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Beijing Key Laboratory of Microanalytical Me

Chemistry, Tsinghua University, Beijing 10

tsinghua.edu.cn; Fax: +86 10 62792343; Tel

† Electronic supplementary information (solvent on the average volume of the droof benzaldehyde in methanol–water usingof watermelon juice generated by10.1039/c3an36404f

Cite this: Analyst, 2013, 138, 2163

Received 29th September 2012Accepted 18th January 2013

DOI: 10.1039/c3an36404f

www.rsc.org/analyst

This journal is ª The Royal Society of

Development and applications of paper-basedelectrospray ionization-mass spectrometry formonitoring of sequentially generated droplets†

Wu Liu, Sifeng Mao, Jing Wu and Jin-Ming Lin*

In this work, sub-microlitre droplets were generated by gravity and electrostatic attraction using a

capillary tube. The parameters affecting the sizes and frequency of the droplets were investigated. The

volume of droplets could be controlled in the range from 0.7 mL to 2.4 mL and the time interval from

15 s to 60 s with appropriate parameters. Combining the droplets with on-line mass spectrometry (MS)

via paper-based electrospray ionization (ESI), a steady flow of solvent was delivered by the capillary

tube to the base-side of the paper, which maintained the consistent state of the electrospray. With this

approach, each droplet produced a peak in the ion chromatogram. Relative standard deviations (RSDs)

not higher than 9% for both the intensities and the time intervals were achieved when using

rhodamine 6G and L-phenylalanine as model analytes. The present method was utilized for the

monitoring of the amine–aldehyde condensation reaction of butylamine and benzaldehyde. Direct

analysis and distribution of molecules in fruits were also performed, which demonstrated the potential

application of this approach.

Introduction

Applications of droplets for studies in small culture volume,especially microdroplets in microuidics, have aroused interestfrom the physical, chemical and biological elds.1–3 During thepast 10 years, various methods for forming and manipulatingsmall droplets have been developed.4–7 Each droplet provides acompartment in which species or reactions can be isolated andtherefore is suitable for quantitative studies. Furthermore,high-throughput experiments with extremely small volumes,single molecules, or single cells can also be achieved throughdroplet-based systems.7–10

Microdroplets with controllable size are typically generatedin T-junctions11 or ow-focusing devices.12 The two well-estab-lished methods both utilize two immiscible uids, generally oiland water, to create droplets in the form of an emulsion. Duringthese droplet-forming processes, the viscous forces, surface/interfacial chemistry and channel geometry are the majorspecies of the driving forces or inuencing factors.13 Besides,researchers have also developed a large number of dropletgeneration techniques, which depend on different mechanisms

thod and Instrumentation, Department of

0084, P. R. China. E-mail: jmlin@mail.

: +86 10 62792343

ESI) available: Effect of Dh, L, and theplets (V); LOD of the method; analysisthe present approach; mass spectrumconventional ESI-MS. See DOI:

Chemistry 2013

such as pressure,14 surface tension,15 electrical,16 and centrif-ugal forces.17 It is not surprising that the topic attracts so muchattention, because it acts as a cornerstone for subsequentapplications.

On the other hand, the development of analytical tools toinvestigate the content of the droplets is also a challenge tofurther researches. So far, the identication and quantitation ofthe matters in the droplets were mainly accomplished by opticalmethods, including uorescence18,19 and absorption spectros-copy,4,20 which requires chromophoric substrates or labelingand thus enormously connes the applications. Since massspectrometry (MS) is a universal, label-free, sensitive, andmolecularly specic method, the integration of MS methodsinto the online analysis of droplets is apparently of greatimportance, yet only a few implementations have beenreported.21,22

Ambient ionization, which aims at direct sampling of analy-tes in the ambient state, has emerged rapidly in recent years.23–26

Among the latest advances in atmospheric pressure electrospray-based ionization (ESI) techniques, paper-based ESI is highlypromising in consideration of its simplied protocol of samplepreparation and equipment.27,28 The capability of paper-basedESI-MS for the analysis of drugs, peptides, nucleotides andphospholipids in complex biological uid samples, such aswhole blood and raw urine, has been demonstrated recently.29–32

And for the transport and ionization mechanisms, the appro-priate substrate and solvent for effective paper-based ESI havebeen investigated. Besides, a recent work reported surfaceacoustic wave as an alternative of electrospray for paper-based

Analyst, 2013, 138, 2163–2170 | 2163

Fig. 1 Generation of the sub-microlitre droplets and direct MS analysis viapaper-based ESI.

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ionization mass spectrometry.33 All these above researchesindicate the potential of paper-based ionization as an immediateanalytical technique for complex samples in their native form.34

In a typical paper-based ESI-MS experiment, the sample wasdirectly loaded onto a paper with a triangular shape, 10 mmlong and 5mmwide at the base. Once the paper was wetted withsome solvent and then supplied with a high voltage, a spray ofcharged droplets would be induced at the tip of the paperwithout pneumatic assistance. In this work, we generate sub-microlitre droplets via gravity and electrostatic attraction andprovide a proof-of-principle experiment to show the utilizationof paper-based ESI-MS in the online analysis of the generateddroplets. A solution including analytes in a centrifuge tube wasconducted by a capillary tube to the upside of a piece of chro-matography paper, which was supplied with a high voltage. Bythe pressure difference between the inlet and outlet of thecapillary tube, together with the electrostatic attraction, sub-microliter droplets spontaneously formed and dropped ontothe paper. With a steady ow of solvent introduced to the base-side of the paper, MS spectra for each droplet were recorded.Thus, chemical reactions taken place in the centrifuge tubecould be monitored. With further modication of the inlet ofthe capillary tube, this technique was applied in depth to thedirect MS analysis and localization of molecules in fruits.

ExperimentalMaterials and chemicals

Whatman no. 1 chromatography paper (Whatman, Maidstone,Kent, UK), a kind of paper made out of pure cellulose, was thesubstrate for paper-based ESI-MS in this study because betterperformance was observed with chromatography paper thanwith lter paper or glass ber paper.28 Additionally, it hasrelatively uniform thickness and wicking properties. Butyl-amine was obtained from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China) and benzaldehyde was obtained fromBeijing Chemical Reagent Company (Beijing, China). Rhoda-mine 6G was purchased from Sigma Chemical Co. (St. Louis,MO, USA). Methanol was purchased from Fisher (New Jersey,USA) and puried water (>18 MU cm) was used. All of the fruitswere purchased from a local market.

Generation of droplets

A hole of diameter 0.5 mm was drilled at the lip of a 1.5 mLpolypropylene centrifuge tube, followed by introducing thesolvent or sample of interest into the centrifuge tube. The endsection of a capillary tube, with a length of about 20 mm, wastwined around by a grounding brass wire and then xing itvertically with the outlet of the capillary below the liquid level inthe centrifuge tube. Directly under the outlet, a piece of chro-matography paper was placed horizontally using a copper clip.This paper, with a triangular shape, was the substrate for thefollow-up electrospray ionization. To initiate the generation ofdroplets, a high voltage was applied to the copper clip, and thenthe inlet of the capillary tube was inserted through the hole andimmersed in the solution, as shown in Fig. 1. The solution in

2164 | Analyst, 2013, 138, 2163–2170

the centrifuge tube would enter the capillary tube based on thecapillary effect, and the droplets were generated at the outlet ofthe capillary.

To measure the ow velocity (v), a small amount of air wasreleased into the capillary tube from the inlet, and the timeinterval between which the small air bubble passed through twopositions marked beforehand was recorded. The forming cycle(T) of the droplets was calculated from the time consumptionfor 20 droplets. The ow rate (u) multiplied by the forming cyclegives the average volume of the droplets (V).

Paper-based ESI and mass spectrometry

Paper-based ESI ionization was performed as reported.28,30

Briey, the paper mentioned above was cut into a triangularshape, typically 10mm long and 5mmwide at the base. The apexof the paper was adjusted to direct at the inlet of the massspectrometer with a distance of 10 mm, followed by applying ahigh voltage to the paper. Then, MS analysis can be accom-plished just by introducing samples onto the paper. To apply thistechnique into the monitoring of the droplets, sufficient solventshould be ensured for a consistent and steady electrospray.Therefore, a ow of methanol–water (7 : 3, v/v) was pumped tothe base-side of the paper with a PHD 22/2000 Syringe Pump(Harvard Apparatus, Holliston, MA, USA). Once a solution ofinterest was conducted by the capillary tube, droplets droppedfrom the outlet were successively sprayed and subsequentlydetected by MS. Mass spectra were recorded using a ShimadzuLCMS-2010A mass spectrometer (Shimadzu, Kyoto, Japan).

Monitoring of amine–aldehyde condensation

1 mL of benzaldehyde at 0.4 mM in methanol–water (7 : 3, v/v)was added to a centrifuge tube and introduced for droplet

This journal is ª The Royal Society of Chemistry 2013

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generation and paper-based ESI as mentioned above. Thevoltage applied to the paper was set at 4.5 kV. The length anddiameter of the capillary tube used here were 330 mm and350 mm, respectively. As soon as the MS response of benzalde-hyde was approximately steady, 20 mL of butylamine at 20 mMwas injected into the solution. The select ions monitoring (SIM)mode was used by the mass spectrometer to record masschromatograms at m/z 74, 121 and 162, which associated withthe amine–aldehyde condensation between benzaldehyde andbutylamine in methanol–water (7 : 3, v/v).

Analysis of fruits

The pointed end of a 10 mL pipette tip, the orice diameter ofwhich was about 460 mm, was cut at about 10 mm. A capillarytube with outer diameter at 500 mm was inserted from the wideend of the pipette tip. The capillary tube and the pipette tip werethus glued together as a probe with a 1.5 mm offset at the tip.The juices were sampled by directly inserting the probe into thefruit eshes and guided to paper-based ESI-MS analysis by thecapillary tube.

For conventional ESI-MS experiments, 1 g of esh washomogenized in 10 mL methanol–water (7 : 3, v/v). The mixturewas centrifuged at 2000 rpm for 5 min and the supernatantliquid was analyzed by ESI-Q-TOF-MS (Bruker Daltonics Inc.,Billerica, MA, USA).

Results and discussionGeneration of sub-microlitre droplets

A home-made generation unit of sub-microlitre droplets isshown in Fig. 1. With the height difference (Dh) between theliquid level in the centrifuge tube and the outlet of the capillarytube, the liquid could ow out when no voltage is applied to thepaper. But the droplets are rather big and the ow rate is ratherslow because the droplets have to overcome the surface tensionto fall off. In this experiment, when the electrostatic attraction(Fe) between the paper and the droplets ought to be taken intoaccount, a droplet is separated from the liquid and it is usuallyelectrically charged. However, it is somewhat uncertain whetherthe droplet is positively or negatively charged. In order to avoidthe difficulty that positively charged droplets fall from the outletof the capillary tube onto the paper surface, where positivecharge accumulated, the capillary tube was twined around witha grounding brass wire. In this case, the pendent droplets werealways negatively charged, induced by the high-voltage electriceld. Otherwise, if a droplet was positively charged, it was evenmore difficult to fall off for the droplet compared to that in theabsence of the high-voltage electric eld, because the electricrepulsion greatly increased the resistance force.

By this strategy, the generation of droplets was facilitatedand the volume of the droplets was much smaller. For example,the average volume of methanol–water (7 : 3, v/v) dropletsgenerated from a capillary tube with external diameters at 350mm (Dh¼ 170 mm) under natural conditions was determined tobe about 4.0 mL, while with a 4.5 kV voltage applied to the paper,this volume decreases to 0.752 mL. This difference also proved

This journal is ª The Royal Society of Chemistry 2013

that the electrostatic interaction, together with the gravity of thedroplets, played a prominent part in the droplet generation.

Volume and time-interval controllability of the droplets

Parameters were investigated to establish the optimal condi-tions for the analysis of controllable and regular droplets.Methanol–water (7 : 3, v/v) was used as the solution. Mainexternal forces applied to a pendent droplet include gravity,electrostatic attraction from the paper, and the surface tension.Accompanied by the growth of the droplet, the gravityincreased, while the electrostatic attraction was positivelycorrelated with the voltage applied on the paper. Takingaccount of the electric eld, together with Tate's law, when thesum of the gravity and electrostatic attraction was just equal tothe surface tension, the droplet would fall off, at the instant ofwhich force analysis of the droplet was carried out:

rgV + Fe ¼ gpD (1)

where r is the density of the solution, g is the acceleration ofgravity, V is the volume of the droplet, D is the external diameterof the capillary tube, and g represents the surface tension. Theparameters r and g are particular physical properties of thesolution, which depend crucially on the solvent. As the majorimpetus for the falling of the pendent droplets, the electric elddetermined the sizes of the droplets, to a large extent. Thediameter of the capillary tube was also a non-ignorable factor.Herein, we xed the distance between the outlet of the capillarytube and the paper at 5 mm, and measured the volumes of thedroplets versus the voltage using several capillary tubes withdifferent diameters. As shown in the results in Fig. 2a, we foundthat a smaller external diameter of the capillary tube and ahigher voltage indeed facilitated the generation of smallerdroplets.

Regarding the liquid ow in the capillary tube as a whole,electrostatic attraction and the pressure difference between theends of the capillary tube are made up of the driving force,which should be balanced with the frictional resistance (f) whenthe ow rate became stable:

rgDhS þ Fe ¼ f ¼ lLv2

2d(2)

where l is the frictional coefficient according to the Fanningequation, and S, L, and d represent the cross-sectional area,length, inner diameter of the capillary tube respectively. Eqn (2)indicates the parameters which may affect the ow velocity,which further involve the forming cycle of the droplets. Thisassumption was demonstrated by examining the ow rates fordifferent Dh and L values with xed voltage and capillarydiameters (Fig. 2b), which aimed to maintain the stability of thedroplet volume (see ESI, Fig. S1†). Through these surveys, acontrollable volume ranging from 0.7 mL to 2.4 mL and a timeinterval from 15 s to 60 s of the droplets can be obtained withappropriate parameters. Meanwhile, the distance between theoutlet of the capillary tube and the paper was also a signicantparameter, due to its effect on the electrostatic attraction, butthe impact was difficult to be quantitatively dened for the

Analyst, 2013, 138, 2163–2170 | 2165

Fig. 2 (a) Volumes of the droplets (V) versus the voltage supplied on the paperfor capillary tubes with different external diameters (D); Dh ¼ 170 mm, L ¼ 330mm. (b) Flow rate in the capillary tube versus Dh for capillary tubes with differentlengths (L); voltage¼ 4.5 kV, D¼ 350 mm.Methanol–water (7 : 3, v/v) was used asthe solvent.

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non-uniform electric elds. Therefore, we xed this value at5 mm to provide comparable results.

Moreover, the nature of the solution, such as the density,viscosity and surface tension, would also have an impact on thedroplets. For example, the volumes of the droplets had a posi-tive correlation with the percentage of the water in the meth-anol–water mixture (Fig. S2†). Methanol–water (7 : 3, v/v) waschosen as the solvent in the following experiments because highmethanol concentrations would facilitate spray ionization,28

and more species of molecules are soluble in this mixturecompared to in pure methanol.

It should be noted that the ow rate of the solvent (u0), whichwas controlled using a syringe pump, must be set at a suitablevalue. Neither too much nor too little liquid on the paperresulted in a continuous and efficient spray. As an example,when the droplets generator was inoperative (namely u¼ 0), theow rate of the methanol–water (7 : 3, v/v) at 4 mL min�1 wasevaluated for our precise need. This data implied that the owrate of the solvent should be set at: u0 ¼ 4 mL min�1 � u, whenmethanol–water (7 : 3, v/v) was employed as the spray solvent.

Application to model analytes

Rhodamine 6G, a commonly used biological dye, was employedas a model analyte to assess the performance of the proposedsystem. Rhodamine 6G at a concentration of 10 ppm was spikedin methanol–water (7 : 3, v/v) and the droplets were analyzedunder the conditions: voltage ¼ 4.5 kV, D ¼ 350 mm, Dh ¼

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150 mm and L¼ 330 mm. The ow rate in the capillary tube wasmeasured at 1.44 mL min�1. Droplets were generated andsprayed, in which the droplets were closely consecutive in therapid process. For each droplet, the rhodamine 6G can beidentied easily (Fig. 3a) and the corresponding MS signal wasdisplayed as an individual peak in the extracted ion chro-matogram (XIC) of ion atm/z 443 (Fig. 3b). Although paper is themost ancient substrate for chromatographic separation, thediffusion of rhodamine 6G on the paper was not notable,because sharp peaks with few overlapping were observed. Therelative standard deviations (RSDs) for the intensities and thetime intervals between two adjacent peaks were calculated at8.97% and 7.35% (n ¼ 20), respectively, which indicated satis-ed reproducibility and robustness of the present method.Time intervals between two adjacent peaks were averaged to be22.3 s, which yielded an average volume of the droplets at about0.53 mL. No red spot was observed on the paper when theproduction of droplets was terminated, reecting rather fewresidual rhodamine 6G molecules on the paper. The limit ofdetection (LOD) was measured to be 30 ppb (or 16 pg absolute)based on a signal/noise (S/N) ratio of 3 (Fig. S3†).

In addition, it is worthwhile to mention that, since thespraying liquid was mixed by the analytes-including solutionand the solvent, by increasing the concentration of methanol inthe solvent, aqueous solutions can also be analyzed using thismethod. Herein, we utilized a solution of L-phenylalanine inwater as a case to illustrate this point. L-Phenylalanine is one ofthe essential amino acids, and its solubility in water is 2.965 gper 100 mL at 25 �C, while it is very slightly soluble in organicsolvents including methanol and alcohol because of the polarcharacteristics of amino acids.35 A ow of pure methanol,instead of methanol–water (7 : 3, v/v), was pumped to the paperas the solvent. As shown in Fig. 3d, the intensities and the timeintervals also exhibited good reproducibilities. Since the anal-ysis lasted for several minutes, an inadequacy was the existenceof a small peak between two peaks, each of which reported theanalytical results of a droplet.

Monitoring of amine–aldehyde condensation reaction

Real-time monitoring of chemical or biological reactions is asignicant direction because knowledge of the kinetics canbring a better understanding of the reaction mechanisms,which is further essential for catalyst screening and drugdevelopment.36–38 Schiff's bases and their metal complexes playimportant roles in medicine, catalysis science and analyticalchemistry.39–41 Schiff's bases are generally synthesized bycondensation reactions of primary amines with active carbonylcompounds. Motivated by the above results, we attempted tomonitor the formation of Schiff's base from butylamine andbenzaldehyde (Fig. 4a) using our system. At 0 min, 20 mL ofbutylamine at 20 mM was injected into 1 mL of benzaldehyde at0.4 mM in methanol–water (7 : 3, v/v). Extracted ion chro-matograms of ions at m/z 74, 121 and 162, corresponding to theinitial reactant butylamine, dehydration product of the methylhemiacetal of benzaldehyde, and the product N-butylbenzyli-denimine respectively, were recorded for 40 min (Fig. 4b).

This journal is ª The Royal Society of Chemistry 2013

Fig. 3 Analytical performances of the present method for solutions of rhodamine 6G in methanol–water (7 : 3, v/v) and L-phenylalanine in water at 10 ppmconcentration. (a) Typical mass spectrum of rhodamine 6G. (b) Extracted ion chromatogram of ion atm/z 443; each peak represents a droplet. (c) Typical mass spectrumof L-phenylalanine. (d) Extracted ion chromatogram of ion at m/z 166.

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Due to the transportation time from the inlet to the outlet ofthe capillary tube, only the dehydration product of the methylhemiacetal of benzaldehyde (m/z 121) was detectable untilabout 3.5 min (Fig. S4†). It was obvious that the peak intensityof ions at m/z 162 increased rapidly until about 20 min, fromwhen it tended to be stable. At 40 min, the inlet of the capillarywas taken out from the reaction vessel and immersed intomethanol–water (7 : 3, v/v) in another centrifuge tube. For eachdroplet, the peak intensity of ions at m/z 162 was much higherthan the two other ions, especially the ions at m/z 121, whichare nearly negligible relative to ions at m/z 162. Because theconcentration of the product cannot be higher than the initialconcentration of the reactant, this phenomenon suggested theranking of response factors (R): R162 > R74 > R121, meaning thatthe Schiff's base was ionized, transmitted and detected betterthan the reactants. It should be related to the higher hydro-philicity and proton affinity of the Schiff's bases. Furthermore,the ratio of the peak intensity of the N-butylbenzylidenimine(product) to that of the butylamine (reactant) was plotted as afunction of the reaction time (Fig. 4c), which could almostentirely eliminate interfere of the spray efficiency and thusgive a more intuitive overview of the reaction process. Thus,by paper-based ESI-MS monitoring of the droplets, informa-tion of chemical kinetics can be obtained. And with thesimultaneous detection of multiple analytes, reduced reagentconsumption and wide-ranging universality, this methodprovides an alternative analytical tool for organic chemists andbiologists.

This journal is ª The Royal Society of Chemistry 2013

Direct proling and localization of molecules in fruits

Another potential application of the proposed method is thedirect analysis and in vivo localization of molecules of interestin plants, which could provide essential evidence for theexplanation of their physiological functions and mechanisms.Herein, a preliminary study with fruits is carried out. As shownin Fig. 5a, the inlet of the capillary tube was employed as theprobe of the ‘MS sensor’. Direct insertion of the capillary tubeinto a fruit would usually cause the blocking of the capillary, soan adaptation was applied with a pipette tip. Before the analysisof a fruit, this probe was immersed inmethanol–water (7 : 3, v/v).Even if the probe was taken out, the liquid in the capillary tubewould continue to ow. Thereupon, the probe would act as astraw to ‘suck’ the sample for MS analysis. When the probe wasinserted into the esh of the fruit, the juice owed into thepipette tip due to the capillary effect and was suctioned into thecapillary tube. Either before or aer the sampling from a fruit, ashort plug of air was released into the probe, which formed the‘barrier’ between the juice and the solvent, and thus largelyreduced Taylor dispersion during the transportation process. A5 mm plug of fruit juice was sampled each time, which gave thesample volume always at 22.1 nL for a capillary tube with aninner diameter at 75 mm.However, the sample could not directlyfall off the capillary tube because the droplets were generated atthe sub-microlitre level. In other words, the juice was diluted bythe solvent at the outlet of the capillary before paper-based ESIand MS analysis. The dilution multiple could be adjusted byregulating the droplet volume as described above.

Analyst, 2013, 138, 2163–2170 | 2167

Fig. 4 (a) Aldol reaction for benzaldehyde and methanol, and Schiff's baseformation from benzaldehyde and butylamine. (b) Extracted ion chromatogramsof ions atm/z 74, 162 and 121. (c) The ratio of ISchiff's base to Ibutylamine versus time.

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Mass spectra for the fruit juice of apple, pear, and water-melon samples were obtained following this protocol (Fig. 5b–d,respectively). The major components of all the juices, namelyoligosaccharides, were detected in the form of K+ or Na+

adducts, but the relative abundances were not alike in differentfruits. In general, the amount of K+ contained in fruit eshsamples is much higher than Na+. The difference was particu-larly observed in the mass spectra of apples: the peaks at m/z219, 381, and 399 were dominant corresponding to [hexose +K]+, [sucrose + K]+ and [2hexose + K]+ respectively, but the Na+

adducts were not evident. However, the mass spectrum of pearswas dominated by [sorbitol + K]+, and Na+ adducts of sorbitolwere also detected as amajor peak. This result was in agreementwith the fact that the concentration of sorbitol is much higherin pears than in apples and watermelons. Comparison between

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the present method and conventional ESI-MS was made by theanalysis of watermelon fruits. Themost abundant ions observedby conventional ESI-MS (Fig. S5†) could be likewise found in themass spectrum generated by paper-based ESI-MS system.Meanwhile, paper-based ESI could tolerate a complex matrix,which allows a much broader scope of application comparedwith conventional ESI. The analysis was accomplished withoutany sample preparation, and thereby the original information ofactive molecules in the fruits was preserved. Additionally,beneting from low sample consumption, the spatial distribu-tion of specic molecules in the fruits could be investigated asdemonstrated below.

Citrulline is an amino acid with a high concentration inwatermelon fruits. The signal of the potassium adduct ofcitrulline could be facilely detected in watermelon esh (Fig. 5d)while no signal was observed in other fruits. To assess theanalytical performance of the method for fruit juice, citrullinewas spiked into fresh pear juice and analyzed using the sameprocedure. The peak area for m/z 214 showed an approximatelylinear correlation with the concentration of citrulline in therange of 5–100 mg L�1 (Fig. 6), and an LOD as low as 0.8 mg L�1

was achieved.On the basis of direct MS analysis of fruits, we attempted to

investigate if the levels of citrulline in different parts of awatermelon fruit are the same. Droplets were generated with atime interval at about 30 s and successively analyzed. A xedvolume (22.1 nL) of juice was sampled from different positionsof a watermelon fruit every 1 min, and the XIC at m/z 214 wasrecorded, as shown in Fig. 7. Every droplet containing the juicewas followed by a ‘clean-up’ droplet, which ushed out theresidue in the capillary tube. This ‘clean-up droplet’ and the ‘airbarrier’ in the capillary tube ensured the elimination of theinterference between adjacent samples. It was obviously dis-played that a difference existed between the abundances ofcitrulline in the outer layer, the middle layer and the inner core.The content was the lowest in the outer layer of the esh, andincreased gradually as the position was close to the inner core.At the same time, no signicant difference between the abun-dances was observed with the points at the same distances fromthe center (e.g. points d–f). These results were in accordancewith the conclusion when studied with UV-Vis spectroscopy.42

For ambient MS analysis of bioactive molecules in planttissues, desorption electrospray ionization (DESI) is the mostcommonly used ionization method.43,44 However, DESI belongsto surface analysis techniques. As a complementary tool, themethod presented here can meet the requirement for depthanalysis. Additionally, the sample volume extracted from thefruits each time was approximately the same, and was dilutedinto the same volume in the form of a droplet at the outlet of thecapillary tube, followed by on-line ionization and MS analysis ofthe droplet under a constant set of conditions. Therefore, thequantitative aspect of the method as an analytical techniquemay be more reliable. Besides, direct comparison could bemade between the intensities of specic molecules at differentpoints of the fruits, while the relative ion abundance to that ofdisaccharide or S/N values were used in similar researchespublished recently.45,46

This journal is ª The Royal Society of Chemistry 2013

Fig. 5 (a) Probe of the ‘MS sensor’ for direct analysis of fruits and typical mass spectra generated from the analysis of (b) apple, (c) pear and (d) watermelon fleshes.

Fig. 6 The intensity of m/z 214 as a function of the spiked concentration ofcitrulline in fresh pear juice.

Fig. 7 Abundances of citrulline at different points of a watermelon fruit sensed b

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Conclusions

In summary, a practical method was developed for the genera-tion of sub-microlitre droplets via gravity and electrostaticattraction. Another feature of the present approach is theremarkable feasibility and matrix-tolerance for immediateanalysis via paper-based ESI. Label-free MS analysis of dropletswas successfully performed with favorable reproducibility androbustness. This method indicates the potential of mass spec-trometry as a ‘sensor’ for complex systems by monitoring of theformation of a Schiff's base. Moreover, direct MS analysis offruits provides a new tool for molecular imaging of biologicalsystems. With the simplied equipment and compatibility withminiature, portable mass spectrometers, it would allow rapidanalysis at point-of-care facilities, even in resource-limitedenvironments. Future work will focus on two aspects: the inte-gration of MS detection into droplet-based systems will greatlyexpand the capacity; and the extension of the application is

y direct MS analysis using SIM mode.

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awaited with the rapid development of paper-based micro-uidic devices.47–49

Acknowledgements

This work was supported by National Natural Science Founda-tion of China (no. 21227006, 20935002). The authors alsoacknowledge Prof. Yu An fromDepartment of Physics, TsinghuaUniversity, for helpful discussion and suggestion relating to thiswork.

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