Analytica Chimica Acta 887 (2015) 245e252

Contents lists available at ScienceDirect

Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

Graphitic carbon nitride embedded hydrogels for enhanced gel electrophoresis Mohammad Zarei a, Hossein Ahmadzadeh a, Elaheh K. Goharshadi a, b, *, Ali Farzaneh c a

Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, P.O. Box 91779, Iran Center of Nano Research, Ferdowsi University of Mashhad, Mashhad, P.O. Box 91779, Iran c Department of Chemical Engineering, Ferdowsi University of Mashhad, Mashhad, P.O. Box 91779, Iran b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 g-C3N4 nanosheets improved the resolution and efficiency in gel electrophoresis.  g-C3N4 loading into polyacrylamide gel increases the thermal conductivity of gel.  g-C3N4 nanosheets increase the resolution between bands.  g-C3N4 nanosheets act a polymerization catalyst and as heat sink.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 March 2015 Received in revised form 9 July 2015 Accepted 12 July 2015 Available online 10 August 2015

Here, we show, for the first time, the use of graphitic carbon nitride (g-C3N4) nanosheets to improve the resolution and efficiency of protein separation in gel electrophoresis. By loading 0.04% (m/v) g-C3N4 nanosheets into the polyacrylamide gel at 25  C, the thermal conductivity increased approximately 80% which resulted in 20% reduction in Joule heating and overall increase of separation efficiency. Also, polymerization of acrylamide occurred in the absence of tetramethylethylenediamine (TEMED) when the polyacrylamide gel contained g-C3N4 nanosheets. Hence, the g-C3N4 act simultaneously as a polymerization catalyst as well as heat sinks to lower Joule heating effect on band broadening. © 2015 Elsevier B.V. All rights reserved.

Keywords: Gel electrophoresis Joule heating Band broadening Graphitic carbon nitride

1. Introduction The potential application of nanomaterials in various fields of science has been recognized and significant advances have been achieved [1e4]. The properties of nanomaterials including thermal,

* Corresponding author. Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, P.O. Box 91779, Iran. E-mail address: [email protected] (E.K. Goharshadi). http://dx.doi.org/10.1016/j.aca.2015.07.022 0003-2670/© 2015 Elsevier B.V. All rights reserved.

electrical, chemical, and mechanical are influenced by their size, shape, and composition [5]. High surface-to-volume ratio of nanoparticles (NPs) has led to their usage in applications such as detection and separation of biomolecules [4,6e9]. Various types of nanostructures such as carbon nanotubes [10], fullerenes [11], silica [12], latex [13], magnetic [14] and non-magnetic metal oxides [15], metal oxide semiconductor [15], silver [16], gold [17,18], ceria [4], and polymer-based NPs have been used successfully for the separation purposes and also for coating in electrophoresis [19,20]. Use of nanomaterials in separation science has been recognized in

246

M. Zarei et al. / Analytica Chimica Acta 887 (2015) 245e252

electrophoresis [4] and capillary electrophoresis [21], capillary electrochromatography [22,23], microchip electrophoresis [24], and chromatography separations [25,26]. Despite the widespread applications of nanomaterials in analytical chemistry, very little research has been devoted to their applications in gel electrophoresis. With respect to the post-genome era and the importance of recombinant DNA technology, there has been revival in the use of gel electrophoresis to identify and characterize biological compounds [27]. Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful techniques for separation of biological samples [28]. However, the efficiency and reproducibility of the separation are limited by some parameters such as band broadening due to the diffusion, Joule heating, adsorption, and other effects [29]. Temperature gradient in separation medium has a major influence on band broadening. For example, the electrophoretic mobility has a strong dependence on temperature, thus the parabolic profile of temperature leads to the parabolic velocity profile. The dispersion caused by a parabolic velocity profile due to flow is given by Ref. [30]:

s2D ¼

R2 n2avg t

(1)

24Dy

R6i E6 c2e U2T m2 1536Dy k2b

t

(2)

where E and ce stand for the electric field strength and the electrical conductivity of the medium, respectively. The kb is the thermal conductivity of the medium, UT is the temperature coefficient of electrophoretic mobility, and m is the solute electrophoretic mobility. The magnitude of s2DT is inversely proportional to square of kb. Therefore, any enhancement in thermal conductivity of medium leads to a dramatic reduction of the total peak variance generated by temperature gradient. When voltage, U is applied on a separation medium, the heat produced, q can be calculated by Eq. (3):

q ¼ UI t

(3)

where I and t stand for electric current and time, respectively. The temperature difference between the center line and the inside line of separation medium can be calculated by Knox equation [32]:

DT ¼

Qr 2 4kb

(4)

where r is radius of separation medium and Q is the heat produced per unit volume and time:



UI whl

2. Experimental 2.1. Chemicals

where navg is the average linear velocity of the analyte across the medium, R is the internal radius of the medium, s2D is the peak variance due to dispersion, t is time, and Dy is the analyte diffusion coefficient. The expression relating the temperature induced electrophoretic velocity profile to band variance [31]; the peak variance due to the temperature gradient, s2DT is given by:

s2DT ¼

separation efficiency. One approach is using lower voltages at the expense of longer separation times. Within the past decade, smart gels such as nanocomposite (NC) gels have been synthesized and used for a wide range of applications [33]. NCs have attracted much attention and are believed to be a revolutionary type of hydrogel [34]. They may improve the gel thermal and mechanical strength [35], interact with the analyte and force the transport of analytes through inter-particle channels [36] and change the gel cross-link density [37]. The NCs gels represent new opportunities for improving the balance between the thermal and mechanical properties [38,39]. Herein, we present a detailed study of graphitic carbon nitride nanosheets (g-C3N4) inclusion to the polyacrylamide (PA) gels. We chose g-C3N4 nanosheets because of their low electrical conductivity (band gap is in the range 3e4 eV) [40,41] as well as high thermal conductivity [42]. The aims of this study are to investigate the influence of g-C3N4 nanosheets in improvement of analytical figures of merit in gel electrophoresis and to study use of g-C3N4 nanosheets as catalyst for acrylamide polymerization in order to eliminate the use of TEMED.

(5)

where w, h, and l are dimensions of gel. As Eq. (4) shows there is an inverse relationship between temperature difference and thermal conductivity of medium. It implies thermal conductivity plays a vital role for improvement of separation. Reduction in Joule heating in slab gel electrophoresis is important for improving the

All reagents were of analytical grade. Tris-hydroxymethyla minomethane (Tris), glycine, silver nitrate, N,N-methylenebisa crylamide (Bis), acrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), sodium carbonate, mercaptoethanol, formaldehyde, TEMED, and sodium thiosulfate were purchased from Merck (Germany). Melamine (99%) was prepared from Khorasan Petrochemical Complex (KPC, Iran) and used without further purification. E. coli protein samples were prepared from Biotechnology Research Center of Ferdowsi University of Mashhad. 2.2. SDS-PAGE of protein mixtures One-dimensional gel electrophoresis of proteins was carried out in a 12% (m/v) vertical PA gel containing sodium dodecyl sulfate (SDS-PAGE) according to the Laemmli protocol [43]. The 30% (m/v) acrylamide/bis-acrylamide stock solution was prepared to produce the specific percentage of PA gel (12%). Acrylamide/bis-acrylamide stock solution 30% m/v (29:1) is based upon the total concentration (T) and cross-linker concentration (C) which are 30% T and 3.3% C, respectively. All samples were prepared in 5 concentrated Laemmli reducing buffer (1.0 mL TriseHCl (0.125 M, pH 6.8), 1 mL glycerol (20%), 1.5 mL SDS (10%), 0.4 mL mercaptoethanol (2%), and 0.2 mL bromphenolblue (0.05%)) and boiled for 4 min before using. The protein concentration of the samples was adjusted so that about 7 mL of protein (0.04e0.09 mg/mL) was loaded per lane. Gels were stained by Coomassie Brilliant Blue and destained according to the standard protocol [44]. Gel images were analyzed using ImageJ [45] and GelAnalyzer [46] softwares. The SDS-PAGE was performed without cooling (thermostatization). 2.3. Synthesis of g-C3N4 nanosheets The g-C3N4 nanosheets were synthesized by a two-step method in a tube furnace. In a typical procedure, a given amount (5.00 g) of melamine in a closed crucible was heated to 380  C and 600  C sequentially for 1 h and 2 h, respectively. 2.4. Characterization methods The X-ray diffraction (XRD) analysis was carried out on Unisantis

M. Zarei et al. / Analytica Chimica Acta 887 (2015) 245e252

XMD 300 diffractometer using Cu Ka (l ¼ 0.15406 nm) radiation. The transmission electron microscopy (TEM) analysis of the samples was performed on a LEO 912 AB transmission electron microscope. The electron beam accelerating voltage was 120 kV. In order to obtain a TEM image, the solid sample was dispersed in water using a sonicating bath. 2.5. Synthesis of PA/g-C3N4 (NCs) hydrogel The PA/g-C3N4 hydrogel was synthesized by means of ex situ NCs method [47] with and without TEMED. Briefly, a proper amount (0.02e0.05%) of g-C3N4 was added to 2.00 mL deionized water and sonicated for 20 min at 30  C using an ultrasound bath. The 30% (m/ v), PA gel stock solution was prepared by dissolving 29.20 g acrylamide and 0.80 g Bis in 100 mL H2O. The 12% (m/v) gel solution was prepared by mixing 5.0 mL gel stock solution, 2.5 mL TriseHCl (1.5 mol L1, pH 8.80), 100 mL (NH4)2S2O8 (10%, m/v), 10 mL TEMED, 100 mL SDS (10%, m/v), and 0.005 g of g-C3N4 (0.05% m/v). The sample buffer (5) was prepared by mixing 1.0 mL TriseHCl (0.125 M, pH 6.8), 1 mL glycerol (20%), 1.5 mL SDS (10%), 0.4 mL mercaptoethanol (2%), and 0.2 mL bromphenolblue (0.05%). Running buffer (5) was prepared by mixing 5.00 g SDS, 94.00 g Tris, and 15.10 g in 1.00 L deionized water. The gel solution used in this work was freshly prepared within 1 day of running the experiment. The gel solution containing g-C3N4 was quite stable till the polymerization at room temperature indicating that the nanosheets were well dispersed in the gel medium. The transparency and optical properties of PA and PA/g-C3N4 gels are very important for detection purposes. As Figs. 1a and 1b show the texts under the gels are quite legible. 3. Results and discussion 3.1. Characterization Fig. 2a shows the XRD pattern of, g-C3N4 nanosheets. The characteristic diffraction peak of g-C3N4 is at 2q ¼ 27.4 (d-spacing of 0.325 nm and crystallite size of 4.2 nm) corresponding to (002) crystallographic plane. As TEM image in Fig. 2b shows, g-C3N4 nanosheets are compacted together and produced non-wrinkled flat sheets. These prepared g-C3N4 nanosheets are used to reduce heat generation induced by Joule heating. 3.2. Joule heating To study the Joule heating effect, Ohm's plot was constructed by

Fig. 1. Prepared (a) PA/g-C3N4, (b) PA gels at room temperature.

247

applying different voltages across the separation medium and monitoring the generated current. Fig. 3a shows IeV measurement (Ohm's plot) for the PA gel and the PA/g-C3N4 gel. A threshold voltage (Vt) above which the current begins to abruptly increase is the maximum separation voltage that should be applied in order to having the shortest separation time. The values of Vt in the pure PA gel and the PA/g-C3N4 gel were measured to be 120 and 130 V, respectively. Fig. 3b displays the Joule heating measurements for the PA gel and the PA/g-C3N4 gel. The Joule heating disrupts the separation process at the threshold of nonlinearity in the IeV values and this happens when the rate of heat generation surpasses the rate of heat dissipation. As Fig. 3b shows, at high voltages, Joule heating for the PA/gC3N4 gel is less than that of the PA gel. Using Eq. (3) could be useful to calculate the Joule heating. The g-C3N4 nanosheets act as heat sinks and increase the rate of heat dissipation. By incorporating the g-C3N4 nanosheets in the PA solution, the amount of Joule heating decreases. By loading 0.04% (m/v) of the gC3N4 nanosheets at applied voltage of 130 V, the value of Joule heating decreased 15% compared to that of PA solution. At 150 V, the reduction in Joule heating is 20%. The temperature difference between the center and the edges of gels was calculated by Knox equation (Equation (4)) and is shown in Fig. 3c. As it can be seen, the temperature gradient in PA/g-C3N4 gel is smaller than that of PA gel due to higher thermal conductivity of PA/g-C3N4. The temperature difference in PA/g-C3N4 gel at 160 V is 1.1  C while for PA gel is 2.4  C. Fig. 3d shows the measured temperature gradient in three different positions of the gel (2, 5, and 8 cm from cathode (0) to anode (10 cm)). The temperature probe (Misonix s-400) was used to measure the temperature in different positions of the gel (from cathode to anode). The measurement was performed at constant voltage (160 V) from 1 to 60 min. By increasing the separation time, temperature of the gel increased gradually from cathode to anode. The positions close to the anode (x ¼ 8 cm) show the highest increase in temperature compared to those of closer to the cathode (x ¼ 2 cm). Fig. 3d shows that overall temperature of PA/g-C3N4 gel is smaller than PA gel which confirms the role of PA/g-C3N4 nanosheets as heat sinks. There are no theoretical formulas to predict the thermal conductivity of the suspensions of NPs, so-called nanofluid, satisfactorily [48]. The Maxwell model [49], a traditional model for thermal conductivity, was proposed for solideliquid mixtures and also for NCs with polymeric matrix [4]:

  lf þ 2lm þ 2f lf  lm   lc ¼ lf þ 2lm  f lf  lm

(6)

where lf and lm stand for the thermal conductivity of composite (PA/g-C3N4 suspension), filler material (g-C3N4 nanosheets), and polymer matrix (PA), respectively and F is volume fraction of the nanosheets. Using equilibrium molecular dynamics simulations, the thermal conductivity of g-C3N4 nanosheets was predicted to be around 3.5 W m1 K1 [50]. The thermal conductivity of PA is 0.56 W m1 K1 [51]. The thermal conductivity of PA/g-C3N4 nanosheets suspensions was calculated according to the Maxwell model as 1.0005, 1.0007, and 1.0105 for 0.01, 0.02, and 0.04% m/v of g-C3N4 nanosheets, respectively. This is equivalent to 78, 79, and 80% enhancement in thermal conductivity of PA/g-C3N4 gel compared with that of PA gel without g-C3N4 nanosheets. Hence, the g-C3N4 nanosheets act as heat sink centers and lower the value of Joule heating. It should be mentioned that Maxwell's formula underestimates the thermal conductivity of PA/g-C3N4 suspension

248

M. Zarei et al. / Analytica Chimica Acta 887 (2015) 245e252

Fig. 2. (a) XRD pattern, (b) TEM image of prepared g-C3N4 nanosheets.

Fig. 3. (a) Current vs voltage curve for the PA gel ( ) and PA/g-C3N4 gels ( ) (b) The magnitudes of Joule heating versus applied voltage for PA gel and PA/g-C3N4 gels, (c) Temperature difference between the center and the edges of gels. The symbols are the same as (a), (d) Temperature gradient in three different positions of the gel from cathode to anode at constant voltage (PA gel: x ¼ 2 cm ( ), x ¼ 5 cm ( ), x ¼ 8 cm ( ) and PA/g-C3N4 gel: x ¼ 2 cm ( ), x ¼ 5 cm ( ), x ¼ 8 cm ( )).

[52], i.e. the thermal conductivity of PA/g-C3N4 suspension should be much higher than the values predicted by this model. 3.3. Separation of protein mixtures The separation profiles and electropherograms of E. coli proteins in pure PA and in PA/g-C3N4 gels are shown in Fig. 4a and b, respectively. The separation was performed at 140 V on pure PA and modified PA gels. The efficiency of the electrophoresis can be evaluated by the number of theoretical plates, N [29]:

N ¼ 5:54

t w1=2

!2 (7)

where t and w1/2 stand for migration time of the proteins and the temporal peak widths at half of the peak heights. The number of theoretical plates for the peaks A to K for both PA and PA/g-C3N4 (m/v ¼ 0.04%) gels are given in Table 1. As this table shows the values of N increases in PA/g-C3N4 gel compared to those of PA gel. The number of theoretical plates (N) is related to the applied voltage, U, by the following equation [53]:



zFU 2qRT

(8)

where z is effective charge, F, the Faraday constant or charge per mole of protons, T is temperature, R is gas constant, and q is dispersion coefficient. In electrophoresis, efficiency (N) increases by increasing voltage U as far as Joule heating allows. By incorporation

M. Zarei et al. / Analytica Chimica Acta 887 (2015) 245e252

249

Fig. 4. The separation electropherograms of E. coli proteins sample versus migration time and distance on the (a) PA gel (b) PA/g-C3N4 gel.

Table 1 Calculated theoretical plate number of E. coli proteins. Enhancement %

14 05 10 160 70 10 30 16 182 11 167

N 0.04% (m/v)

0.00% (m/v)

180.44 282.54 647.43 1794.06 3402.22 6099.38 2385.31 5093.70 7999.32 5251.74 4602.51

157.57 268.74 719.97 691.83 1999.01 5573.12 1840.28 4387.36 2830.65 4739.15 1720.82

Table 3 Measured full width at half-maximum. Peak

Reduction %

W1/2 0.04% (m/v)

0.00% (m/v)

A B C D E F G H I J K

8 6 6 38 25 5 11 6 39 7 37

2.86 3.52 3.72 2.51 2.32 2.21 4.02 3.12 3.00 3.80 5.12

3.11 3.32 3.51 4.03 3.12 2.32 4.50 3.31 4.92 4.09 8.16

of g-C3N4 nanosheets into the PA gel, the Joule heating threshold voltage expands in order to maximize the separation efficiency and lowers the separation time. For example, the number of theoretical plates for baseline resolved peaks such as E, F, and I improves by 70, 80, and 182%, respectively. Reduction of Joule heating in PA/g-C3N4 gel causes the W1/2 to be lower for almost all baseline resolved peaks. This is tabulated in Table 3 and also in visualized by increase in signal intensity when comparing Fig. 4a and b. This improvement in separation parameters when loading 0.04% (m/v) g-C3N4 nanosheets into gel is better visualized by the inset electropherograms in top panels of Fig. 4a and b. The resolution, Rs is an important criterion of success for the analytical separation of two specific components. The resolution between two peaks in electropherogram can be calculated as [29]:

Rs ¼

2ðt2  t1 Þ w1 þ w2

(9)

where t1 and t2 are the migration times for components 1 and 2, respectively. w1 and w2 stand for the temporal peak width of

Table 2 Measured resolution between separated bands of E. coli protein sample. Enhancement %

10 41 24 33 31

Peak

Rs 0.04% (m/v)

0.00% (m/v)

1.28 1.05 3.61 2.01 0.88

1.16 0.74 2.90 1.50 0.67

AB CD EF FG IJ

Peak

A B C D E F G H I J K

components 1 and 2, respectively. The appropriate resolution between components requires a combination of good selectivity and good separation efficiency (Rs ~ N1/2 ~ U1/2) [54,55]. Resolution between peaks was also measured in PA/g-C3N4 and PA gels. In general, the resolutions increased in PA/g-C3N4 gel compared to those of the PA gel (Fig. 4). For example, the resolution between peaks I and J for PA and PA/g-C3N4 gels improves from 1.50 to 2.01, respectively. Close examination of the unidentified bands between peaks J and K, shows one broad peak for PA gel (Fig. 4a) whereas this broad peak splits into four partially resolved peaks in PA/gC3N4 gel. This shows the enhancement in resolution where the gC3N4 is embedded in PA gel. In PA/g-C3N4 gel, resolution between AB, CD, EF, and FG increased 10, 41, 24, and 33%, respectively (Table 2). Reduction in peak widths and also staining process in the presence of g-C3N4 cause increase in contrast and increase in signal intensity from 200 for PA gel to 250 for PA/g-C3N4 gel as it is shown in Fig. 4a and b. Table 3 shows the W1/2 for both PA gel and PA/gC3N4 gel. W1/2 is narrower for almost all peaks in PA/g-C3N4 gel. To better compare the results, we might also focus on the resolution of unidentified peaks before peak A and before peak K in which is shown the improvement in resolution for PA/g-C3N4 gel. W1/2 was used to calculate N and Rs to lower the possible error in defining baseline. We also used Originpro8 software to determine the baselines of electropherograms manually and automatically. The Gaussian function (constant mode) was used to fit the peaks. The peaks observed in the PA/g-C3N4 gel do actually show higher intensity than those observed on pure PA gel. This phenomenon was observed for modified capillaries with gold NPs but no interpretation was reported [56]. We believe that this enhancement in peak height is related to the Joule heating reduction in separation medium because of high thermal conductivity of g-C3N4 nanosheets.

250

M. Zarei et al. / Analytica Chimica Acta 887 (2015) 245e252

Hence, incorporation of g-C3N4 nanosheets into PA gel distributed uniformly in gel matrix causes: (1) increasing the Joule heating threshold voltage and this leads to lowering migration time. (2) the uniform dissipation of heat in the gel. Thus, it lowers the uneven distribution of migration and causes the sharpness of peaks. Table 3 shows the measured full width at half maximum of peaks. As it can be seen, the W1/2 values for separated proteins in PA/g-C3N4 gel are smaller than those of PA gel because of higher thermal conductivity of gel.

3.4. Influence of g-C3N4 concentration In order to study the relationship between the g-C3N4 nanosheets loading and the degree of improvement in separation parameters, different concentrations of g-C3N4 nanosheets (0.01, 0.03, and 0.04% (m/v)) were incorporated into PA gels. When the concentration of g-C3N4 nanosheets was set at 0.01% (m/v), no obvious improvement in efficiency was observed. The optimum separation efficiency was obtained for 0.04% (m/v) of g-C3N4 nanosheets. However, when the concentration of the g-C3N4 nanosheets was higher than 0.04% (m/v), no significant improvement in separation parameters was achieved. The excess g-C3N4 nanosheets increased the background of stained gels and lowered the S/N ratio. 3.5. Probing nanosheeteprotein interaction Evaluation of protein-nanosheet interaction may be insightful for promoting the understanding of the nanosheets mechanism in the gel electrophoresis. If there is an interaction between standard proteins and g-C3N4 nanosheets, the molecular masses of the standard proteins will change and the change in electrophoretic mobility should be observed. Fig. 5a shows the separation profiles of standard proteins (lane A) and standard protein-nanosheets mixture with two different concentrations (lane B and C). The gel was imaged just before and after applying electric field. Comparison of panels A, B, and C in Fig. 5a shows there is no obvious interaction of g-C3N4 nanosheets with standard proteins. By applying electric field, proteins moved without moving g-C3N4 nanosheets because of its poor electrical conductivity. Fig. 5a shows, there is no shift in separation bands when g-C3N4 nanosheets mixed with proteins. There is no difference between the patterns of standard proteins (lane A) and protein-nanosheets (lane B and C). The effect of increase in g-C3N4 nanosheets concentration is illustrated in lanes A, B, and C (Fig. 5b). It can be seen that the intensity of the bands remains unchanged with increasing concentration of g-C3N4 nanosheets. The reason may be related to hydrophobicity of g-C3N4 nanosheets and hydrophilicity of protein, i.e. they are not compatible. In Fig. 5b, the standard protein bands have unaltered migration distance. Zeta potential, z, has been also used to study cell biological activities, agglutination, and adhesion which are related to cell surface charges [57e59]. The direction and velocity, ne, of the particle depends on the applied field (E):

ne ¼ m e E

(10)

where me is the electrophoretic mobility of protein. By directly measuring the electrophoretic mobility of a colloid, the zeta potential may be calculated using the Henry equation [60]:

Fig. 5. Gel electrophoretic analysis of standard protein-g-C3N4 nanosheets interaction (a) the gel electrophoresis pattern of standard proteins in the presence of different concentrations of g-C3N4 nanosheets. Lanes are as follows: lane A ( ), standard proteins (0.5 mg/mL); lane B ( ), standard proteins (0.5 mg/mL) þ g-C3N4 nanosheets (0.6 ppm); lane C ( ), standard proteins (0.5 mg/mL) þ g-C3N4 nanosheets (0.9 ppm) (b) relative mobility for different molecular weights, the symbols are the same as (a), (c) zeta potential measurements for different molecular weights.

me ¼

2εxf ðkrÞ 3h

(11)

where ε is the relative permittivity, f(ka) is Henry's function, and h is the viscosity. Henry's function has value of either 1.5 or 1.0. The changes in Zeta potential values are related to protein surface charge, nanosheet surface charge, and the interaction between protein and nanosheets. Fig. 5a and b shows that the relative mobility, electrophoretic velocity, and zeta potential of proteins did not change by inclusion

M. Zarei et al. / Analytica Chimica Acta 887 (2015) 245e252

251

of nanosheets. Also, protein and nanosheet did not produce stable protein-nanosheet corona. The standard proteins show no aggregative effect in the presence of g-C3N4 nanosheets, i.e. the standard proteins remain practically unaffected. This suggests that the gC3N4 nanosheets have no stable interactions with proteins. Therefore, nanosheets did not move by movement of proteins during electrophoresis because of their low electrical conductivity. 3.6. Influence of catalytic behavior of g-C3N4 nanosheets Elimination of TEMED could be beneficial for synthesis of hydrogel which needs to be biocompatible. TEMED has a disruptive effect on proteins because of sulfhydryl groups [61]. Also, TEMED influences the buffer pH [62]. The excess amount of TEMED has a negative impact on the pattern of bands [63]. The disadvantages can be partially removed when g-C3N4 nanosheets are used as a polymerization catalyst. The polymerization of acrylamide in the absence of TEMED and presence of g-C3N4 nanosheets was investigated. Polymerization of acrylamide occurred even in the absence of TEMED when the PA contains g-C3N4 nanosheets. There was no polymerization for PA solution in the absence of TEMED when g-C3N4 nanosheets were not included. The influence of g-C3N4 nanosheets might be explained by the proposed polymerization mechanism: At first, persulfate ions are adsorbed on the surface of g-C3N4, and then persulfate-adsorbed g-C3N4 nanosheets act as the initiators for radical polymerization of monomers. Some reports suggested the role of persulfate adsorbed-carbon based materials on the polymerization of polymer monomers [64e66]. In adsorption process, the g-C3N4 nanosheets act as seeds to induce the grafting polymerization. The g-C3N4 nanosheets have porous structure which allow selective adsorption [67]. Free radical initiators can be absorbed by g-C3N4 nanosheets which might act as a supporter surface or macro initiator to perform free polymerization. Surface adsorption of initiator species, initiation, and growth of the polymerization from surface of g-C3N4 nanosheets are two important steps in polymerization process of acrylamide monomers. The K2S2O8/H2O2eFeSO4 initiating system have been reported to initiate the polymerization of polymers onto the carbon nanotube (CNT) surface [64]. When the g-C3N4 with absorbed initiators were dispersed into the suspension containing acrylamide monomers, the monomer and reductive initiator moved from the aqueous phase to the gC3N4 nanosheet surface. It leads to initiating the polymerization, growth of the polymer's molecular mass, and deposition of polymer onto the nanosheets surface. Some of the polymer chains graft onto or permeate into the sheets and tangle with the nanosheets. As a result, the nanosheets are plated with a single layer of polymers (Fig. 6). The oxidative initiators are immobilized by the porous nanosheets. Only a few oxidative initiators diffuse into the aqueous phase and initiate the free radical polymerization to form PA homopolymer [64]. It is clear that persulfate adsorption is the first step in the polymerization process followed with catalysis by TEMED. Reaction of TEMED with persulfate ions produces free radicals which then react with monomers in order to initiate polymerization. 4. Conclusions Graphitic carbon nitride embedded PAGE improved the analytical figures of merit by reducing Joule heating and lowering band broadening. Number of theoretical plate, resolution, and detection of separated proteins improved by embedding graphitic carbon nitride into the gel. As far we know, this is the first report for using g-C3N4

Fig. 6. Grafting polymerization on the surface of g-C3N4 nanosheet ( free radical, polymer, and monomer).

nanosheets to improve the separation efficiency by taking advantages of high throughput capability of slab gel electrophoresis and the high heat dissipation efficiency, resolution, and number of theoretical plates in capillary gel electrophoresis. By loading 0.04% (m/v) g-C3N4 nanosheets in PA solution at 25  C, the thermal conductivity increased approximately 80%, which resulted in 20% reduction in Joule heating and increase in separation efficiency. Our results showed polymerization of acrylamide occurs even in the absence of TEMED when the PA contains g-C3N4 nanosheets. On the other hand, the polymerization in the absence of TEMED did not occur when g-C3N4 nanosheets were not included. This paper presents a future potential to improve the separation efficiency and resolution of separation for other electrophoretic methods which suffer from Joule heating. Acknowledgments The authors acknowledge Ferdowsi University of Mashhad for supporting this project (3/23032). HA thanks ATF Committee (100671) for financial support. Authors thank to the FUM college of agriculture and Dr. M. R. Nasiri. References [1] P.S. Weiss, Nanoscience and nanotechnology: present and future, ACS Nano 4 (2010) 1771e1772. [2] M. Goldsmith, L. Abramovitz, D. Peer, Precision nanomedicine in neurodegenerative diseases, ACS Nano 8 (2014) 1958e1965. [3] X. Chen, S.S. Mao, Titanium dioxide nanomaterials: synthesis, properties, modifications, and applications, Chem. Rev. 107 (2007) 2891e2959. [4] M. Zarei, H. Ahmadzadeh, E.K. Goharshadi, Embedded ceria nanoparticles in gel improve electrophoretic separation: a preliminary demonstration, Analyst 140 (2015) 4434e4444. [5] K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, The optical properties of metal nanoparticles: the influence of size, shape, and dielectric environment, J. Phys. Chem. B 107 (2003) 668e677. [6] C. Nilsson, S. Nilsson, Nanoparticle-based pseudostationary phases in capillary electrochromatography, Electrophoresis 27 (2006) 76e83. [7] E. Guihen, Nanoparticles in modern separation science, Trac-trend. Anal. Chem. 46 (2013) 1e14. [8] F. Li, R.J. Hill, Nanoparticle gel electrophoresis: bare charged spheres in polyelectrolyte hydrogels, J. Colloid. Interface. Sci. 394 (2013) 1e12. [9] J. Gao, N. Latep, Y. Ge, J. Tian, J. Wu, W. Qin, Polyamidoamine-grafted silica nanoparticles as pseudostationary phases for capillary electrochromatographic separation of proteins, J. Sep. Sci. 36 (2013) 1575e1581. [10] H. Luo, Z. Shi, N. Li, Z. Gu, Q. Zhuang, Investigation of the electrochemical and electrocatalytic behavior of single-wall carbon nanotube film on a glassy carbon electrode, Anal. Chem. 73 (2001) 915e920. [11] M. Gallego, Y. Petit de Pena, M. Valcarcel, Fullerenes as sorbent materials for metal preconcentration, Anal. Chem. 66 (1994) 4074e4078. [12] C.L. Arthur, J. Pawliszyn, Solid phase microextraction with thermal desorption using fused silica optical fibers, Anal. Chem. 62 (1990) 2145e2148.

252

M. Zarei et al. / Analytica Chimica Acta 887 (2015) 245e252

[13] P. Zakaria, J.P. Hutchinson, N. Avdalovic, Y. Liu, P.R. Haddad, Latex-coated polymeric monolithic ion-exchange stationary phases. 2. Micro-ion chromatography, Anal. Chem. 77 (2005) 417e423. [14] K. Aguilar-Arteaga, J. Rodriguez, E. Barrado, Magnetic solids in analytical chemistry: a review, Anal. Chimi. Acta 674 (2010) 157e165. [15] A. Kolmakov, M. Moskovits, Chemical sensing and catalysis by onedimensional metal-oxide nanostructures, Annu. Rev. Mater. Res. 34 (2004) 151e180. [16] A.D. McFarland, R.P. Van Duyne, Single silver nanoparticles as real-time optical sensors with zeptomole sensitivity, Nano. Lett. 3 (2003) 1057e1062. [17] C.S. Wu, F.K. Liu, F.H. Ko, Potential role of gold nanoparticles for improved analytical methods: an introduction to characterizations and applications, Anal. Bioanal. Chem. 399 (2011) 103e118. , Z. Zmatlíkov kov l, D. Sýkora, [18] I. Miksík, K. Lacinova a, P. Sedla a, V. Kra  P. Rezanka, V. Kasi cka, Open-tubular capillary electrochromatography with bare gold nanoparticles-based stationary phase applied to separation of trypsin digested native and glycated proteins, J. Sep. Sci. 35 (2012) 994e1002. [19] C. Gelfi, M. Curcio, P.G. Righetti, R. Sebastiano, A. Citterio, H. Ahmadzadeh, N.J. Dovichi, Surface modification based on Si-O and Si-C sublayers and a series of N-substituted acrylamide top-layers for capillary electrophoresis, Electrophoresis 19 (1998) 1677e1682. [20] Y. Wang, J. Ouyang, W.R. Baeyens, J.R. Delanghe, Use of nanomaterials in capillary and microchip electrophoresis, Expert. Rev. Proteomics 4 (2007) 287e298. [21] N. Na, Y. Hu, J. Ouyang, W.R. Baeyens, J.R. Delanghe, Y.E. Taes, M. Xie, H. Chen, Y. Yang, On the use of dispersed nanoparticles modified with single layer bcyclodextrin as chiral selecor to enhance enantioseparation of clenbuterol with capillary electrophoresis, Talanta 69 (2006) 866e872. [22] L. Yang, E. Guihen, J.D. Holmes, M. Loughran, G.P. O'Sulliva, J.D. Glennon, Gold nanoparticle-modified etched capillaries for open-tubular capillary electrochromatography, Anal. Chem. 77 (2005) 1840e1846.  ruba, P. Mate jka, V. Kr [23] D. Sýkora, V. Kasi cka, I. Miksík, P. Rezanka, K. Za al, Application of gold nanoparticles in separation sciences, J. Sep. Sci. 33 (2010) 372e387. [24] A.J. Wang, J.J. Xu, Q. Zhang, H.Y. Chen, The use of poly (dimethylsiloxane) surface modification with gold nanoparticles for the microchip electrophoresis, Talanta 69 (2006) 210e215. [25] G. Zhao, S. Song, C. Wang, Q. Wu, Z. Wang, Determination of triazine herbicides in environmental water samples by high-performance liquid chromatography using graphene-coated magnetic nanoparticles as adsorbent, Anal. Chimi. Acta 708.1 (2011) 155e159. [26] N. Li, J. Guo, B. Liu, Y. Yu, H. Cui, L. Mao, Y. Lin, Determination of monoamine neurotransmitters and their metabolites in a mouse brain microdialysate by coupling high-performance liquid chromatography with gold nanoparticleinitiated chemiluminescence, Anal. Chimi. Acta 645 (1) (2009) 48e55. [27] B.D. Hames, Gel Electrophoresis of Proteins: A Practical Approach: A Practical Approach, Oxford University Press, 1998. [28] C.R. Martin, D.T. Mitchell, Peer reviewed: nanomaterials in analytical chemistry, Anal. Chem. 70 (1998) 322Ae327A. [29] R. Kuhn, S. Hoffstetter-Kuhn, Capillary Electrophoresis: Principles and Practice, SpringereVerlag, Berlin, 1993, p. 37 (chapter 3). [30] G. Taylor, Dispersion of soluble matter in solvent flowing slowly through a tube, Proc. R. Soc. Lond. Ser. A. Math. Phy. 219 (1953) 186e203. [31] P.D. Grossman, J.C. Colburn, Capillary Electrophoresis: Theory and Practice, Academic Press, 1992, p. 31 (chapter 1). [32] J. Knox, I. Grant, Miniaturisation in pressure and electroendosmotically driven liquid chromatography: some theoretical considerations, Chromatographia 24 (1987) 135e143. [33] I. Galaev, B. Mattiasson, Smart Polymers: Applications in Biotechnology and Biomedicine, CRC Press, 2012. [34] K. Haraguchi, Nanocomposite hydrogels, Curr. Opin. Solid. St. M. 11 (2007) 47e54. [35] T. Agag, T. Koga, T. Takeichi, Studies on thermal and mechanical properties of polyimideeclay nanocomposites, Polymer 42 (2001) 3399e3408. [36] J.J. Simhadri, H.A. Stretz, M. Oyanader, P.E. Arce, Role of nanocomposite hydrogel morphology in the electrophoretic separation of biomolecules: a review, Ind. Eng. Chem. Res. 49 (2010) 11866e11877. [37] T.R. Hoare, D.S. Kohane, Hydrogels in drug delivery: progress and challenges, Polymer 49 (2008) 1993e2007. [38] K. Haraguchi, T. Takehisa, S. Fan, Effects of clay content on the properties of nanocomposite hydrogels composed of poly (N-isopropylacrylamide) and clay, Macromolecules 35 (2002) 10162e10171.

[39] K. Haraguchi, R. Farnworth, A. Ohbayashi, T. Takehisa, Compositional effects on mechanical properties of nanocomposite hydrogels composed of poly (N, N-dimethylacrylamide) and clay, Macromolecules 36 (2003) 5732e5741. [40] D.M. Teter, R.J. Hemley, Low-compressibility carbon nitrides, Science 271 (1996) 53e55. [41] S. Muhl, J.M. Mendez, A review of the preparation of carbon nitride films, Diam. Relat. Mater. 8 (1999) 1809e1830. [42] F. Zhao, H. Cheng, Y. Hu, L. Song, Z. Zhang, L. Jiang, L. Qu, Functionalized graphitic carbon nitride for metal-free, flexible and rewritable nonvolatile memory device via direct laser-writing, Sci. Rep. 4 (2014). [43] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680e685. [44] R.J. Simpson, Rapid Coomassie Blue Staining of Protein Gels, Cold Spring Harbor Protocols, 2010. [45] M.D. Abr amoff, P.J. Magalh~ aes, S.J. Ram, Image processing with Image, J. Biophot. Int. 11 (2004) 36e43. [46] I. Lazar, I. Lazar, Gel Analyzer 2010a: Freeware 1D Gel Electrophoresis Image Analysis Software, 2012. [47] L. Nicolais, G. Carotenuto, Metal-polymer Nanocomposites, John Wiley & Sons, 2004. [48] E. Goharshadi, H. Ahmadzadeh, S. Samiee, M. Hadadian, Nanofluids for heat transfer enhancement e a review, Phys. Chem. Res. 1 (2009) 1e33. [49] R. Kochetov, A. Korobko, T. Andritsch, P. Morshuis, S. Picken, J. Smit, Modelling of the thermal conductivity in polymer nanocomposites and the impact of the interface between filler and matrix, J. Phys. D. Appl. Phys. 44 (2011) 395401. [50] B. Mortazavi, G. Cuniberti, T. Rabczuk, Mechanical properties and thermal conductivity of graphitic carbon nitride: a molecular dynamics study, Comp. Mater. Sci. 99 (2015) 285e289. [51] S.R.H. Davidson, M.D. Sherar, Measurement of the thermal conductivity of polyacrylamide tissue-equivalent material, Int. J. Hyperther 19 (2003) 551e562. [52] M. Hosseini, A. Mohebbi, S. Ghader, Prediction of thermal conductivity and convective heat transfer coefficient of nanofluids by local composition theory, J. Heat. Transf. 133 (2011) 052401. [53] J. Giddings, Unified Separation Science, John Wiley and Sons, New York, 1991, p. 166 (chapter 8). [54] Y. Ito, J. Cazes, Encyclopedia of Chromatography, Taylor & Francis, 2001. [55] J.C. Reijenga, Applied Voltage: Effect on Mobility, Selectivity, and Resolution in Capillary Electrophoresis, Encyclopedia of Chromatography, second ed., Taylor & Francis, 2005, p. 121. [56] M. Pumera, J. Wang, E. Grushka, R. Polsky, Gold nanoparticle-enhanced microchip capillary electrophoresis, Anal. Chem. 73 (2001) 5625e5628. [57] B. Veronesi, C. de Haar, L. Lee, M. Oortgiesen, The surface charge of visible particulate matter predicts biological activation in human bronchial epithelial cells, Toxicol. Appl. Pharm. 178 (2002) 144e154. [58] D.Q. Lin, L.N. Zhong, S.J. Yao, Zeta potential as a diagnostic tool to evaluate the biomass electrostatic adhesion during ion-exchange expanded bed application, Biotechnol. Bioeng. 95 (2006) 185e191. [59] Y. Zhang, M. Yang, N.G. Portney, D. Cui, G. Budak, E. Ozbay, M. Ozkan, C.S. Ozkan, Zeta potential: a surface electrical characteristic to probe the interaction of nanoparticles with normal and cancer human breast epithelial cells, Biomed. Microdevices 10 (2008) 321e328. [60] R.J. Hunter, Zeta Potential in Colloid Science: Principles and Applications, Academic Press, 2013, p. 79 (chapter 3). [61] M. Dirksen, A. Chrambach, Studies on the redox state in polyacrylamide gels, Separ. Sci. 7 (1972) 747e772. [62] A. Chrambach, D. Rodbard, T. Jovin, P. Svendsen, Analytical and Preparative Polyacrylamide Gel Electrophoresis, Methods of Protein Separation, Springer, 1976, pp. 27e144. [63] C. Gelfi, P.G. Righetti, Polymerization kinetics of polyacrylamide gels I. Effect of different cross-linkers, Electrophoresis 2 (1981) 213e219. [64] Y. Liu, J. Tang, J. Xin, Fabrication of nanowires with polymer shells using treated carbon nanotube bundles as macro-initiators, Chem. Commun. (2004) 2828e2829. [65] K. Papagelis, M. Kalyva, D. Tasis, J. Parthenios, A. Siokou, C. Galiotis, Covalently functionalized carbon nanotubes as macroinitiators for radical polymerization, Physica. Status. Solidi. B 244 (2007) 4046e4050. [66] J.S. Yang, X. Jing, W.J. Li, X.K. Hu, W. Wei, Z.Z. Wang, A new potential radiosensitizer-multi-walled carbon nanotubes modified by ammonium persulfate, Gene. Ther. Mol. Biol. 12 (2008) 247e325. [67] M. Arab, F. Picaud, M. Devel, C. Ramseyer, C. Girardet, Molecular selectivity due to adsorption properties in nanotubes, Phys. Rev. B 69 (2004) 165401.

Graphitic carbon nitride embedded hydrogels for enhanced gel electrophoresis.

Here, we show, for the first time, the use of graphitic carbon nitride (g-C3N4) nanosheets to improve the resolution and efficiency of protein separat...
2MB Sizes 2 Downloads 23 Views