Full color emitting fluorescent carbon material as reversible pH sensor with multicolor live cell imaging

doi:10.1016/j.jphotobiol.2018.04.006

Journal of Photochemistry and Photobiology B: Biology

Volume 182, May 2018, Pages 137-145

https://doi.org/10.1016/j.jphotobiol.2018.04.006 Get rights and content

Highlights

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One-step, green and quick synthesis of hetero-atom doped fluorescent carbon material.
•
Highly selective pH sensing in wide pH range of 09–14
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Interesting full range emission is exploited for multicolour bioimaging.
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Confocal and flow cytometry based authentication of generality of cell-staining.

Abstract

Carbon-based nano materials are developed as a cytocompatible alternative to semiconducting quantum dots for bioimaging and fluorescence-based sensing. The green alternatives for the synthesis of carbon materials are imminent. The present study demonstrates microwave based one step quick synthesis of fluorescent carbon material (FCM) having three variants: (i) un-doped fluorescent carbon material (UFCM) (ii) nitrogen doped FCM (N@FCM), and (iii) nitrogen & phosphorus co-doped FCM (N-P@FCM) using sugarcane extract as a carbon source. The N doping was performed using ethylenediamine and phosphoric acid was used for P doping. The heteroatom doped FCM were synthesized due to insolubility of UFCM in water. Unlike, UFCM, the N@FCM and N-P@FCM were found to be highly soluble in water. The N-P@FCM shows highest quantum yield among the three. The N-P@FCM was explored for alkaline pH sensing and it shows a quenching of fluorescence in the pH range 09–14. The sensing behaviour shows reversibility and high selectivity. Further, the sensor was also investigated for their biocompatibility and hence employed as a promising multicolour probe for cancer cell imaging. The generality in cell imaging was investigated by flow cytometry. The hetero-atom doped green carbon-dots may open new avenues for sensing and selective cellular targeting.

Graphical Abstract

This work demonstrates a one-step, quick and green synthesis of fluorescent carbon materials for reversible pH sensing in the range 9–14 along with interesting full color emission for intracellular employability.

Introduction

The benign synthesis process by employing biomolecules and plant tissues are being explored for the synthesis of nanomaterials and are shown to have added advantages over chemically synthesized materials [1]. Ever since the inception of semiconducting quantum dots for bioimaging applications, their higher toxicity remains a concern [2], while poor chemical stability and photo-bleaching limits the use of conventional organic fluorophores. The carbon-based nanomaterials have emerged as a promising fluorescent alternative to unstable organic dyes and toxic semiconducting quantum dots [3]. There are several methods reported so far for the synthesis of carbon dots such as electrochemical [4], microwave [5], hydrothermal [6,7], arc discharge [8], laser ablation [9] and refluxing [10]. However, due to the use of harsh chemicals, tedious reaction conditions and time-consuming synthesis, there is a compelling requirement for an eco-friendly, quick and economical route for synthesis of fluorescent carbon material with large-scale synthesis capability [[11], [12], [13], [14]]. The eco-friendly synthesis of carbon nanodots using abundant carbohydrates and proteins present in different biomass are recently explored and is an emerging field [[15], [16], [17], [18], [19], [20]]. The quantum yield is an important parameter to assess the quality of a fluorophore and efforts have been made to increase quantum yield and tune the emission wavelengths using heteroatom doping [[21], [22], [23], [24], [25]].

The pH sensors have found numerous applications in the fields ranging from environmental science to medical diagnosis and are an essential part of biological research. The electrochemical pH sensor is well established but suffers from disadvantages like the requirement of frequent calibrations, electrode corrosion and electrical interference [26]. On the other hand, recently growing fluorescence-based pH sensors offer advantage of high sensitivity, ease of miniaturization and contactless measurement [27,28].

The bioimaging using carbon dots is an exciting and growing area [[29], [30], [31], [32]]. The lipophilicity of cell staining agent is of the utmost importance that can derive these materials into the cells.

Herein, we report a one-step benign synthesis of fluorescent carbon material (FCM) using sugarcane extract as a precursor. The hetero-atom doped variant N@FCM and N-P@FCM were also synthesized. The N-P@FCM was employed as a pH-responsive material with the added advantage of promising bio-imaging application along with excellent cytocompatibility.

Section snippets

Reagents

The sugarcane was procured from local market. Phosphoric acid and ethylenediamine were purchased from Sigma-Aldrich. Different metal salts of analytical grade were procured from Sigma-Aldrich and Merck and were used without further purification.

Instrumentation

Anton-Paar, Monowave 300 Microwave reactor was used for synthesis. The absorption studies were performed on a Varian Cary 100 Bio UV–Visible spectrophotometer. A Fluoromax spectrofluorometer was used for fluorescence spectrophotometric studies. The

Results and Discussion

A series of fluorescent carbon material (FCM) was synthesized by a single step, benign approach using sugarcane extract as a carbon source under microwave irradiation. The FCM consists of: (i) Undoped/As synthesized FCM (UFCM), (ii) Nitrogen-doped FCM (N@FCM) using ethylenediamine, and (iii) Nitrogen and Phosphorus co-doped FCM (N-P@FCM) using ethylenediamine and phosphoric acid (Scheme 1). The challenge with UFCM was its solubility in water and hence water-soluble alternatives N@FCM and N-P@FCM

Conclusions

In conclusion, the green carbon precursors can be used as a source for low cost, high yield c-dots. The heteroatom doping results in enhanced fluorescence properties and the inherent non-toxicity of biomass could assist excellent biological applicability of c-dots.

In summary, we demonstrate a green synthesis of fluorescent carbon material (FCM) using sugarcane as the sole carbon precursor. The doping of nitrogen and phosphorus (N@FCM and N-P@FCM) offers better solubility, high quantum yield and

Conflicts of Interest

There are no conflicts of interest to declare.

Acknowledgements

We are grateful to the Sophisticated Instrumentation Centre, IIT Indore for all the characterization facilities. V. S. and N. K. gratefully thanks UGC, New Delhi for research fellowship. P. T. thanks MHRD for research fellowship. SMM thanks SERB-DST, Govt. of India for a research grant. We sincerely acknowledge ACMS, IIT Kanpur for providing XPS facility. We are grateful to Prof. G. Hundal and Dr. Sanyog Sharma, Gurunanak Dev University for helping in TEM analysis.

References (54)

J.R. Bhamore et al.
One-step green synthetic approach for the preparation of multicolor emitting copper nanoclusters and their applications in chemical species sensing and bioimaging
Biosens. Bioelectron.
(2016)
Y.-J. Yan et al.
Nitrogen-doped graphene quantum dots-labeled epitope imprinted polymer with double templates via the metal chelation for specific recognition of cytochrome c
Biosens. Bioelectron.
(2017)
T.N.J.I. Edison et al.
Microwave assisted green synthesis of fluorescent N-doped carbon dots: cytotoxicity and bio-imaging applications
J. Photochem. Photobiol. B
(2016)
T.N.J.I. Edison et al.
Turn-off fluorescence sensor for the detection of ferric ion in water using green synthesized N-doped carbon dots and its bio-imaging
J. Photochem. Photobiol. B
(2016)
P. Roy et al.
Photoluminescent carbon nanodots: synthesis, physicochemical properties and analytical applications
Mater. Today
(2015)
S. Godavarthi et al.
Nitrogen doped carbon dots derived from Sargassum fluitans as fluorophore for DNA detection
J. Photochem. Photobiol. B
(2017)
X. Sun et al.
Green synthesis of carbon dots originated from Lycii Fructus for effective fluorescent sensing of ferric ion and multicolor cell imaging
J. Photochem. Photobiol. B
(2017)
V. Arul et al.
Biological and catalytic applications of green synthesized fluorescent N-doped carbon dots using Hylocereus undatus
J. Photochem. Photobiol. B
(2017)
J. Kudr et al.
Carbon dots based FRET for the detection of DNA damage
Biosens. Bioelectron.
(2017)
C. Zhang et al.
Algae biomass as a precursor for synthesis of nitrogen-and sulfur-co-doped carbon dots: a better probe in Arabidopsis guard cells and root tissues
J. Photochem. Photobiol. B
(2017)

A.-M. Alam et al.

Synthesis of carbon quantum dots from cabbage with down- and up-conversion photoluminescence properties: excellent imaging agent for biomedical applications

Green Chem.

(2015)

W. Shi et al.

N, S co-doped carbon dots as stable bio-imaging probe for detections of intracellular temperature and antibiotic

J. Mater. Chem. B

(2017)

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View full text

Full color emitting fluorescent carbon material as reversible pH sensor with multicolor live cell imaging

Highlights

Abstract

Graphical Abstract

Introduction

Section snippets

Reagents

Instrumentation

Results and Discussion

Conclusions

Conflicts of Interest

Acknowledgements

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