- Email: [email protected]
Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot
Accepted Manuscript Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot Morteza Hosseini, Hossein Khabbaz, Amin Dezfoli, Mohammad RezaGanjali, Mehdi Dadmehr PII: DOI: Reference:
S1386-1425(14)01614-X http://dx.doi.org/10.1016/j.saa.2014.10.117 SAA 12932
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
21 August 2014 15 October 2014 27 October 2014
Please cite this article as: M. Hosseini, H. Khabbaz, A. Dezfoli, M. RezaGanjali, M. Dadmehr, Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.10.117
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot Morteza Hosseini a,∗∗ ,Hossein Khabbaza,Amin Dezfolib,Mohammad RezaGanjalib,c,Mehdi Dadmehra,d a
Department of life science engineering, Faculty of New Sciences & Technologies, University of
Tehran, Tehran, Iran b
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran,
Iran c
Endocrinology& Metabolism Research Center, Tehran University of Medical Sciences, Tehran,
Iran d
Payame Noor University, Tehran, Iran
*
Corresponding authors. Tel.: +98-21-61112788; Fax: +98-21-66495291 Email addresses:[email protected] (M. Hosseini)
1
ABSTRACT Graphene quantum dots (GQDs) have successfully been utilized as an efficient nano-sized fluorescence chemosensor to detect selectively Glutamate (Glu) in Tris-HCl buffer solution (pH=9). The fluorescence emission spectrum of graphene quantum dots was at about 430 nm. The study showed that fluorescence intensity of the quantum dot gradually enhanced with increase in concentration of Glutamate and any change in fluorescence intensity was directly proportional to the concentration of Glutamate. Under optimum conditions, the linear range for the detection of Glutamate was 1.6×10-7M to 1.0×10-5 M with a detection limit of 5.2×10-8 M. The sensor showed high selectivity toward Glutamate in comparison with otheramino acids. Keywords:Graphene; Quantum dot; Glutamate; Fluorescence Enhancement; Amino acid
Introduction
2
To date, graphene which is composed of a single layer of carbon atoms with closely packed honeycomb structure [1], has received more considerable attention due to its individual and novel properties [2] such as large surface area, resistance property, chemical stability, and
improved mechanical flexibility, thermal environmentally friendly nature., These
characteristics introduce it as a promising alternative agent may show broad application in biosensors[3]. Lack of a band gap in pristine graphene results to failure of
its optical
luminescence observation. [4] Therefore, many attempts has been made to convert the 2D graphene sheets into 0D graphene quantum dots (GQDs) [5] In order to facilitate the application of graphene in nanoscale devices and also effectively tuning the band gap of graphenes. Graphene quantum dots (GQDs) are single or few-layer graphenes with a tiny size of only several nanometers [6], they show some remarkable properties, such as chemical stability, excellent water solubility and suitability. Importantly, the presence of carboxylic acid groups at the edge of GQDs make it easy for functionalization with various organic, polymeric, inorganic or biological species [7]. L- Glutamate (anion of glutamic acid) is one of the 20 standard amino acids used by all organisms. It plays an important role in food processing, clinical applications and is also well known as a flavour enhancer, commonly found in various foods. The excessive intake of this flavour enhancer can cause allergic effects such as headache and stomach pain [8,9]. LGlutamate is also an excitatory neurotransmitter in the central nervous system of vertebrates, and is a potent neuroexcitatory amino acid associated with certain behavior patterns such as aggressive behavior visual task learning, morphine-induced muscular rigidity and retrograde amnesia [10-12].
3
Aspartate and glutamate are major excitatory amino acids (EAAs) in the central nervous system. In recent years, some researchers have focused on the correlation between altered levels of EAAs in humans and some pathology, including diabetes and cancer [13]. The analysis of EAAs in biological samples is particularly applicale in biochemistry and clinical chemistry [14,15]. Several methods for determination of a trace amount of glutamate have been reported: amperometric method [16], Chromatography [17] and capillary electrophoresis [18]. However, most of the above mentioned methods do not seem to satisfy the requirements for monitoring of Glu in the terms of facility, sensitivity, matrix toughness and cost effectiveness. Among the various detection techniques, fluorescent chemosensors have been developed quickly due totheir simplicity and high sensitivity [19-23]. Fluorescent chemosensors are of great importance owing to their high sensitivity and low detection limit. Most literatures report use of fluorescence quenching as the readout mechanism for the sensor response. The greatest advantage of fluorescence enhancement sensors, in comparison with fluorescence quenching sensors, is the ease of measuring low concentration contrast relative to a “dark” background. This reduces the likelihood of false positive signals and increases the sensitivity [24].
Consequently, there is an urgent need to develop new turn-on fluorescent sensors which can be advantageous. However, to the best of our knowledge, fluorescent sensors for Glu, especially with fluorescence enhancement technique, have not been reported. Experimental Reagents All chemicals were of the reagent–grade purity from Aldrich and Merck chemical companies. The amino acids tested (L-Alanine(Ala), L-Methionine(Met), L-Serine(Ser), L4
Leucine(Leu), L-Glutamic Acid(Glu), L-Threonine(Thr), L-Valine(Val), L-Arginine(Arg), LProline(Pro),
L-Aspartate(Asp), L-Phenylalanine(Phe), L-Cysteine(Cys), L-Pyroglutamic
acid(Pyr) were purchased from Aldrich Company.
Apparatus All fluorescence measurements were carried out on a Perkin-Elmer LS50 luminescence spectrometer. Synthesis procedure of GQDs Graphene oxide (GO) was synthesized from graphitic powder according to Hummer’s method [25]. In brief, 0.5 g of graphite powder, 0.5 g of NaNO3 and 23 mL of H2SO4 were added into a three-necked flask with stirring in an ice bath. Then, 3 g of KMnO4 was slowly added to the mixture. After mixed together, the flask was transferred to 35±5 oC water bath for 1 h with stirring and then a brown paste was obtained. Next, 40 mL of ultrapure water was added, and the solution was stirred for 30 min. Another 100 mL of ultrapure water was added, and then addition of 3 mL H2O2 (wt 30%) made the color of the solution change to bright yellow. The warm GO solution was filtered and washed with ultrapure water until pH to 7.0. The precipitates were dried for 24 h in a freeze-dryer.
Preparation of GQDs The GQDs were prepared from graphene sheets (GSs) by a hydrothermal approach as the literature procedure [26]. Graphene sheets (GSs) were produced by thermal deoxidization of dried GO at 250oC for 2h in a tube furnace with a heating rate of 5 oC/min. The condition was in a nitrogen atmosphere. 0.05 g of GSs were oxidized in 40 mL concentrated H2SO4 and HNO3 5
(volume ratio 1:3) for 18 h under mild sonication. The solution was diluted with 250 mL of water, and then filtered to remove the acids. Purified oxidized GSs were redispersed in 40 mL of ultrapure water and the pH was adjusted to 8 with NaOH solution. Then, the suspension was transferred into a Teflon-lined autoclave (50mL) and heated at 200 oC for 10 h. After cooling to room temperature, the resulting suspension was filtered and the brownish filtrate was dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 24 h.
Result and discussion Characterization of GQDs During the oxidation, oxygen-containing functional groups, including C=O/COOH, OH, and C-O-C, were introduced at the edge and on the basal plane, as shown in the Fourier transform infrared (FTIR) spectrum (Fig. 1). Due to the presence of these groups, the chemically synthesized GQDs are readily water-dispersible. The peak at about 1363 cm-1 indicate the existence of COO-, while the peak at 3320 cm-1 corresponds to the OH stretching mode. Furthermore, the observed peaks at 1018, 1200, 1720, and about 1660 cm-1, are assigned to C– O–C stretching, C-OH stretching, C=O stretching, and C=C stretching mode of vibration, respectively. Fig.2 shows a transmission electron microscope (TEM) image of GQDs. Their diameters are mainly distributed in the range of 5–20 nm. Fig. 1 Fig. 2
6
Preliminary studies In order to evaluate whether GQDs could be used as a selective fluorescent chemosensor for amino acids, the interaction of GQDs with a number of amino acids was investigated spectrofluorometrically in Tris-HCl buffer solution at 25.0±0.1 ºC. The resulting fluorescence intensities for amino acids are shown in Fig. 3. It can be seen clearly from this Fig. 3, no significant fluorescence changes were observed when Ala, Cys, Trp, Arg, Asp, Ser, Val, Tyr, His, Pro, and Lys were added. In contrast, the addition of Glu, affords a very high-intensity emission centering around 430 nm. Fig. 3 Fluorescence titration Fluorescence properties of GQDs(5.0×10-7M) were examined in Tris-HCl buffer solution at room temperature. It exhibited emission with the emission maxima at 430 nm when they are excited at 365 nm. Considering the appreciable changes in fluorescence properties of the GQDs toward addition of Glu, the potential for developing a novel fluorescence probe for determination of Glu was assessed. As shown in Figs. 4 and 5, upon addition of increasing concentrations of Glu to the GQDs solution, a gradual increase in the fluorescence intensity was clearly detected. Furthermore, as shown in Fig. 4B, the fluorescence emission spectra of GQDs was sensitive to Glu. In the presence of various concentration of Glu ranging from 1.6×10−7 to1.0×10−5mol/L, significant fluorescence enhancement of fluorescent probe was observed. The detection limit was estimated as 5.2×10−8mol/L (calculated as three times standard deviation of blank solution). The binding constant value has been determined from the emission intensity data following the modified Benesi–Hildebrand equation [27]: 7
1/F=1/Fmax+(1/K[C])( 1/Fmax) where F=Fx–F0 and Fmax= F–F0, and where F0, Fx and F are the emission intensities of GQDs considered in the absence of Glu, at an intermediate Glu concentration, and at a concentration of complete interaction, respectively. K is the binding constant for the interaction and [C] is the Glu concentration. From the plot of (F–F0)/(Fx–F0) against [C]-1 for L, the value of K extracted from the slope is 4.2×106 M -1. Fig. 4 Fig. 5 A comparison between this work and other previous reported methods for Glu determination is listed in Table 1. In comparison with these methods, this sensor has a wider linear range than previously methods. The detection limit of the proposed sensors is 5.2 ×10-8 mol L−1 which is lower than electrochemical sensor and electrochemical biosensor. In term of the linear range and detection limit, it can be seen that the proposed sensor displays even more sensitivity than most of the reported methods [16, 18, 28-30]. pH effect In order to investigate the effect pH on the emission GQDs, the change of intensity were measured at a fixed concentration of Glu (5.0×10-5 M) having different pH values (Fig.6). As it is seen (Fig.6) in the presecnce of Glu, response of the fluorescent sensor was dependent on the pH of the test solution and a pH value of 8.4, maximum emission of the GQDs in the presence Glu was obtained. The pH was altered from 3- 10 by adding HNO3 or NaOH. Fig. 6
8
Selectivity The selectivity behavior is obviously one of the most important characteristics of a chemosensor, which is the relative sensor response for the primary amino acid over other amino acids present in solution. To examine the selectivity of one, we investigated its affinity for other amino acids. The influence of various amino acids on the fluorescence behaviour of GQDs was shown in Fig. 7. As can be seen from Fig. 7, except for Cys and Try, other amino acids made no obvious changes in the fluorescence intensity. The existence of oxygen-containing groups ( carboxylic (_COOH) and hydroxyl(–OH) ) at the edges of GQDs was expected to increase both stability and selectivity and provide a binding site for the α-amino group of glutamate
as
reported previously[31,32]. The possible mechanism of the enhancement of GQDs with addition glutamate could arise H-bond interaction from the alcohol or carboxylic group of the GQDs and the amino group of the glutamate[31-33]. Furthermore, competition experiments were also performed for GQDs in the presence of Glu at 50µM mixed with 200µM background amino acids such as Val, Met, Cys, Trp, Glu, Tyr, Ser, Asp, Na+ and K+ (Fig. 7). In the presence of other amino acids, no obvious variation in the fluorescence was observed except of Trp have quenching effect. All these results implied that GQDs could be used as a potential candidate of Gluchemosensor with very high selectivity. Fig. 7
Conclusion In summary, we have demonstrated a convenient and novel type of GQDs based sensor for the detection of Glu with high sensitivity and selectivity. Presence or addition of Glu show enhancement of fluorescence emission intensity of GQDs. The remarkable properties for this
9
method such as simplicity, rapidness and selectivity will help to extend its applications for determination of Gluin samples. Acknowledgements: The authors are grateful to the Research Council of University of Tehran for the financial support of this work.
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[10] D. Compagnone, G. Federici, R. Massoud, L. Santoro, M. Anichini, M. Palleschi, Clin. Chem. 38 (1992) 2306-2310. [11] J.A. Gordon, Nature Neurosci. 13 (2010) 2-4. [12] M. Jamal, M. Hasan, A. Mathewson, K.M. Razeeb, Biosens.Bioelectron. 40 (2013) 213– 218. [13] G.A. Qureshi, A.R. Qureshi,J.Chromatogr. 491(1989) 281–289. [14] A.V. Hemelrijck, S. Sarre, I. Smolders, Y. Michotte,J.Neurosci. Method. 144 (2005) 63– 71. [15] H.J. Zhang,J.S. Li,H. Wang, Y.Q.Feng, Anal.Bioanal. Chem. 386 (2006) 2035–2042. [16] M. Jamal, M. Hasan, A. Mathewson, K.M. Razeeb,Biosens. Bioelectron. 40 (2013) 213– 218. [17] E. Swanepoel, M.M. De Villiers, J.L. Du Preez, J. Chromatogr. A 729 (1996) 287-291. [18] J. Chapman, M. Zhou, Anal. Chim. Acta 402(1999) 47-52. [19] S. Tucci, C. Pinto, J. Goyo, P. Rada, L. Herna´ndez,Clin. Biochem. 31 (1998) 143. [20] M. Hosseini, Z. Vaezi, M.R. Ganjali,F.Faridbod, S.D. Abkenar, K Alizadeh, M. SalavatiNiasari, Spectrochim. Acta part A 75 (2010) 978-982. [21] M.R. Ganjali, M. Hosseini, A. Karimi, H. Haji-hashemi, M. Salavati-Niasari, P. Norouzi,Spectrochim. Acta Part A 121 (2014) 224-229. [22] M. Hosseini, M.R. Ganjali, B. Veismohammadi, F. Faridbod, P. Norouzi, S. DehghanAbkenar, Sens. Actuators B 147 (2010) 23-30. [23] M.Hosseini,, V.K. Gupta, M.R.Ganjali, Z.Rafiei-Sarmazdeh, F. Faridbod, H. Goldooz, A.R. Badiei, P.Norouzi,Anal. Chim.Acta 715 (2012) 80–85. [24] M.R. Ganjali, M. Hosseini, M. Hariri, F. Faridbod, P. Norouzi, Sens. Actuators B 141 (2009) 90–96. [25] W. S. Hummers, Jr. Richard, E. Offeman, J.Am. Chem. Soc 80 (1958) 1339-1339 [26] D. Pan, J.C. Zhang, Z. Li, M.H. Wu, Adv. Mater. 22 (2010) 734-738. [27] H.A. Benesi, J.H. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703-2707.
[28] V. M. Tolosa, K. M. Wassum, N. T. Maidment, H. G. Monbouguette, Biosens. Bioelectron. 42 (2013) 256-260 11
[29] H. J. Zhang, J. S. Li, H. Wang, Y. Q. Feng, Anal. Bioanal. Chem. 386 (2006) 2035-2042
[30] S. L. Alvez-Crespo, M. J. Lobo-Caston, A. J. Miranda-Ordieres, P. Tunon-blanco, Biosens. Bioelectron. 12 (1997) 739-747.[31] H. Charkraborti, S. Sinha, S. Ghosh, S. K. Pal, Mater. Lett. 97 (2013) 78-80. [32] H. Sun, L. Wu, W. Wei, X. Qu, Mater. Today 16 (2013) 433-442. [33] X. b. Zhou, Y. W. Yip, W. H. Chan , A. W. M. Lee, Beilstein J. Org. Chem. 7 (2011,) 75-81
Detection Limit
Dynamic Range
(M)
(M)
method
Ref.
Table 1 Comparison of the characteristics of the proposed sensor with those of the previously reported for Glu.
12
electrochemical sensor
9.6×10-6
1.0×10-3-8.0× 10-3
16
electrochemical biosensor
3.2×10-7
5.0×10-6-3.0×10-4
28
HPLC- Fluorescence detector
5.3×10-9
Fluorometric
1. 8×10-6
Amperometric
3.8 ×10-6
Fluorescence sensor
5.2 ×10-8
8.0×10-9-9.6×10-7 5.0×10-9-1.2×10-6
5.0×10-6-7.8×10-5 1.6×10-7-1.0×10-5
29 18 31 This work
Figure Caption: Fig.1.FT-IR spectra of GO and GQDs Fig.2. TEM image of GQDs Fig. 3. Fluorescence responses of GQDsTris-HCl buffersolution upon addition of amino acids (5×10-4M) (λex= 365 nm). 13
Fig. 4.a) Fluorescence titration of GQDs inTris-HCl buffersolution in the presence of varying concentrations of Glu(λex= 365nm). b) Visual fluorescence changes of sensor GQDs in the absence and excess presence of Glu ion (1.0×10-2M). The photo was taken under a handheld UV (365 nm) lamp. Fig.5. Calibration curve of Glu fluorescent chemosensor. Fig.6. Effect of pH of the test solution on the fluorescence response of GQDs in the presence Glu (5.0×10-5M) Fig. 7.Relative fluorescence intensity ofGQD the presence of various ions and containing 30 µM Glu and the background amino acids 200 µM (λex: 365 nm).
14
Fig.1
15
Fig.2.
16
300
250 Fluorescence Intensity (A.U)
Glu
200
Valine, Methionine, Histidine, Asparate , Tryptophan, Glycine, Lysine, Cysteine, other amino acid
150
100 GOD
50
0 370
420
470
520
Wavelength(nm)
Fig.3.
17
570
Fig.4.
18
700
Fluorescence Intensity(A.U)
600 500 400 300 200 100 0 0
5
10 -6
10 M [Glu]
Fig.5.
19
15
20
Fluorescence Intensity(A.U)
490 420 350 280 210 140 70 0 3
5
7 pH
Fig.6
20
9
2 1.8 1.6 1.4
I/I0
1.2 1
0.8 0.6 0.4 0.2 0 GOD Val
Met
Cys
Trp
Glu
Fig.7.
21
Tyr
His
Ser
Asp Na+
K+
Highlights •
A novel fluorescence chemosensor for glutamate in aqueous buffered solution has been developed.
•
The fluorescence of graphene dot enhanced significantly in the presence of Glutamate.
•
Linear detecting range of fluorescent chemosensor for glutamate was 1.6×10-7M to 1.0×105
M with a detection limit of 5.2×10-8 M .
22
Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot
23
S1386-1425(14)01614-X http://dx.doi.org/10.1016/j.saa.2014.10.117 SAA 12932
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
21 August 2014 15 October 2014 27 October 2014
Please cite this article as: M. Hosseini, H. Khabbaz, A. Dezfoli, M. RezaGanjali, M. Dadmehr, Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.10.117
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot Morteza Hosseini a,∗∗ ,Hossein Khabbaza,Amin Dezfolib,Mohammad RezaGanjalib,c,Mehdi Dadmehra,d a
Department of life science engineering, Faculty of New Sciences & Technologies, University of
Tehran, Tehran, Iran b
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran,
Iran c
Endocrinology& Metabolism Research Center, Tehran University of Medical Sciences, Tehran,
Iran d
Payame Noor University, Tehran, Iran
*
Corresponding authors. Tel.: +98-21-61112788; Fax: +98-21-66495291 Email addresses:[email protected] (M. Hosseini)
1
ABSTRACT Graphene quantum dots (GQDs) have successfully been utilized as an efficient nano-sized fluorescence chemosensor to detect selectively Glutamate (Glu) in Tris-HCl buffer solution (pH=9). The fluorescence emission spectrum of graphene quantum dots was at about 430 nm. The study showed that fluorescence intensity of the quantum dot gradually enhanced with increase in concentration of Glutamate and any change in fluorescence intensity was directly proportional to the concentration of Glutamate. Under optimum conditions, the linear range for the detection of Glutamate was 1.6×10-7M to 1.0×10-5 M with a detection limit of 5.2×10-8 M. The sensor showed high selectivity toward Glutamate in comparison with otheramino acids. Keywords:Graphene; Quantum dot; Glutamate; Fluorescence Enhancement; Amino acid
Introduction
2
To date, graphene which is composed of a single layer of carbon atoms with closely packed honeycomb structure [1], has received more considerable attention due to its individual and novel properties [2] such as large surface area, resistance property, chemical stability, and
improved mechanical flexibility, thermal environmentally friendly nature., These
characteristics introduce it as a promising alternative agent may show broad application in biosensors[3]. Lack of a band gap in pristine graphene results to failure of
its optical
luminescence observation. [4] Therefore, many attempts has been made to convert the 2D graphene sheets into 0D graphene quantum dots (GQDs) [5] In order to facilitate the application of graphene in nanoscale devices and also effectively tuning the band gap of graphenes. Graphene quantum dots (GQDs) are single or few-layer graphenes with a tiny size of only several nanometers [6], they show some remarkable properties, such as chemical stability, excellent water solubility and suitability. Importantly, the presence of carboxylic acid groups at the edge of GQDs make it easy for functionalization with various organic, polymeric, inorganic or biological species [7]. L- Glutamate (anion of glutamic acid) is one of the 20 standard amino acids used by all organisms. It plays an important role in food processing, clinical applications and is also well known as a flavour enhancer, commonly found in various foods. The excessive intake of this flavour enhancer can cause allergic effects such as headache and stomach pain [8,9]. LGlutamate is also an excitatory neurotransmitter in the central nervous system of vertebrates, and is a potent neuroexcitatory amino acid associated with certain behavior patterns such as aggressive behavior visual task learning, morphine-induced muscular rigidity and retrograde amnesia [10-12].
3
Aspartate and glutamate are major excitatory amino acids (EAAs) in the central nervous system. In recent years, some researchers have focused on the correlation between altered levels of EAAs in humans and some pathology, including diabetes and cancer [13]. The analysis of EAAs in biological samples is particularly applicale in biochemistry and clinical chemistry [14,15]. Several methods for determination of a trace amount of glutamate have been reported: amperometric method [16], Chromatography [17] and capillary electrophoresis [18]. However, most of the above mentioned methods do not seem to satisfy the requirements for monitoring of Glu in the terms of facility, sensitivity, matrix toughness and cost effectiveness. Among the various detection techniques, fluorescent chemosensors have been developed quickly due totheir simplicity and high sensitivity [19-23]. Fluorescent chemosensors are of great importance owing to their high sensitivity and low detection limit. Most literatures report use of fluorescence quenching as the readout mechanism for the sensor response. The greatest advantage of fluorescence enhancement sensors, in comparison with fluorescence quenching sensors, is the ease of measuring low concentration contrast relative to a “dark” background. This reduces the likelihood of false positive signals and increases the sensitivity [24].
Consequently, there is an urgent need to develop new turn-on fluorescent sensors which can be advantageous. However, to the best of our knowledge, fluorescent sensors for Glu, especially with fluorescence enhancement technique, have not been reported. Experimental Reagents All chemicals were of the reagent–grade purity from Aldrich and Merck chemical companies. The amino acids tested (L-Alanine(Ala), L-Methionine(Met), L-Serine(Ser), L4
Leucine(Leu), L-Glutamic Acid(Glu), L-Threonine(Thr), L-Valine(Val), L-Arginine(Arg), LProline(Pro),
L-Aspartate(Asp), L-Phenylalanine(Phe), L-Cysteine(Cys), L-Pyroglutamic
acid(Pyr) were purchased from Aldrich Company.
Apparatus All fluorescence measurements were carried out on a Perkin-Elmer LS50 luminescence spectrometer. Synthesis procedure of GQDs Graphene oxide (GO) was synthesized from graphitic powder according to Hummer’s method [25]. In brief, 0.5 g of graphite powder, 0.5 g of NaNO3 and 23 mL of H2SO4 were added into a three-necked flask with stirring in an ice bath. Then, 3 g of KMnO4 was slowly added to the mixture. After mixed together, the flask was transferred to 35±5 oC water bath for 1 h with stirring and then a brown paste was obtained. Next, 40 mL of ultrapure water was added, and the solution was stirred for 30 min. Another 100 mL of ultrapure water was added, and then addition of 3 mL H2O2 (wt 30%) made the color of the solution change to bright yellow. The warm GO solution was filtered and washed with ultrapure water until pH to 7.0. The precipitates were dried for 24 h in a freeze-dryer.
Preparation of GQDs The GQDs were prepared from graphene sheets (GSs) by a hydrothermal approach as the literature procedure [26]. Graphene sheets (GSs) were produced by thermal deoxidization of dried GO at 250oC for 2h in a tube furnace with a heating rate of 5 oC/min. The condition was in a nitrogen atmosphere. 0.05 g of GSs were oxidized in 40 mL concentrated H2SO4 and HNO3 5
(volume ratio 1:3) for 18 h under mild sonication. The solution was diluted with 250 mL of water, and then filtered to remove the acids. Purified oxidized GSs were redispersed in 40 mL of ultrapure water and the pH was adjusted to 8 with NaOH solution. Then, the suspension was transferred into a Teflon-lined autoclave (50mL) and heated at 200 oC for 10 h. After cooling to room temperature, the resulting suspension was filtered and the brownish filtrate was dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 24 h.
Result and discussion Characterization of GQDs During the oxidation, oxygen-containing functional groups, including C=O/COOH, OH, and C-O-C, were introduced at the edge and on the basal plane, as shown in the Fourier transform infrared (FTIR) spectrum (Fig. 1). Due to the presence of these groups, the chemically synthesized GQDs are readily water-dispersible. The peak at about 1363 cm-1 indicate the existence of COO-, while the peak at 3320 cm-1 corresponds to the OH stretching mode. Furthermore, the observed peaks at 1018, 1200, 1720, and about 1660 cm-1, are assigned to C– O–C stretching, C-OH stretching, C=O stretching, and C=C stretching mode of vibration, respectively. Fig.2 shows a transmission electron microscope (TEM) image of GQDs. Their diameters are mainly distributed in the range of 5–20 nm. Fig. 1 Fig. 2
6
Preliminary studies In order to evaluate whether GQDs could be used as a selective fluorescent chemosensor for amino acids, the interaction of GQDs with a number of amino acids was investigated spectrofluorometrically in Tris-HCl buffer solution at 25.0±0.1 ºC. The resulting fluorescence intensities for amino acids are shown in Fig. 3. It can be seen clearly from this Fig. 3, no significant fluorescence changes were observed when Ala, Cys, Trp, Arg, Asp, Ser, Val, Tyr, His, Pro, and Lys were added. In contrast, the addition of Glu, affords a very high-intensity emission centering around 430 nm. Fig. 3 Fluorescence titration Fluorescence properties of GQDs(5.0×10-7M) were examined in Tris-HCl buffer solution at room temperature. It exhibited emission with the emission maxima at 430 nm when they are excited at 365 nm. Considering the appreciable changes in fluorescence properties of the GQDs toward addition of Glu, the potential for developing a novel fluorescence probe for determination of Glu was assessed. As shown in Figs. 4 and 5, upon addition of increasing concentrations of Glu to the GQDs solution, a gradual increase in the fluorescence intensity was clearly detected. Furthermore, as shown in Fig. 4B, the fluorescence emission spectra of GQDs was sensitive to Glu. In the presence of various concentration of Glu ranging from 1.6×10−7 to1.0×10−5mol/L, significant fluorescence enhancement of fluorescent probe was observed. The detection limit was estimated as 5.2×10−8mol/L (calculated as three times standard deviation of blank solution). The binding constant value has been determined from the emission intensity data following the modified Benesi–Hildebrand equation [27]: 7
1/F=1/Fmax+(1/K[C])( 1/Fmax) where F=Fx–F0 and Fmax= F–F0, and where F0, Fx and F are the emission intensities of GQDs considered in the absence of Glu, at an intermediate Glu concentration, and at a concentration of complete interaction, respectively. K is the binding constant for the interaction and [C] is the Glu concentration. From the plot of (F–F0)/(Fx–F0) against [C]-1 for L, the value of K extracted from the slope is 4.2×106 M -1. Fig. 4 Fig. 5 A comparison between this work and other previous reported methods for Glu determination is listed in Table 1. In comparison with these methods, this sensor has a wider linear range than previously methods. The detection limit of the proposed sensors is 5.2 ×10-8 mol L−1 which is lower than electrochemical sensor and electrochemical biosensor. In term of the linear range and detection limit, it can be seen that the proposed sensor displays even more sensitivity than most of the reported methods [16, 18, 28-30]. pH effect In order to investigate the effect pH on the emission GQDs, the change of intensity were measured at a fixed concentration of Glu (5.0×10-5 M) having different pH values (Fig.6). As it is seen (Fig.6) in the presecnce of Glu, response of the fluorescent sensor was dependent on the pH of the test solution and a pH value of 8.4, maximum emission of the GQDs in the presence Glu was obtained. The pH was altered from 3- 10 by adding HNO3 or NaOH. Fig. 6
8
Selectivity The selectivity behavior is obviously one of the most important characteristics of a chemosensor, which is the relative sensor response for the primary amino acid over other amino acids present in solution. To examine the selectivity of one, we investigated its affinity for other amino acids. The influence of various amino acids on the fluorescence behaviour of GQDs was shown in Fig. 7. As can be seen from Fig. 7, except for Cys and Try, other amino acids made no obvious changes in the fluorescence intensity. The existence of oxygen-containing groups ( carboxylic (_COOH) and hydroxyl(–OH) ) at the edges of GQDs was expected to increase both stability and selectivity and provide a binding site for the α-amino group of glutamate
as
reported previously[31,32]. The possible mechanism of the enhancement of GQDs with addition glutamate could arise H-bond interaction from the alcohol or carboxylic group of the GQDs and the amino group of the glutamate[31-33]. Furthermore, competition experiments were also performed for GQDs in the presence of Glu at 50µM mixed with 200µM background amino acids such as Val, Met, Cys, Trp, Glu, Tyr, Ser, Asp, Na+ and K+ (Fig. 7). In the presence of other amino acids, no obvious variation in the fluorescence was observed except of Trp have quenching effect. All these results implied that GQDs could be used as a potential candidate of Gluchemosensor with very high selectivity. Fig. 7
Conclusion In summary, we have demonstrated a convenient and novel type of GQDs based sensor for the detection of Glu with high sensitivity and selectivity. Presence or addition of Glu show enhancement of fluorescence emission intensity of GQDs. The remarkable properties for this
9
method such as simplicity, rapidness and selectivity will help to extend its applications for determination of Gluin samples. Acknowledgements: The authors are grateful to the Research Council of University of Tehran for the financial support of this work.
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Detection Limit
Dynamic Range
(M)
(M)
method
Ref.
Table 1 Comparison of the characteristics of the proposed sensor with those of the previously reported for Glu.
12
electrochemical sensor
9.6×10-6
1.0×10-3-8.0× 10-3
16
electrochemical biosensor
3.2×10-7
5.0×10-6-3.0×10-4
28
HPLC- Fluorescence detector
5.3×10-9
Fluorometric
1. 8×10-6
Amperometric
3.8 ×10-6
Fluorescence sensor
5.2 ×10-8
8.0×10-9-9.6×10-7 5.0×10-9-1.2×10-6
5.0×10-6-7.8×10-5 1.6×10-7-1.0×10-5
29 18 31 This work
Figure Caption: Fig.1.FT-IR spectra of GO and GQDs Fig.2. TEM image of GQDs Fig. 3. Fluorescence responses of GQDsTris-HCl buffersolution upon addition of amino acids (5×10-4M) (λex= 365 nm). 13
Fig. 4.a) Fluorescence titration of GQDs inTris-HCl buffersolution in the presence of varying concentrations of Glu(λex= 365nm). b) Visual fluorescence changes of sensor GQDs in the absence and excess presence of Glu ion (1.0×10-2M). The photo was taken under a handheld UV (365 nm) lamp. Fig.5. Calibration curve of Glu fluorescent chemosensor. Fig.6. Effect of pH of the test solution on the fluorescence response of GQDs in the presence Glu (5.0×10-5M) Fig. 7.Relative fluorescence intensity ofGQD the presence of various ions and containing 30 µM Glu and the background amino acids 200 µM (λex: 365 nm).
14
Fig.1
15
Fig.2.
16
300
250 Fluorescence Intensity (A.U)
Glu
200
Valine, Methionine, Histidine, Asparate , Tryptophan, Glycine, Lysine, Cysteine, other amino acid
150
100 GOD
50
0 370
420
470
520
Wavelength(nm)
Fig.3.
17
570
Fig.4.
18
700
Fluorescence Intensity(A.U)
600 500 400 300 200 100 0 0
5
10 -6
10 M [Glu]
Fig.5.
19
15
20
Fluorescence Intensity(A.U)
490 420 350 280 210 140 70 0 3
5
7 pH
Fig.6
20
9
2 1.8 1.6 1.4
I/I0
1.2 1
0.8 0.6 0.4 0.2 0 GOD Val
Met
Cys
Trp
Glu
Fig.7.
21
Tyr
His
Ser
Asp Na+
K+
Highlights •
A novel fluorescence chemosensor for glutamate in aqueous buffered solution has been developed.
•
The fluorescence of graphene dot enhanced significantly in the presence of Glutamate.
•
Linear detecting range of fluorescent chemosensor for glutamate was 1.6×10-7M to 1.0×105
M with a detection limit of 5.2×10-8 M .
22
Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot
23