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* E-mail: jingwu.kang@sioc.ac.cn; Tel.: 0086-021-54925385; Fax: 0086-021-54925481 Received March 12, 2014; accepted April 11, 2014; published online April 22, 2014. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201400148 or from the author. Chin. J. Chem. 2014, 32, 293—297 © 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 293

DOI: 10.1002/cjoc.201400148

Preparation of Durable Emitter of Electrospray Mass Spec-trometry by Covalently Coating the Fused-Silica Capillary Tip

with Carbon-Nanotube Sol-Gel Composite Material

Hanzhi Zhang, Chao Liu, Xuepei Zhang, and Jingwu Kang*

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China

A simple approach for the preparation of emitter of electrospray ionization mass spectrometry by covalently coating the fused-silica capillary tip with the conductive carbon-nanotube sol-gel composite material (CNTSCM) is described. The CNTSCM was prepared by dispersing single-walled carbon nanotubes in the sol composed of a mixture of 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltrimethoxysilane, and ethanol. The long-term sta-bility of the prepared ESI emitters was demonstrated by at least 180 h of continuous use. Signal intensity obtained by the prepared emitter was mass-flux sensitive when the flow rate was lower than 500 nL/min, while the signal in-tensity performed a concentration dependence when the flow rate was in the range of 500－800 nL/min. The use-fulness of such a prepared emitter was demonstrated by the analysis of various types of samples such as organic small molecular drugs, oligosaccharide, peptide, and protein.

Keywords electrospray emitter, mass spectrometry, carbon-nanotube sol-gel composite material

Introduction Electrospray ionization (ESI) represents the most

widely used ionization technique in chemical and bio-chemical analysis by mass spectrometry (MS).[1] It has become an essential interface for coupling MS with separation techniques such as high-performance liquid chromatography[2] and capillary electrophoresis.[3-6] The introduction of the nano-electrospray ionization (nano- ESI) by Wilm and Mann has considerably extended the application of ESI-MS for biological analysis.[7,8] The nano-ESI is based on a sheathless electrospray emitters. Compared with the conventional ESI,[9] the use of the sheathless electrospray emitters not only significantly reduces sample consumption, but also improves the ionization efficiency and sensitivity.[7,10] Fused-silica capillaries with an orifice in the micrometre range are commonly used as the sheathless electrospray emitters. An electrical contact coating at the tip of the silica cap-illary is necessary to produce ESI. The contact coating can be prepared by depositing gold,[7,11] silver,[12] cop-per,[13] or nickel[14] on the tip of fused- silica capillary. Although the gold coating is electrochemically stable, it has poor physical stability because the coating does not adhere well to the silica surface. Moreover, the silver, copper, and nickel coatings are easily destroyed under the high applied voltage owing to poor chemical stabil-ity. Various efforts have been made to improve the du-

rability of the conductive coating. Kriger et al.[11] pro-posed an approach to reinforce the conductive coating by pretreating the fused-silica capillary tip with (3-mercaptopropyl)trimethoxysilane. The thiol groups of the silane provide strong binding force to the gold coating to improve the physical stability of the coating. Trapp et al.[15] proposed another approach to prepare a stable metal coating for the sheathless electrospray emitters by using the silver mirror reaction followed by electrochemical deposition of gold onto the silver layer. In addition to the metal materials, conductive polymers and carbon materials have also been used to prepare the sheathless ESI. A method for preparing ESI emitters using conductive polyaniline was introduced by Maziarz III et al.[16] A polypropylene or graphite mixture was used by Wetterhall et al.[17] as a conductive coating to prepare the emitter of nano-ESI. Chang et al.[18] re-ported the preparation of the ESI emitter by coating the beveled fused-silica capillary emitter with a soft pencil.

Carbon nanotubes (CNTs) possess remarkable physical and chemical properties such as high thermal stability and electrical conductivity. Carbon-nanotube sol-gel composite material (CNTSCM) has been exten-sively investigated because it integrates the unique characteristics of both CNTs and silica.[19] CNTs impart good mechanical and electrical properties to the com-posites, while silica sol-gel chemistry allows the com-posites to be fabricated easily.[19] Therefore, CNTSCM

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has been used to construct electrochemical electrodes and sensing devices.[19-25] Logically, it should be a good conductive material for preparing the emitters of ESI-MS. To our best knowledge, such an application of CNTSCM has not been reported yet.

The aim of the present study is to utilize CNTSCM as the conductive material for the preparation of the sheathless ESI emitter. The prepared emitters are much more durable because the CNTSCM is covalently at-tached on the surface of the fused-silica tip. They can be used continuously for at least 180 h without a signifi-cant reduction of the signal intensity. Moreover, the emitters are easily renewed, and the preparation method is very simple and cost-effective.

Experimental Chemical and reagents

Single-walled carbon nanotubes (SWCNTs) were purchased from Shenzhen Nanotech (Shenzhen, China); 3-aminopropyltrimethoxysilane (APTMOS) was ob-tained from Fluka (Buchs, Switzerland); 3-glycidyl- oxypropyltrimethoxysilane (GPTMOS) was obtained from TCI (Tokyo, Japan); reserpine and cytochrome C were purchased from Sigma Aldrich (St. Louis, MO, USA). 5-Carboxyfluorescein-labeled peptide (5-FAM- RRGRTGRGRRGIFR, 5-CFLP) was purchased from AnaSpec (San Jose, CA, USA). Fused-silica capillaries with 50 μm internal diameter (i.d.) and 375 μm outer diameter (o.d.) were purchased from Polymicro Tech-nologies (Phoenix, AZ, USA); concentrated HNO3 was obtained from Shanghai Lingfeng (Shanghai, China); methanol, acetonitrile, and acetic acid were purchased from Merck (Shanghai, China); ultrapure water was prepared by a MilliQ purification system from Millipore (Bedford, MA, USA).

Stock solution of reserpine was prepared by dissolv-ing 6.1 mg of reserpine in 10 mL of acetonitrile to give a concentration of 1 mmol/L, the desired concentration was diluted by the stock solution. Sulfated tetrasccharide was dissolved in a methanol/water (V∶V＝80∶20) solution to give a concentration of 3 nmol/L. 5-CFLP was dis-solved in methanol/0.1% acetic acid (V∶V＝50∶50,) to give a concentration of 2 μmol/L. Cytochrome c was dissolved in methanol/1% acetic acid (V∶V＝75∶25) to give a concentration of 6 μmol/L.

Instrumentation An LCQ-Fleet ion-trap mass spectrometer equipped

with a nano-ESI ion source (Thermo Scientific, CA, USA) was used for all the experiments. The position of the capillary emitter was adjusted with an x-y-z transla-tion stage.

Preparation of CNTSCM-coated ESI emitter The SWCNTs were treated with concentrated HNO3

(65%－68%) to produce hydroxyl and carboxyl groups on the surface of the nanotubes.[26] Briefly, 110 mg

SWCNTs were dispersed in 50 mL concentrated HNO3 by sonication for 20 min, followed by refluxing at 120 ℃ for about 7 h. After removing the HNO3 solution, the SWCNTs were rinsed with deionized water until the pH turned to be neutral. Then the SWCNTs were washed with methanol and dried under vacuum. To the mixture of 0.2 mL GPTMOS and 0.3 mL APTMOS, 1.2 mg oxidized SWCNTs were added and dispersed into the sol by sonication until a homogenous black suspen-sion was formed. Subsequently, 0.7 mL ethanol and 25 μL acetic acid were added, followed by sonication for 10 min to initiate the sol-gel process. The addition of ethanol was necessary to achieve a desirable viscosity.

A long fused-silica capillary was cut into pieces with a length of 10.5 cm. An approximate length of 0.5 cm polyimide coating at one end of the capillaries was burned off and sharpened manually to a symmetrical tip using a home-made grinding device. Subsequently, the tip was etched with 1 mol/L NaOH solution for 30 min to produce more silanol groups available for chemically immobilizing the CNTSCM. After flushing with deion-ized water and acetone, the surface of the capillary tip was coated with the CNTSCM suspension to produce a thin film. This process should be done carefully to avoid clogging the orifice. The gelation of the CNTSCM pro-ceeded for 24 h at 60 ℃ until a glassy solid film was formed.

Evaluation of the prepared ESI emitters Continuous infusion of a 2 μmol/L reserpine solution

was used for evaluation of the prepared ESI emitters mounted on the LCQ mass spectrometer. The sample solution was conveyed to the fused-silica capillary emitter by a syringe pump with a 500 μL syringe (Ham-ilton, NV, USA). The temperature of the heated transfer capillary was kept at 200 ℃ and the capillary voltage was set at 10 V. The ESI spray voltage ranged from 1.5 to 4.5 kV in the positive ion mode and from −1.5 to −3.0 kV in the negative ion mode.

Results and Discussion Several organic silane reagents, including tetra-

methoxysilane, methyltriethoxysilane, 3-aminopropyl-trimethoxysilane (APTMOS), and 3-glycidyloxypropyl-trimethoxysilane (GPTMOS) were tried out as the sol-gel precursors. However, it was hard to generate a uniform suspension of CNTSCM when a single organic silane was used. We found that a stable suspension of SWCNTs could be obtained when the sol was made of a mixture of GPTMOS and APTMOS. Effect of the GPTMOS/APTMOS molar ratio on the stability of the suspension was investigated (see Supporting Informa-tion). A very stable suspension of CNTSCM was ob-tained when the molar ratio between GPTMOS and APTMOS was 1∶2. Such a prepared suspension could generate a homogeneous gel (Figure S1). The resulting sol could be easily brushed on the tip of the capillary to

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form a thin conductive coating. The scanning electron microscopy (SEM) images of the CNTSCM-coated tip of the silica capillary are shown in Figure 1A. The glassy surface of the coating is smooth (Figure 1B) and hard. A symmetric Taylor cone and plume was pro-duced as shown in Figure 1C.

Figure 1 (A) SEM image of a tapered, fused-silica capillary emitter (50 μm i.d., 360 μm o.d.) coated with CNTSCM. (B) Close-up image of the surface of SWCNT sol-gel composite ma-terial on emitter tip. (C) Formation of a symmetrical Taylor cone and plume from the tip of emitter is visible.

Evaluation of the prepared ESI emitters Effect of the ESI voltage on MS signal intensity was

investigated by continuously injecting 2 μmol/L reser-pine solution at a flow rate of 800 nL/min. As shown in Figure 2, the MS signal intensity decreased slightly with increase of the voltage ranging from 1.8 to 4.5 kV. The signal intensity at each voltage was monitored for 5 min. Ten data points in the total ion chromatogram (TIC) were collected in an interval of 30 s to calculate the av-erage ion intensity and the relative standard deviation (RSD). The RSD was used for evaluating the stability of the ESI. At the applied voltages of 1.8, 2.2, 2.8 and 3.2 kV, the corresponding RSDs were determined as 2%, 2%, 1% and 2%, respectively, implying a very stable electrospray. However, the signal became unsteady when the applied voltage was lower than 1.5 kV or over 3.2 kV owing to the unstable static Taylor cone or dis-charge.

Figure 2 Dependence of MS signal intensity on applied voltage under continuous infusion of 2 μmol/L reserpine in acetonitrile at a flow rate of 800 nL/min.

Effect of flow rate on MS signal intensity Effect of the injection flow rate on the MS signal in-

tensity was investigated by varying the flow rate rang-ing from 100 to 800 nL/min under a voltage of 2.2 kV. The TIC was recorded for 5 min under each flow rate.

Figure 3 Dependence of MS signal intensity on flow rate of sample, when 2 μmol/L reserpine in acetonitrile was infused at an applied voltage of 2.2 kV.

As shown in Figure 3, a typical mass-flux sensitive signal intensity pattern was observed when the flow rate was lower than 500 nL/min; while the signal intensity reached a plateau when the flow rate was in the range 500－800 nL/min, implying a concentration dependence manner. The RSDs of the signal intensities obtained at the flow rate of 100, 200, 400, 500, 700 and 800 nL/min were determined as 3.1%, 1.6%, 2.8%, 2.5%, 2.7% and 2.4%, respectively, indicating a stable status of the ESI emitter.

Method reproducibility and durability of the CNTSCM-coated emitters

The reproducibility of the method for the preparation of the CNTSCM-coated emitter was evaluated. Three emitters were prepared under the identical conditions, and the ESI signal intensity was recorded under the same ESI conditions: 2 μmol/L reserpine was injected at

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400 nL/min; ESI voltage was kept at 2.5 kV. The aver-age signal intensities for each of the three emitters were 1.3E6, 1.1E6, and 1.2E6. The RSDs for the correspond-ing emitters were 1.6%, 1.0%, and 1.5%, respectively. The batch-to-batch RSD for the three different emitters was determined as 6.8%. Therefore, it was concluded that there was no significant difference among the three CNTSCM-coated emitters. That is to say, the method for the preparation of the capillary emitter is robust.

The durability of the emitter was also evaluated. Figure 4 shows the TICs obtained after continuously running for 22, 55, 106 and 180 h, respectively. The signal intensity decayed very slowly with the running time. During the period of first 55 h, the signal intensity kept almost constant (Figure 4A and B). After 106 h, the signal intensity was dropped to 2.0×105 (Figure 4C). After 180 h, the signal intensity was 1.1×104 (Figure 4D). The decay of the signal intensity resulted from the coating being gradually destroyed by the high voltage.

Figure 4 TICs obtained with CNTSCM-coated emitter after running continuously for (A) 22 h, (B) 55 h, (C) 106 h, and (D) 180 h. TICs were obtained by infusing 2 μmol/L reserpine in acetonitrile at a voltage of 2.2 kV and a flow rate of 300 nL/min.

Durability comparison with the gold-coated emitter The performance of the CNTSCM-coated emitter

was compared with that of a gold-coated emitter (Pico-TipTM EMITTER) (Supporting Information, Figure S2). The CNTSCM-coated emitter could produce very stable electrospray for at least 180 h; while, the gold coated emitter only can be used for 20 h. The CNTSCM can be covalently attached onto the surface of the fused-silica capillary tip through the Si—O—Si bonds. Therefore, the durability of the CNTSCM-coated emitters is much better than that of the gold-coated emitters.

Applications The CNTSCM-coated emitters were applied for ESI

MS of various compounds including reserpine, a sul-fated oligosaccharide, a peptide, and a protein. Figure 5A shows the mass spectrum of 2 μmol/L reserpine ob-tained in the positive ion mode. The limit of detection for reserpine was determined as 10 nmol/L (S/N＝3). The spectrum of the sulfated tetrasaccharide (m/z＝

Figure 5 Typical mass spectra obtained with CNTSCM-coated emitter: (A) 2 μmol/L reserpine in acetonitrile, voltage＝2.2 kV, flow rate＝300 nL/min; (B) 3 nmol/L sulfated tetrasccharide in CH3OH/H2O (V∶V＝80∶20), voltage＝−2.2 kV, flow rate＝500 nL/min; (C) 2 μmol/L peptide 5-CFLP in CH3OH/H2O (V∶V＝50∶50) containing 0.1% (φ) acetic acid, voltage＝2.5 kV, flow rate ＝ 100 nL/min; (D) 6 μmol/L cytochrome c in CH3OH/H2O (V∶V＝65∶35) containing 1% (φ) acetic acid, voltage＝3.0 kV, flow rate＝600 nL/min.

1030.1) obtained in the negative ion mode is shown in Figure 5B. Two deprotonated ion peaks ([M−3H]3−, m/z

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＝343.4; [M−2H]2−, m/z＝514.7) were observed. The emitter was also used for analysis of peptide and protein. Figure 5C shows the mass spectrum of peptide 5-CFLP ([M＋6H]6＋, m/z＝344.3; [M＋5H]5＋, m/z＝412.8). Figure 5D shows the spectrum of cytochrome c. Multi-ple charged ions ranging from [M＋9H]9＋ to [M＋17H]17＋ were observed.

Conclusions We demonstrated that the CNTSCM can be a desir-

able conductive coating material for the preparation of ESI emitters. Such prepared emitters display much longer durability than the gold-coated nano-ESI emitters. The CNTSCM-coated emitters can produce very stable MS signals in a wide range of flow rates. Moreover, the protocol for the preparation of the CNTSCM-coated emitter is very simple and inexpensive.

Acknowledgement This work was financially supported by the National

Natural Science Foundations of China (No. 21175146).

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