This work was supported by the National Research Foundation of Korea (2013R1A1A1061387) and KU-KIST research fund.

1. Preparation of Precursor
<ol>
	<li>Preparation of graphene oxide (GO):
	<ol>
		<li>GO preparation using modified Hummer&#8217;s method14
		<ol>
			<li>Add 3.0 g of graphite flakes to a mixture of concentrated H2SO4/H3PO4 (360:40 ml) at RT. (Note: Special care has to be taken while using strong acids H2SO4 and H3PO4.)</li>
			<li>Add KMnO4 (18.0 g) slowly with stirring and cooling in an ice bath to maintain the temperature of reaction mixture at &#60; 35 &#176;C. (The solution become sticky with increased reaction time, need to use proper method to maintain efficient stirring.) (Note: Special care has to be taken while adding KMnO4 due to exothermic reaction.)</li>
			<li>Stir for 12 hr at 50 &#176;C and then cool to RT and pouring reaction mixture onto ice (400 ml) containing 30% H2O2 (3 ml).</li>
			<li>Filter the reaction mixture using a metal U.S. Standard testing sieve (300 &#181;m) to remove unreacted graphite and centrifuge the filtrate (4,722 x g speed for 2 hr) to remove the supernatant.</li>
			<li>Repeat the centrifugation step with 200 ml of water, 200 ml of 30% HCl, 200 ml of ethanol, and distilled water again until pH of solution reach at 5.0&#8211;6.0.</li>
			<li>Lyophilize the final solutions to produce a fluffy GO powder.</li>
			<li>In order to make nanosized GO solution, dissolve 20 mg of GO powder in 40 ml of triple distilled water (&#62; 18 M&#937;), and then exfoliate by prolonged sonication (35% amplitude, 500 W, 2 hr) until the entire size distribution become below 150 nm, then centrifuge it two times (10,625 x g speed, 15 min) to remove precipitates (un-exfoliated large GO sheets).&#160;&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of plasmonic nanoparticle
	<ol>
		<li>Preparation of AuNPs
		<ol>
			<li>Citrate-stabilized spherical shape gold nanoparticle (AuNPs, O.D. = 1.0) of 30 nm particles size was used for r-GO reduction.</li>
		</ol>
		</li>
		<li>Preparation of AuNRs15
		<ol>
			<li>Prepare the seed solution by adding a freshly prepared 0.6 ml ice-cold solution of NaBH4 solution (0.01 M) into an aqueous mixture solution compose of 0.25 ml of HAuCl4 (0.01 M) and 9.75 ml of cetyltrimethylammonium bromide (CTAB, 0.1 M).</li>
			<li>Stir the resulting mixture vigorously for 0.5 min and then keep it at 28 &#176;C for 3 hr.</li>
			<li>Prepare the growth solution by mixing 475 ml of CTAB (0.1 M), 3 ml of AgNO3 (0.01 M) and 20 ml of HAuCl4 (0.01 M).</li>
			<li>Then add freshly prepared 3.2 ml of ascorbic acid (0.01 M) to the mixture followed by the addition of 0.8 ml of an aqueous HCl (1.0 M) solution.</li>
			<li>In the final step add 3.2 ml of seed solution to the growth solution at 28 &#176;C and subject the reaction mixture to quick inversion for few seconds. Finally, keep the resulting mixture undisturbed for at least 6 hr.</li>
			<li>Analyze the prepared AuNRs with UV-Visible spectroscopy for absorption maxima (&#955;max) and TEM analysis (typically the &#955;max and aspect ratio was found to be 730 nm and 3.5, respectively).</li>
		</ol>
		</li>
		<li>Preparation of AuNSs16
		<ol>
			<li>Prepare aqueous stock solution of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) with concentration of 100 mM, and adjust the pH to 7.4 at 25 &#176;C by adding 1.0 M NaOH solution.</li>
			<li>Mix 20 ml of phosphate buffer (100 mM) with 30 ml of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM).</li>
			<li>Then add 500 &#181;l of gold(III) chloride trihydrate (20 mM) to the above mixture and keep at 28.5 &#176;C for 30 min in water bath. Solution color changes from light yellow to greenish blue after 30 min could be observed.</li>
			<li>Centrifuge the solution at 8,928 x g speed for 30 min and disperse the precipitates in distilled water.</li>
			<li>Finally, analyze the prepared AuNSs with UV-Visible spectroscopy for absorption maxima (&#955;max) and TEM analysis for particles size confirmation which is found to be 740 nm and 30 nm, respectively.&#160;&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparation of r-GO Using Visible Light and AuNPs
<ol>
	<li>Add 1 ml of plasmonic nanoparticles (Abs 1.0 at 520 nm for AuNPs, Abs 1.0 at 750 nm for AuNRs, and Abs 1.0 at 730 nm for AuNSs, respectively) and 100 &#181;l of ammonium hydroxide (28%, w/v%) to 10 ml of GO solution (OD 1.0 at 230 nm, 0.125 mg ml-1) placed in a Pyrex glass reactor equipped with a water-circulating jacket.</li>
	<li>Irradiate the mixture with Xe lamp (power density of 1.56 W cm-2) for 30 min with water circulation through water-circulating jacket to maintain the temperature at 25 &#176;C and then centrifuge the solution at 10,625 x g speed for 15 min to remove gold nanoparticles.</li>
	<li>Take the supernatant containing the prepared r-GO to analyze with UV-Visible spectrophotometer (r-GO should show the distinctive absorption band at 270 nm) in the range of 200&#8211;900 nm.</li>
</ol>
3. Target DNA Detection Using r-GO Solution17
<ol>
	<li>For fluorescence quenching, add 20 &#181;l of 10-6 M Cy3-modified ssDNA (5&#39;-ATC CTT ATC AAT ATT TAA CAA TAA TCC CTC-Cy3-3&#39;) into GO or r-GO solution containing 25 &#181;l of GO (0.125 mg ml-1) or r-GO (0.125 mg ml-1) in 1,955 &#181;l of 0.3 M PBS solution (10 mM phosphate buffer, 0.3 M NaCl) and incubate for 10 min at RT.</li>
	<li>Measure the fluorescence intensity of these samples with spectrofluorometer (&#955;ex= 529 nm).</li>
	<li>For Target Detection, add 200 &#181;l of target oligonucleotide solution (5&#39;- GAG GGA TTA TTG TTA AAT ATT GAT AAG GAT- 3&#39;) in three different concentrations (10-6 M, 10-7 M, 10-8 M) into the GO or r-GO solution containing 20 &#181;l of 10-6 M ssDNA-Cy3, 25 &#181;l of GO or r-GO (0.125 mg ml-1), and 1,755 &#181;l of 0.3 M PBS for fluorescence recovery experiment.17 
	Notes: 
	Light Sources &#38; Reactor 
	Visible light (400&#8211;780 nm) source. Visible light irradiate through Pyrex glass reactor (window diameter = 1.1 cm) containing GO solution using Xe lamp (1.56 W/cm2 power). The photon energy applied to the reactor is calculated to be 4.8 &#215; 1021 photons per min (Figure 2A-2C). 
	Near-infrared (NIR) laser. NIR laser (window diameter = 13.2 cm) with power density of 0.36 W/cm2, and operating wavelength of 808 nm has been used as the source of near-infrared light for GO reduction reactions (Figure 2E). The photon energy is calculated to be 2.43 &#215; 1021 photons per min. 
	Reactor: Pyrex glass reactor (window diameter = 1.1 cm; reaction volume = 10 ml) equipped with a water-circulating jacket is used for both visible light and NIR light irradiated GO reduction reactions (Figure 2F).</li>
</ol>

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We have nothing to disclose.

Visible light irradiation onto GO solution for 30 min with gold nanoparticles (AuNPs, AuNSs &#38; AuNRs) showed the rapid color changes from light yellow-brown to black color (Figure 1). To obtain highly pure r-GO product in high yield, there are two important factors need to follow. One is the use of AuNPs as an efficient plasmonic catalyst, since AuNPs can strongly absorb the visible light among other structures (i.e., AuNRs, AuNSs). Another is the use of nanosized GO solution to obtain nanopa...

The first developed scotch-tape based method1 and chemical vapor deposition2 were excellent methods to produce the pristine state of a graphene, but the large scale graphene synthesis or graphene layer formation on the surface with wide area have been regarded as a key limitation of previous methods.3 One of possible solution for large scale r-GO synthesis will be wet-chemical synthetic method which first requires the reactions with strong oxidants, extensive physical treatment such as sonication to produce GO sheet, and finally the reduction of oxygen functionalities such as hydroxy, epoxide and carbonyl groups in GO is essential in order to recover its original physical properties.4 Mostly, the reduction of GO was carried out with either chemical method using hydrazine or its derivatives5 or by thermal treatment method (550&#8211;1,100 &#176;C) in an inert or reducing atmosphere.6
These processes require the toxic chemicals, long reaction time and high temperature which increased total energy demand for r-GO synthesis.7 While the photo-irradiating reduction processes such as UV-induced,8 photo-thermal process using a pulsed xenon flash,9 pulsed laser assisted10 and photo-thermal heating with camera flash lights11 have also been reported for the preparation of r-GO. In general, the low conversion efficiency of the photo-induced methods propagated to the use of UV or pulsed laser irradiation that can deliver high photon energy. The low photon energy of visible light limits its use and not attracted much for r-GO synthesis. Excellent light absorption properties of plasmonic nanoparticles in the visible and/or NIR regions can greatly improve the current drawbacks of the use of visible light for r-GO synthesis.12,13 Mild reaction conditions, short reaction time and limited use of toxic chemicals could make the visible light induced plasmon assisted photocatalytic reduction of GO as a useful alternative method.
In present method, we describe the efficient and simple r-GO synthetic method using plasmonic nanoparticles and visible light. The reaction progress was found to be strongly dependent on the structures of plasmonic nanoparticles such as spherical gold nanoparticles (AuNPs), gold nanorods (AuNRs), and gold nanostars (AuNSs). The use of AuNPs showed the most efficient reduction of GO and the nanoparticles are easily removable and recyclable for the repeated use (Figure 1). The r-GO synthesized using visible light and AuNPs showed almost equal quality compared with the r-GO prepared by well-known chemical method (hydrazine) as demonstrated by use of various analytical measurements and the fluorescence quenching/recovery based DNA detection method.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cy3 modifeid ssDNA</td>
			<td>IDT(Iowa, USA)</td>
			<td></td>
			<td>HPLC purified by IDT</td>
		</tr>
		<tr>
			<td>Gold nanoparticles (30 nm)</td>
			<td>Ted Pella, Inc(Redding, CA, USA).</td>
			<td>15706-20</td>
			<td>colloidal solution</td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) (99.5%)</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold(III)Chloride Hydrate (99.999%)</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>&#160;27988-77-8</td>
			<td>strongly hygroscopic</td>
		</tr>
		<tr>
			<td>Sodium Borohydride (99.99%)</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>16940-66-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadecyltrimethylammonium bromide (&#8805;99%)</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>57-09-0</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid(&#8805;99.0%)</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>50-81-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (99.5%)</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Nitrate&#160; (&#8805;99.0%)</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>7761-88-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphite</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>7782-42-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>7664-93-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Phophoric acid</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>7664-38-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium permanganate</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>7722-64-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>JUNSEI</td>
			<td>23150-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>1336-21-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Xe-lamp</td>
			<td>&#160;Cermax, Waltham, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIR Laser</td>
			<td>Class-IV, Sanctity Laser, Shanghai, China&#160;</td>
			<td></td>
			<td>6W (output power)</td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer&#160;</td>
			<td>S-3100, SINCO, South Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy</td>
			<td>H-7650, Hitachi, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectro Fluorometer</td>
			<td>Jasco FP-6500, Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray Photoelectron Spectrometer</td>
			<td>AXIS&#8211;NOVA, KRATOS Inc., UK</td>
			<td></td>
			<td></td>
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visible-light induced reduction of graphene oxide using plasmonic nanoparticle

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using visible light irradiation with plasmonic nanoparticles. The plasmonic nanoparticle is used to improve the reduction efficiency of GO. It only takes 30 min&#160;at RT by illuminating the solutions with Xe-lamp, the r-GO solutions can be obtained by completely removing gold nanoparticles through simple centrifugation step. The spherical gold nanoparticles (AuNPs) as compared to the other nanostructures is the most suitable plasmonic nanostructure for r-GO preparation. The reduced graphene oxide prepared using visible light and AuNPs was equally qualitative as chemically reduced graphene oxide, which was supported by various analytical techniques such as UV-Vis spectroscopy, Raman spectroscopy, powder XRD and XPS. The reduced graphene oxide prepared with visible light shows excellent quenching properties over the fluorescent molecules modified on ssDNA and excellent fluorescence recovery for target DNA detection. The r-GO prepared by recycled AuNPs is found to be of same quality with that of chemically reduced r-GO. The use of visible light with plasmonic nanoparticle demonstrates the good alternative method for r-GO synthesis.

Figure 1 shows the overall scheme for visible light and plasmonic nanoparticle based r-GO reduction reaction. Figure 2 shows the instrumental setup for the reactions. After reaction, it is required the centrifugation step to remove the used photocatalyst (AuNSs, AuNRs, or AuNPs) as shown in Figure 3A. The HRTEM analysis shows the complete removal of nanoparticles in the supernatant (r-GO) (Figure 3B), which is also possible to confirm with UV-Visible ana...

Watch this Scientific Journal Video about Visible-light Induced Reduction of Graphene Oxide Using Plasmonic Nanoparticle at JoVE.com

Visible-light Induced Reduction of Graphene Oxide Using Plasmonic Nanoparticle

A simple protocol for the preparation of reduced graphene oxide using visible light and plasmonic nanoparticle is described.

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using ...

visible-light-induced-reduction-graphene-oxide-using-plasmonic

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JoVE Journal

Engineering

14.3K Views.  Korea University. A simple protocol for the preparation of reduced graphene oxide using visible light and plasmonic nanoparticle is described.