Graphite-Coated Paper as Substrate for High Sensitivity Analysis in

Article pubs.acs.org/ac

Graphite-Coated Paper as Substrate for High Sensitivity Analysis in Ambient Surface-Assisted Laser Desorption/Ionization Mass Spectrometry Jialing Zhang, Ze Li, Chengsen Zhang, Baosheng Feng, Zhigui Zhou, Yu Bai, and Huwei Liu* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, P.R. China S Supporting Information *

ABSTRACT: In this work, an extremely simple and quite sensitive mass spectrometric method termed ambient surfaceassisted laser desorption/ionization mass spectrometry (ambient SALDI-MS) was developed to analyze different kinds of compounds, just using a piece of graphite-coated paper for the sample introduction. This provides great advantage in simplifying the analysis process. The method is quite easy to use, and there is no need to worry about the source of graphite, that is, the brands or the types of pencil. And the whole process was carried out under atmospheric pressure, offering all the merits that could occur in ambient MS. The improved sensitivity of this method is mainly because of the graphite, which serves as energy-transfer medium to absorb the energy of the photons and release it to the analytes that are adsorbed on the graphite surface. Also, three different laser wavelengths (1064, 532, and 355 nm) was tested to investigate the desorption mechanism. Fifty-one compounds, with varied chemical structures, were tried to prove that this new method possessed universal applicability to detect different kinds of small organic molecules.

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pressure ionization techniques like atmospheric pressure graphite-assisted laser desorption/ionization,9 enable ionization in the absence of enclosures. In past decades, the search for more sensitive, versatile, and robust ionization techniques, such as desorption electrospray ionization (DESI),10 direct analysis in real time (DART) ionization,11 electrospray laser desorption/ionization (ELDI),12 low-temperature plasma probe (LTP),13 paper spray ionization,14 infrared laser ablation metastable-induced chemical ionization (IR-LAMICI),15 and electrospray ionization using a wooden tip,16 have paved the way for detecting small molecules and biomolecules through a more simple procedure. The development of AMS opens the door to realizing high-throughput,17−19 nondestructive,20,21 in situ,22−24 and reaction-monitoring25,26 analysis. Therefore, there will be great prospects when applying SALDI-MS in ambient conditions, the advantages lying in simplified analysis procedures and improved operation efficiency. Herein, we present a novel ambient SALDI-MS method in which a piece of filter paper serves as the medium for sample loading and graphite from a pencil functions as “energy converter”, realizing extremely simple, cost-efficient, and practical utilization of graphite as a tool for greatly improving

raphite, which has been discovered since the 16th century and is rich in natural resources, has a layered, planar structure. Owing to the special surface property, graphite was reported to be a near-blackbody, making it suitable for absorbing any wavelength of light.1,2 For instance, graphite surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS), which was originally proposed by Sunner et al. in 1995,3 substitutes the chemical matrix of matrix-assisted laser desorption/ionization (MALDI) for an active surface to enhance absorption efficiency of light and eliminate the matrix interference in the low-mass region of the mass spectrum.4 The equipment used in SALDI-MS is very similar to that of MALDI-MS, with only a few modifications.3,5,6 The main limitation of these techniques then lies in the lack of simplicity because of the surface constraints to be sampled under vacuum environment. Samples might be disrupted or damaged when placed under reduced pressure. Also, it would take a long time to attain the desired vacuum level. Moreover, graphite has to be used in forms of suspension, which extends the sample preparation steps, making the method time-consuming and laborious. Ambient mass spectrometry (AMS) is becoming more and more attractive because of its simple sample pretreatment process, the ability to operate in atmospheric pressure, and capability to probe the surface of samples of any size and shape.7,8 AMS approaches, distinguished from atmospheric © 2012 American Chemical Society

Received: January 1, 2012 Accepted: March 1, 2012 Published: March 1, 2012 3296

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Figure 1. Experimental setup and configuration of the ambient SALDI-MS, illustrating that the device is composed of four parts: (i) a plasma producer; (ii) laser system; (iii) mass spectrometer; (iv) automated-3D mobile platform. (a) Full view of the device. (b) Top view. (c) Side view.

Figure 2. Mass spectra of tetradecanoic acid and 4-aminoantipyrine on two bands under 532 nm of laser (0−0.3 min was the white band; 0.3−0.6 min was the black band). (a) Extracted ion chromatography of tetradecanoic acid (Mr = 228) in negative ion mode; (b) averaged mass spectrum of the white band on which no graphite was drawn; (c) averaged mass spectrum of the black band on which graphite was painted; (d) extracted ion chromatography of 4-aminoantipyrine (Mr = 203) in positive ion mode; (e) averaged mass spectrum of the white band; (f) averaged mass spectrum of the black band.

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the sensitivity of MS. The analysis procedure is so simple and convenient that even a neophyte without any experience can operate well. In the whole process, all one should do is just draw a line on the paper with a common pencil, introducing the sample with a capillary, immobilizing the paper on a robotic 3D-mobile platform, and then analyzing it with the ambient SALDI-MS system. The analysis of one sample can be completed within 1 min. The use of graphite greatly increases the detection sensitivity, which was confirmed by comparing the results obtained with and without graphite on the paper.

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Article

RESULTS AND DISCUSSION

As shown in Figure 1, a helium plasma producer is mounted onto an ion trap mass spectrometer to ionize the species desorbed/ablated by laser. To test the effect of graphite in the desorption/ablation process, two equilong bands (∼20 mm each) were made on the paperone was drawn by a pencil, which can be referred to as “black band”, and the other was not, which can be called “white band”. Then analytes were introduced onto both of the two bands. The paper at the white band was first scanned under 532 nm of laser, and then the black band, so that the difference of the signal intensity can be distinguished easily. Here, we take the tetradecanoic acid as an example (Figure 2a−c): when the laser examined the white band, no ion could be detected on the mass spectrum; however, once the laser exposed on the black band, an increased signal of ion current ([M − H]− = 227) was observed and recorded. After the process was repeated at least three times, we supposed that graphite was necessary for the detection of tetradecanoic acidno graphite, no signal. Under positive ion mode, 4-aminoantipyrine ([M + H]+ = 204) showed the same tendency (Figure 2d−f). Then 51 compounds with different chemical structures were tested under positive or negative ion mode, depending on the properties of the compounds, to see whether this phenomenon is common for different compounds (Supporting Information, Table S1). No matter what the structure is, straight chain compounds like tetradecanoic acid, cyclic compounds like methyltestosterone, aromatic compounds like 2-(phenylamino) benzoic acid, or large conjugated systems like rhodamine B all have one thing in common graphite can greatly improve the sensitivity for all compounds. For compounds like decanedioic acid ([M − H]− = 201) and octadecylamine ([M + H]+ = 270), the signal-to-noise ratios (S/N) were both less than 1 (basically not detected) in the white band, but in the black band, S/N can be reached as high as S/N = 352 and S/N = 593, respectively (Supporting Information, Table S1). Although detection limits have not been thoroughly studied, some compounds have been detected at relatively low levels. For example, 100 pg of anthranone could be detected with S/N of 12, indicating a sensitivity of 25 pg detected by the proposed ambient SALDI-MS method. Different types of pencils, 4H, 2H, 2B, 4B, and 6B, were tried to investigate their influences on the performance of this method. No obvious difference was observed among the five types of pencils (Supporting Information, Figure S1). Three different brands of pencil were also tested and they performed almost the same (Supporting Information, Figure S2). In addition, the thickness of graphite coated onto paper had no proven effect on the sensitivity either (Supporting Information, Figure S3). Therefore, the results above showed that the method was quite easy for use; no need for caring about the type, the brand, or the thickness when drawing a graphite line with a pencil. Apart from the filter paper, a microcrystalline cellulosecoated TLC plate was selected to prove that the graphite effect was surface-independent (Supporting Information, Figure S4) in the ambient SALDI-MS. Extracted solution of Chinese tea was used to test the applicability of this methodology for analyzing complex samples. The experiments were conducted under positive and negative ion mode, respectively. At least 30 compounds were detected because of the existence of graphite (data not shown).

EXPERIMENTAL SECTION

Chemicals and Reagents. Methanol of HPLC-grade and purified water were obtained from Dikma Technologies Inc. (Lake Forest, CA) and Hangzhou Wahaha Group Co., Ltd. (Zhejiang, China), respectively. Cellulose-coated thin-layer chromatography (TLC) glass sheets (thickness, 0.25 mm, with fluorescent indicator, G/UV 254) were purchased from Anhui Liangchen Silicon Material Co., Ltd. (Anhui, China). Different pencils and filter papers were purchased from a local market. Fifty-one compounds were obtained from commercial sources. Fabrication of Ambient SALDI-MS System. The mass spectrometer used was Agilent XCT ion trap (Agilent Technologies, Palo Alto, CA), equipped with a DART ion source (Ionsense, Saugus, MA) as the plasma generator. Helium was used as the discharge gas at the flow rate of 0.10 m3/h. The pulsed Nd:YAG laser (Lai Yin Opto-Electronics Technology, Beijing, China) can be operated at wavelengths of 1064, 532, and 355 nm, with a pulse length of 10 ns at 10 Hz. The laser energy of different laser wavelengths, 1064, 532, and 355 nm, was set to 4.8−5.1, 1.8−2.0, and 1.0−1.2 mJ, respectively. The angle between laser beam and paper surface was 45°. An x,y,z-robotic platform, with a homemade plate holder, was used to manipulate the position of the paper. For the introduction of sample solution, graphite was drawn onto the paper first; then a capillary was used to introduce the sample with about 1 μL at one time; finally, the surface of paper was fixed onto the plate holder and scanned at a rate of 1.5 mm/s. The mass spectrometer operates in the positive or negative ion mode, depending on the nature of the compounds. The parameters were as follows: capillary voltage −3500 or +3500 V, end plate offset −500 V, dry gas temperature 325 °C, and dry gas flow rate 5 L/min. Full scan was carried out at the range of m/z 100−600 with a maximum ion accumulation time of 200 ms. The LC/MSD Trap Data Analysis (Agilent in-house version) was utilized to analyze the obtained data. The working gas of DART ion source was helium (99.999%, Beijing AP beifen gases industry company. Limited, Beijing, China) at 2.3 L/min, which was controlled by a flow meter (Flow Meter Factory, Yuyao, Zhejiang, China). The potential on the discharge needle electrode was set to 6000 V and the grid voltage was at 350 V. Preparation of Longjing Tea Sample. One gram of ground Longjing tea leaves was ultrasonic-extracted for 20 min with 10 mL of methanol/H2O (70/30, v/v). Then the extract was filtered, concentrated, and introduced onto the graphitecoated paper. Safety Considerations. The use of IR, visible, and UV laser goggles is highly recommended. 3298

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Figure 3. Optical microscopy and Raman spectrometry of filter paper: (a) Optical image of clean paper without drawing graphite. (b) Optical image of paper with graphite attached on it. (c) Raman spectrum of clean paper. (d) Raman spectrum of graphite-attached paper.

Figure 4. SEM images of graphite-attached paper and clean paper with different magnification times. Graphite-attached paper: (a) 100 μm, (b) 50 μm, and (c) 10 μm. Clean paper: (d) 100 μm, (e) 50 μm, and (f) 10 μm.

for example: it can be detected by the ambient SALDI-MS under room temperature with very good response; nevertheless, there is no MS signal when it was detected by DARTMS under the same temperature. To acquire the MS response of methyltestosterone, DART-MS requires increasing the temperature to at least 200 °C. Hence, it can be concluded that the ambient SALDI-MS holds the ability to detect certain compounds at room temperature, preventing the possibility from hurting some precious samples by heat.

In this experiment, comparisons of the performance between our ambient SALDI-MS and DART-MS under different temperatures were conducted. It was found that when analyzing the 51 compounds with the ambient SALDI-MS, 30 of them can be detected without heating up the plasma. However, when the analytes were detected by DART-MS directly in which the analytes were desorbed through thermal desorption, most of the compound could only be desorbed and ionized at the temperature range of 200−400 °C. Take methyltestosterone, 3299

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Figure 5. Schematic illustration of the performance of the ambient SALDI-MS under different laser wavelengths.

of visible and IR laser. Anthranone and 1-benzoylnaphthalene can absorb the photon energy directly from UV laser; nevertheless, this does not work when visible and IR laser were used. Therefore, there should be varied desorption paths in this methodology, depending on different compounds and different wavelengths of laser (Figure 5). For most of the detected compounds, such as tetradecanoic acid, octadecylamine, and methyltestosterone, it could be concluded that during the desorption process, graphite played an important role as an “energy converter” in absorbing the energy from UV, visible,32 and IR laser and transferring the energy to the analytes adsorbed on the surface. For the compounds possessing conjugated systems, such as rhodamine B, anthranone, and 1benzoylnaphthalene, they can directly take the energy of UV photons, without the assistance of graphite; but they can only slightly absorb the energy of visible and IR laser photons. In this case, graphite can still make a contribution as the energy mediator. It is noteworthy that, in the best cases, many more neutral species than ions are produced from a typical laser desorption. After desorption of the analytes from graphite surface, a distinct secondary ionization process, mainly proton transfer reaction, happens between the gas-phase desorbed molecules and water ion clusters, which were generated by electronic excited helium atoms (23S).11 So there should be generally four steps during the desorption and ionization process of the ambient SALDI-MS: first, analytes were adsorbed on the surface of graphite flakes; second, the energy of laser photons was absorbed by graphite and transferred to the adsorbed analytes; third, the excited analytes released energy and went into the gas phase; finally, gas-phase analyte molecules reacted with photon-donor or -acceptor and generated molecular ions.

Optical microscopy and Raman spectrometry were conducted to study the graphite surface on paper (Figure 3a−d). We can see that graphite was attached to the cotton fiber of filter paper. Scanning electron microscope (SEM) micrographs (Figure 4a−f) show that after drawing graphite onto the filter paper, graphite flakes with different sizes can be easily observed. This is the same when analyzing the graphite surfaces on cellulose-coated TLC plates (Supporting Information, Figure S5). Graphite can be defined as an infinite 3D crystal consisting of sp2-hybridized carbon atoms tightly bonded in hexagonal rings and the adjacent graphene sheets interact weakly through van der Waals forces.27,28 It has been reported that graphite holds the ability in adsorbing simple molecules such as alkanes, alcohols, and carboxylic acids on the graphite terraces and at the steps.29,30 Therefore, the adsorption of organic compounds on graphite should favor the followed desorption and ionization process. Complex optical and mechanical phenomena as well as thermodynamic and physicochemical processes of phase transition and ionization should be involved in the desorption and ionization processes of the ambient SALDI-MS.31 To investigate the desorption/ionization mechanism of this system, three different wavelengths of laser (1064, 532, and 355 nm) were all tested to survey the performance of each compound. Interestingly, the performance of ultraviolet (UV) laser was quite different from that of visible and infrared (IR) laser. For the compounds like anthranone and 1-benzoylnaphthalene, which possessed large conjugated π systems, both of the MS responses from the white band and the black band were quite high and basically the same (Supporting Information, Figure S6). This means UV laser can desorb some specific analytes from the paper directly without the assistance of graphite. However, with the help of graphite, the sensitivity of anthranone and 1-benzoylnaphthalene could be enhanced obviously under visible and IR laser by comparing the performance of the two bands (Supporting Information, Table S1). The phenomenon above should be attributed to the different energies of photons stored in the UV, visible, and IR laser. Photons of UV laser own the higher energy than that

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CONCLUSION

In summary, the results presented here are the first application of an ambient graphite-coated paper-based MS method for high-sensitivity detection, without any complicated synthesis. With the assistance of graphite, the MS signal can be improved 3300

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as high as 600-fold. Also, the method provides great availability in simplifying the whole analysis process and has the potential in applications of detecting complex samples with high throughput. The desorption and ionization mechanism of the ion source were proposed, showing graphite from a pencil exhibits optical properties that are very close to those of the ideal blackbody in the spectral range from UV to infrared. Recent reports about utilizing graphite-assisted LDI MS for imaging of plant tissues1,2 might benefit from the present methodology since size of the desorption site of the DART method could be reduced to 150 μm in width.33 In addition, because of the use of a plasma-based ion source in this ambient SALDI-MS, the mass range of this method was limited to 1000 Da. Therefore, an electrospray-based ionization technique, which is under study, could be a substitute for the plasma ion source to acquire the ability of analyzing biomolecules like peptides and proteins.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 86-10-62754976. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grants 21027012, 20975005) and the fundamental research funds for the central universities.

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