focused on the design of a DNA-based cellular probe for multiplexed monitoring of cancer cells. The entire assembly process of the probe was illustrated in Scheme 1B. Firstly, QDs (5 mg mL−1) stabilized with mercaptoacetic acid, which coated amino-functionalized MSNs by succinimide coupling method. Subsequently, amino-modified DNA was covalently immobilized on
Conclusions
In conclusion, we combined the fluorescent technology with semiquantitative visual analysis for simple, sensitive, highly selective, rapid, and multiplexed monitoring of cancer cells using three-color fluorescence output signals on μ-PADs. The system showed high specificity, which was provided by the aptamers immobilized on the surface of the NPs. More importantly, it could be detected under the same excitation wavelength, which made the assay much easier to achieve multiplex detection than the
Acknowledgements
This work was financially supported by the Special Fund for Shandong independent innovation and achievements transformation (No. 2014ZZCX02703), National Natural Science Foundation of China (21575051, 21475052), Natural Science Foundation of Shandong Province, China (ZR2015JL006).
Linlin Liang studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student.
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Linlin Liang studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student.
Min Su studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student.
Li Li studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student.
Feifei Lan studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student.
Guangxin Yang studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student.
Shenguang Ge received his PhD degree in Chemistry and Chemical Engineering in 2013 from Shandong University, completed his master degree studies in University of Jinan in 2006. He joined University of Jinan, where currently he is a lecturer of chemistry. His research interests are in the area of biosensor and chemsensor.
Jinghua Yu received her PhD degree in analytical chemistry in 2003 from Lanzhou Institute of Chemical Physics, China. She is currently positioned as a professor at University of Jinan. She spends most of her time investigating biomedical engineering, especially for the development of biosensor devices and analytical tools.
Xianrang Song works in Cancer Research Center, Shandong Tumor Hospital in China.