Authors: Kun Qian, Xiaodan Huang & Chengzhong Yu
The graphene sheets prepared by chemical reduction of graphene oxide were utilised as the precursor and the graphene film can be obtained by vacuum filtration of the as-synthesised graphene suspension. A pulsed laser engineering approach is developed to prepare the functional graphene film with graphitic nanospheres homogeneously decorated on the surface. As a result of laser engineering process, the as-made graphene film enjoys micro- and nano-scale surface roughness with better stability. Meanwhile, the surface hydrophobicity is enhanced and electric conductivity is improved after laser treatment. The structural parameters of the functional graphene films may contribute to advanced applications in mass spectrometry detection, where laser desorption/ionization mass spectrometry (LDI MS) detection of analytes was performed on the engineered graphene films without using conventional organic matrix. The graphene film is (1) stable with minimum matrix interference; (2) able to provide sufficient surface area and desirable interaction with analyte molecules; (3) electric conductive with high electron mobility and efficient in laser absorbance for desorption/ionization. Consequently, the detection of limit can reach femtomolar level towards diverse molecules in MS analysis.
Graphene materials are rapidly attracting interest due to their unique properties and widely spread applications. (1-4) With recent advances in nanotechnology, various nano-structured materials have been used in irradiation involved area, including graphene based nano-materials. (5-12) Nevertheless, graphene is not stable under strong irradiation conditions. (4, 13-17) For example, the in situ formation of C60 on the surface of graphene under 80 keV electron beam irradiation was observed by Chuvilin et al., (14) and carbon clusters with larger sizes (Cn, n<500) were also observed in the mass spectrometry (MS) spectra of graphene with high intensity during the laser desorption/ionization (LDI) process, (16) which may decrease the detection sensitivity in MS based analysis. Considering the fact that carbon clusters produced from graphene during irradiation may have lower atomic energy (~ -0.26 eV/atom) and thus are more stable compared to graphene, (14) we hypothesise that by introducing stable carbon species homogenously distributed on the surface in situ, engineered graphene products can meet the needs of applications designed in extreme conditions, e. g. intense irradiation for MS detection and imaging. Recently it was found that the functional sites on engineered graphene induced by laser irradiation could be utilized for enhanced electric capacitance. (2, 3) In this protocol, we show that the surface of graphene film can be engineered to provide micro- and nano-scale surface roughness with the increased stability through a pulsed-laser engineering process by forming densely packed graphitic nanospheres (stable under 300 keV electron beam) on the film surface. Moreover, the surface hydrophobicity is enhanced and the electric conductivity is improved.
Matrix assisted laser desorption/ionization (MALDI) MS is one of the fundamental analytical tools in modern bio-analysis, such as early stage disease diagnosis and forensic applications. (18, 19) Despite the substantial progress, the application of MALDI MS towards small molecules is limited because the matrices (normally small organo molecules like α-cyano-4-hydroxycinnamic acid, CHCA) introduce significant background signals during laser ablation in the low molecular range (< 1000 Da). In addition, the sweet-spot effects limit the imaging spatial resolution and detection efficiency. (20, 21) To date, overcoming the low sensitivity of MS based imaging towards small molecules and the preparation of stable functional materials under irradiation represent the key challenges to the field. To address those problems, matrix-free MS techniques have been developed. (5-12, 20-23)
The oxidation of graphite may take up to 4 days; the exfoliation and reduction of graphene oxide may take 26 hours; the preparation of graphene film may take 24 hours and the engineering may take 3-24 hours depending on the area to be processed. Despite the preparation of functional graphene films, the rest should be completed within 2 hours.
As shown in the digital image (Figure 1c), the engineered area of the graphene paper turns to dark black after laser treatment with 50% laser intensity. The engineered graphene paper is freestanding and highly flexible, which can be bent and twisted into large angles without breaking as displayed in the inset of Figure 1d. Consequently, the engineered graphene paper (1.5 cm × 0.75 cm) can be easily integrated on the top of a glass slide (Figure 1d). The pristine graphene paper shows a relatively smooth surface (Figure 1c left down). In contrast, the engineered graphene paper possesses a very rough surface with ordered concaves and convexes (Figure 1c right down). The periodic concaves have a uniform distance of ~ 30 µm, consistent with the spot size of the laser beam. Such a rough surface may be responsible for enhanced light absorbance and diffuse reflection, leading to the dark colour of the engineered graphene paper. The contact angle (CA) of graphene paper was measured (inset of Figure 1c) and used as the quantitative indicator of changing surface hydrophobicity. The CA of the untreated graphene paper is 30º, which increases dramatically to 131º for the engineered paper, indicative of enhanced surface hydrophobicity. Due to the high surface tension, a water droplet tends to minimize the surface. Although the surface of a hydrophobic substrate is covered with an aqueous solution, area between ridges on the rough surface may not be filled by the liquid, and so a composite interface forms. As a result, air bubbles are entrapped into micro-/nano-sized pores at the solid surface, where a mix of solid–liquid and solid–gas interfaces is created. Compared to the plain graphene paper, engineered graphene paper with enhanced surface roughness increases the volume of air bubbles entrapped in the interface and the extent of solid–gas interface is proportional to the degree of hydrophobicity of the material. Meanwhile, after the laser treatment, the electrical resistance of the paper is reduced from 1.5 to 0.3 kΩ/cm. The enhanced electrical conductivity is attributed to the elimination of oxygen atoms caused by the laser irradiation process (the oxygen atomic content reduced from 19.5% to 14.1% after laser treatment according to the X-ray photoelectron spectroscopy in Figure 2), a phenomenon consistent with previous literatures. The changes in surface chemistry and the introduction of surface roughness at the micro-/nano-scale (see details in Figure 3) by the laser irradiation may both contribute to the enhanced hydrophobicity of engineered graphene papers.
Efficient detection of analytes can be achieved on the engineered graphene films. In the case of AONB, the ion peak of AONB at m/z of 392 [C26H38BrN3-Br]+ was observed with signal strength over 100,000 (8.5 fmol, Figure 4a) and the signal can still be detected consuming 0.85 fmol analytes (Figure 4b). In the case of DOTAP, two peaks with strong intensity at m/z of 662 ([C42H80NO4]+) and 380 ([C42H80NO4-C18H33O2]+) are observed (Figure 4c). The DOTAP signal can be detected on the engineered film comsuming 30 fmol lipids (Figure 4d). The engineered graphene film was also successfully applied to the matrix-free detection of two phosphorous lipids and the detection of limit reached femtomolar level.
The authors acknowledged the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland. The authors also acknowledged Amanda Nouwens for the helpful discussions.
Figure 1: Schematics of laser engineering graphene film.
(a) A digital image of the graphene film obtained by filtration of graphene sheets. (b) The graphene film is engineered by pulsed laser irradiation in a selected area. (c) The engineered area (darker black) and untreated area show rough and smooth surface, high and low contact angle, respectively. (d) Digital images of the engineered paper after fabrication, which can be used as a substrate.
Figure 2: X-ray photoelectron spectroscopy characterisation of the graphene paper before and after laser engineering.
(a) Untreated graphene paper. (b) Engineered paper with 50% laser intensity.
Figure 3: SEM and TEM images of the nanospheres on the engineered graphene paper.
(a) is the high resolution SEM image of the nanospheres in Fig. 2h. (b) is obtained under 300 keV electron beam and the scale bar is 10 nm. The high resolution image of the circled area in b is shown as the inset.
Figure 4: Matrix-free LDI MS detection of molecules.
MS spectra of (a) 8.5 fmol and (b) 0.85 fmol AONB on the engineered graphene film. MS spectra of (c) 300 fmol and (d) 30 fmol DOTAP on the engineered graphene film. Asterisks indicate the identified ions from the target molecules.
Laser Engineered Graphene Paper for Mass Spectrometry Imaging. Kun Qian, Liang Zhou, Jian Liu, Jie Yang, Hongyi Xu, Meihua Yu, Amanda Nouwens, Jin Zou, Michael J. Monteiro, and Chengzhong Yu. Scientific Reports 3 () 11/03/2013 doi:10.1038/srep01415
Kun Qian, Xiaodan Huang & Chengzhong Yu, Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, QLD 4072, Australia.
Correspondence to: Chengzhong Yu ([email protected])
Source: Protocol Exchange (2013) doi:10.1038/protex.2013.029. Originally published online 12 March 2013.