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Direct Surface and Droplet Microsampling for Electrospray Ionization Mass Spectrometry Analysis with an Integrated Dual-Probe Microfluidic Chip Cong-Min Huang, Ying Zhu, Di-Qiong Jin, Ryan T. Kelly, and Qun Fang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01679 • Publication Date (Web): 05 Aug 2017 Downloaded from http://pubs.acs.org on August 5, 2017

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Analytical Chemistry

Direct Surface and Droplet Microsampling for Electrospray Ionization Mass Spectrometry Analysis with an Integrated Dual-Probe Microfluidic Chip Cong-Min Huang,1 Ying Zhu, 1, * Di-Qiong Jin, 1 Ryan T. Kelly, 2 and Qun Fang1, *

1

Institute of Microanalytical Systems, Department of Chemistry and Innovation

Center for Cell Signaling Network, Zhejiang University, Hangzhou, 310058, China 2

Environmental Molecular Sciences Laboratory, Pacific Northwest National

Laboratory, Richland, Washington 99354, United States

Corresponding Author *

E-mail: [email protected]; [email protected]; Phone: +86-571-88206771.

1

ACS Paragon Plus Environment


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ABSTRACT Ambient mass spectrometry has revolutionized the way of MS analysis and broadens its application in various fields. This paper describes the use of microfluidic techniques to simplify the setup and improve the functions of ambient MS by integrating sampling probe, electrospray emitter probe, and online mixer on single glass microchip. Two types of sampling probes including a parallel-channel probe and a U-shaped channel probe were designed for dry-spot and liquid-phase droplet samples, respectively. We demonstrated the microfabrication techniques not only enhanced the capability of ambient MS methods in analysis of dry-spot samples on various surfaces, but also enabled new applications in the analysis of nanoliter-scale chemical reactions in an array of droplets. The versatility of the microchip-based ambient MS method was demonstrated in multiple different applications including evaluation of pesticide residual on fruit surfaces, sensitive analysis of low ionizable analytes using post-sampling derivatization, and high throughput screening of Ugi-type multi-component reactions.

INTRODUCTION Since the pioneering work of desorption electrospray ionization (DESI) by Cooks and coworkers,1 ambient ionization mass spectrometry has received widespread attention and has undergone rapid progress. Ambient ionization method revolutionized the way of MS analysis, where analytes are directly sampled, ionized, and MS identified from their original and native locations with little to no sample preparation. It allows rapid and high throughput sample analysis from solid surfaces (glass, polymers, drug tablets, plant leaves, biological tissues, and biofilms), liquid surfaces (reaction mixtures, gels, bacterial communities), and vapors. Ambient ionization techniques have greatly broadened the application of MS in various fields including tissue imaging, cell communication, reaction monitoring, high throughput screening, explosives detection, and single cell analysis.2-4 As one major category of ambient ionization methods, liquid extraction-based techniques provide unique advantages and a high degree of flexibility for direct MS analysis.5 In liquid extraction techniques, liquid solvent is first applied to a solid surface to dissolve or extract sample, 2


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which is followed by direct ionization or delivery to a conventional electrospray emitter. Selected sample extraction could be achieved by using different solvent systems to selectively extract molecules of interest from complex matrices. The analytical sensitivity can be improved by, e.g., minimizing matrix effects and introducing sample derivation reagents in the extraction solvent. Isotope-labeled reagents can also be added to the solvent to achieve quantitative sample analysis. The coupling of liquid extraction-based sampling methods with highly efficient nanoelectrospray ionization (nanoESI) has facilitated the extension of ambient MS to trace analyte identification, such as single cell analysis6 and high-resolution MS imaging7. Various types of liquid extraction-based ambient ionization methods have been developed, such as desorption electrospray ionization (DESI),1 liquid extraction surface analysis (LESA),8 liquid microjunction surface sampling probe (LMJ-SSP),9 and nanospray desorption electrospray ionization (nano-DESI).10 With DESI, a stream of electrosprayed droplets is directed to a surface to desorb sample and generate sample-containing secondary droplets for ionization. LESA is achieved by allowing a dispensed droplet from pipette tip to contact a sample surface, and then drawing the droplet back for ESI-MS analysis with a microchip-based emitter. Multiple parameters including extraction time and solvent composition can be optimized to improve performance. LMJ-SSP is developed based on two coaxial capillaries embedded in a tee connector. ESI solvent is delivered through the outer capillary, and the sample is extracted and transported to an ESI emitter through the central receiving capillary. Adjusting the relative positions of the two capillaries can result in distinct sampling modes including continuous sampling and plug formation. Nano-DESI uses two capillaries with a “V”-shaped configuration to form a sampling spot. The sensitivity of nano-DESI is greatly improved due to the use of a shorter receiving capillary and low flow-rate electrospray. To avoid the need to precisely align two capillaries, two approaches based on integrated sampling probes have been developed. Single probe was fabricated by pulling a dual-core capillary to generate a sampling probe with a size of 6−10 μm.6 The tip size is small enough to be punched into single cell for direct sampling. Recently, our group developed the Swan probe, which was fabricated from a single capillary based on pulling, bending, and grinding procedures.11 A sampling hole was produced at the bottom of Swan probe for sample introduction. The Swan probe can directly analyze liquid samples on the microchip surface or in a multi-well plate with high throughput. Although the single probe and Swan probe significantly 3



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simplified the structure of liquid extraction devices, fabrication of these probes with reproducible performance is very challenging, requiring a high degree of skill and practice. We ask if microfluidic technique could be an alternative approach to implement liquid extraction-based ambient MS. Integrated microfluidic conduits and probes offer outstanding advantages in performing in-situ chemical and biological reactions.12 For example, Momotenko et al.13 developed a microfluidic pull-push probe for scanning electrochemical microscopy imaging with high spatial resolution. The integration of fluidic channels with electrodes on single probe allowed direct electrochemical measurement of dry surfaces. The Delamarche group reported a series of microfluidic probes for surface processing with hydrodynamic focusing.14-16 They demonstrated that the combination of microfabrication with fluidic confinement can enable local processing of biological interfaces with high spatial and temporal resolution. Microfluidic probes were applied to micro-immunohistochemistry on tissue sections,17 single cell stimulation on cultured adherent cells,18 and sampling single cells for enzyme activity measurements.19 The coupling of droplet techniques with sampling probes further improved sensitivity and temporal resolution by reducing sample dispersion before detection.20 In the above-mentioned systems, microscopic imaging or offline mass spectrometry methods were commonly used to read the chemical information of the processed or sampled analytes, which could limit their applications requiring label free and high throughput detection. In this paper, we have developed an integrated dual-probe microchip to couple microfluidic surface sampling with electrospray ionization mass spectrometry (ESI-MS). Two probes with orthogonal configuration were monolithically fabricated on a glass microchip. One probe with pull-push channels was used to supply ESI buffer to the sample extraction orifice, while the other probe was used as electrospray emitter to ionize the sample for MS detection. The microfluidic techniques simplified the setup and increased the functionality of liquid extraction-based ambient MS, which were demonstrated using three different microfluidic designs including a parallel-channel probe for surface sampling, a U-shaped channel probe for nanoliter droplet sampling, and a TEE mixer for online derivatization. Such multiplex designs not only allowed to sample from dry solid surface under liquid extraction ambient MS mode, but also enabled the analysis of nanoliter droplet reactions covered by oil phase. The microchip-based ESI-MS system was applied to analysis of dry samples including pesticide residues on fruit surfaces, glucose and 4


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fructose with online derivatization, and liquid-phase samples from Ugi-type combinatorial reactions.

EXPERIMENTAL Reagents and materials All chemicals were used as received unless stated otherwise, and water was purified using a Milli-Q system (Millipore, Bedford, MA). HPLC-grade methanol, glacial acetic acid and betaine aldehyde chloride (BA) were purchased from Sigma-Aldrich (St. Louis, CA). Reserpine was a product of Aladdin Reagent (Shanghai, China). Glucose and fructose were purchased from Sinopharm Chemical (Beijing, China). Thiabendazole was from Beina Chuanglian Biotechnology Institute (Beijing, China). The stock solution of 5 mM thiabendazole was prepared in water. Glucose and fructose (1 μM each) were prepared in 50% methanol. BA was prepared in acetonitrile/chloroform (1:2, v/v) at a concentration of 196 μM. All reagents (amines, aldehdehyde, and isocyanide) for Ugi-type reactions were purchased from J&K Scientific (Shanghai, China). Amine solutions with a concentration of 100 mM were prepared in 70% methanol containing 100 mM triethylamine. Aldehdehyde and isocyanide were dissolved in 70% methanol with a concentration of 100 mM. Fabrication of the dual-probe microchip Standard photolithographic patterning followed by wet etching, room-temperature pre-bonding and finally high-temperature (560 °C) bonding were used to fabricate microchips on glass substrates as described previously.21,22 Unless mentioned otherwise, all the microchannels were patterned at 20 μm in width and etched for 20 min to generate a depth of ~30 μm and a final width of ~80 μm. Multi-step grinding was used to fabricate integrated sampling probes and electrospray emitters on the glass microchip.23 Briefly, microchannels with open ends were first filled with a plug (~2 mm) of epoxy to avoid clogging by grinding particles. Emery drill bits were used to grind the microchip surrounding microchannels to produce relatively large (~1 mm) probes, after which the probes were ground with a 400-grit waterproof sandpaper followed by polishing with a 2000-grit sandpaper until pyramid-shaped tips were formed. A stereomicroscope (ST60-24T2, Sunny Instruments Co., Ningbo, China) equipped with a CCD camera (UMD200, Superimage Digital Technology Exploitation Co., Hangzhou, China) was used to monitor the 5



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grinding process. The epoxy in the channel end was removed by heating the microchip in a muffle furnace (540 °C) for 1 h. Finally, the outer surface of the two probes was treated with Aquapel glass treatment solution (Pittsburgh Glass Works LLC, Pittsburgh, PA) to render the surface hydrophobic. Figure 1 shows the final appearance of a dual-probe microchip in front of a mass spectrometer, as well as a magnified view of the integrated probes with microchannels. Setup of the integrated dual-probe microchip-based ESI-MS system The integrated dual-probe microchip-based ESI-MS system contains the following five parts: 1) an integrated dual-probe microchip for sampling and electrospray ionization, 2) a syringe pump (PHD 2000, Harvard Apparatus, Holliston, MA) with a 100 μL syringe (1700 series, Hamilton, Reno, NV) for ESI buffer delivery, 3) an ion trap mass spectrometer (LCQ DECA XP, Thermo-Fisher, Waltham, MA) for sample detection, 4) a sample-containing plate (glass slide, nanowell microchip, etc.), and 5) an automated x-y-z translation stage (PSA series, Zolix, Beijing, China) for precisely aligning and switching sample spots relative to sampling probe. The microchip was fixed on a stage with a distance of 2 mm between the microchip emitter probe end and the MS inlet orifice. The sample-containing plate was mounted to the translation stage, which was controlled with a home-built Labview program (Labview 8.0, National Instruments, TX). The sample-containing plate was electrically isolated from the translation stage using a glass insulating plate. Two CCD cameras (Daheng Image, Beijing, China) were focused on the sampling probe and the emitter probe to monitor the sampling and electrospray processes, respectively. The microchip was connected to the syringe pump using a 15-cm-long PTFE tubing (0.40 mm i.d., 0.76 mm o.d.). High voltages for electrospray in the range of 2.5–4.0 kV were applied at the steel needle of the syringe (Figure 1a). Procedures Sampling from dry sample spots. Dry sample spots were formed on a hydrophobic glass slide using the nanoliter-scale Sequential Operation Droplet Array (SODA) robotic dispensing platform.24 This sample-containing slide was mounted on the translational stage. A stable electrospray was first established by adjusting the ESI buffer-infusing flow rate and the ESI voltage. Under this stable condition, a liquid bridge connecting the parallel channels was formed at the end of the sampling probe. Sample introduction was achieved by rising up the translation stage to allow the liquid bridge to touch the sample spot, dissolve it, and be transferred to the 6


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electrospray emitter. The distance between the slide surface and the probe end was ~30 μm to maintain efficient sampling performance. Unless mentioned otherwise, the ESI buffer was 80% methanol with 0.1% acetic acid (v/v) and the flow rate was 500 nL/ min. Measurement of pesticide residual on apples. Thiabendazole at a concentration of 5 mM was applied on apple surfaces using a spray bottle. After drying, the apples were cleaned with the following different treatments: (1) soaking in 500 mL water for 5 min; (2) flushing with flowing water for 1 min; (3) soaking with 500 mL detergent for 5 min, following by water; (4) flushing with flowing detergent for 1 min, following by water. The apples were mounted on the translation stage and measured with the parallel-channel probe microchip. Each apple sample was sampled at seven different spots, and the maximum and minimum points were excluded for calculation. To quantify the amount of residue, a series of thiabendazole standard solutions with different concentrations were sprayed on apple surfaces. After spraying, the apples were dried in air by leaving it in a fume hood for 30 min, and then measured to construct a calibration curve. A semi-quantitative analysis was performed by comparing the peak areas of apple samples with different treatments with those of thiabendazole standard samples.25 Online derivatization of glucose and fructose. A 1 μL aliquot of 1 μM glucose or fructose solution was pipetted onto a hydrophobic glass slide and allowed to dry. An additional syringe pump was used to infuse derivatization reagent (betaine aldehyde chloride, BA) into the TEE junction of the microchip. The solvent flow rates for ESI buffer and derivatization reagent were 400 nL/min and 200 nL/min, respectively, to maintain stable sampling, mixing, and electrospray. Sampling from droplet array. The SODA robotic dispensing platform was used to generate a nanoliter-scale droplet array on a nanowell microchip. The droplets were covered with a layer of immiscible oil (mineral oil or FC40) to reduce droplet evaporation during reaction incubation and sample processing. Spontaneous injection was employed to introduce sample into the U-shaped channel probe by allowing the probe to be inserted into a sample droplet (contact time < 1 s) and then removed from it.11 Ugi reaction. The starting material for three-component Ugi reaction was chosen on the basis of prior work.26 One isocyanide (isocyanodiphenylmethane), two amines (L-arginine methyl ester dihydrochloride, 4-aminobenzamidine dihydrochloride), and four aldehydes (4-nitrobenzaldehyde, isovaleraldehyde, valeraldehyde, 3-Benzyloxybenzaldehyde) were used to form 2 × 4 7



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combinatorial reactions. Using the SODA robot, 30 nL of each starting material was deposited into each well to generate a droplet reactor. Each reaction condition was performed in triplicate. The reaction was conducted at room temperature for 24 h. The U-shaped channel probe was used to sample the reaction products for ESI-MS analysis. A washing step was incorporated between different droplet reactions to minimize cross contamination.

RESULTS AND DISCUSSION Design of the integrated dual-probe microchip The main objective of this work was to develop an integrated and compact device for microscale sampling and ESI-MS analysis. We were inspired by liquid extraction-based ambient MS techniques including LMJ-SSP,9 nano-DESI,10 single probe,6 and Swanprobe11, where precisely-assembled capillary devices were used for in-situ sampling and delivery to ESI-MS. We reasoned that microfluidic technique can improve these techniques in at least two ways. First, all fluidic channels can be integrated onto a single microchip with photolithography-based microfabrication techniques, which is in favor of simplifying system setup by eliminating manual capillary alignment procedures and improving the repeatability of different devices. Second, multiple functions can be incorporated by microfluidic designs without introducing evident dead volume. Proof-of-concept study of the chip-integrated microfluidic solutions for microscale ESI-MS analysis was implemented as following: (1) Both sampling probe and electrospray emitter probe were monolithically fabricated on a single microchip with a orthogonal configuration. (2) Two types of sampling probes, a parallel-channel probe and a U-shaped channel probe, were designed for efficiently and reliably transferring dry and liquid-phase samples, respectively. (3) A TEE mixer was integrated to online derivatize low-ionization-efficiency compounds to improve sensitivity after they were sampled. Glass substrates instead of polydimethylsiloxane (PDMS) were employed to fabricate the dual-probe microchip, because glass is inert to most organic solvents used in liquid extraction-based ambient MS techniques, such as high percentage of methanol, acetonitrile, and chloroform. Our previous experiments showed PDMS microchips swelled in these solvents and generated significant MS background due to extracted oligomers. The main challenge using glass substrates lies in the fabrication of sharp probes on planar glass microchips. In our previous work, 8


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we developed a multiple-step grinding procedure to directly fabricate electrospray emitter tips on glass microchip23 and used it for high sensitive droplet detection.27 Using the optimized procedures, the average post-fabrication time for each dual-probe microchip was approximately 30 min. Similar to the above-mentioned liquid extraction-based ambient MS techniques,6,

9-11

the

sampling process were driven by the self-aspiration force of electrospray. The aspiration flow rates were affected by multiple parameters such as spray voltages, solvent and sample compositions, back pressure in the fluidic channels, and temperatures. To achieve stable sampling and electrospray performance, the infusion flow rate was tuned to allow the device to work under the non-leaking mode (no drops dripping from the sampling hole), while keeping other parameters fixed. Parallel-channel probe for dry-spot sample analysis We first employed fluorescence imaging method to investigate the sampling process of the parallel-channel probe using sodium fluorescein as a model sample. As shown in Figure 2a, the sampling process consisted of two steps including extraction of dry-spot sample (a1–a2) and sample transferring into sampling channels (a2–a6). A complete sampling process required ~10 s for each spot under a flow rate of 500 nL/min. To evaluate the system performance, an array of dried spot samples was prepared on a hydrophobic slide by dispensing 5 nL of reserpine solutions with concentration ranging from 1 μM to 100 μM, corresponding to 3 pg to 300 pg per spot. We observed that the sizes of the dried spots of 5 nL reserpine solutions were typically smaller than 150 μm in diameter, ensuring complete sampling with the parallel-channel probe (~250 μm × 250 μm). Figure 2b and 2c show the extracted ion chromatogram (m/z 609–610) and linear response curve of the reserpine samples. A linear relationship having an R2 value of 0.99 was observed in the tested range. The peak area RSDs were in the range of 8.9% to 31.5% (n=3). The detection limit was calculated as 0.4 pg, showing the high sensitivity of the present ESI-MS platform, despite the use of a dated mass spectrometer (LCQ ion trap). The high sensitivity could be ascribed to the minimal dispersion of the sample plug before it arrived at the ESI emitter (transferring distance of ~2 cm). Rapid evaluation of pesticide residual on apple surface The use of pesticides is necessary to protect fruits and vegetables from the damage of insects 9



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and microbes. However, the residual pesticide may cause potential health risks. In-situ-sampling combined with mass spectrometry provides a rapid way to evaluate such risk, compared with other enzyme reaction or liquid chromatography-based methods.28-30 We applied the microchip-based ESI-MS platform with parallel-channel probe to evaluate pesticide residual on apple surface and compare the performance of different cleaning methods for removing pesticide residuals. Thiabendazole, a commonly used fungicide to control fruit and vegetable diseases, was employed as a model sample. We first generated a standard curve by spraying thiabendazole with different concentrations on the surface of an apple and directly measured with the present system (Figure S1). A linear relationship with an R2 value of 0.98 was obtained in the tested range. The relative higher signal variation could be attributed to the heterogeneous distribution of thiabendazole on apple surface during spraying and drying process. If necessary, an internal standard could be included in sample solution to improve the detection repeatability and thus obtain more precise quantification result. We evaluated four different cleaning methods including soaking in water for 5 min, flushing with water for 1 min, soaking in 0.2% detergent for 5 min, and flushing with 0.2% detergent for 1 min. As shown in Figure 3, both water-based cleaning methods only removed ~50% pesticide, compared with uncleaned one. Detergent-based methods significantly removed pesticide from apple surfaces. Long time soaking in detergent solution generated the best performance, resulting in only 2.3% residual, which agreed well with the previous study.25 The present results demonstrated the dual-probe chip-based ESI-MS system could be an efficient way to measure chemical information on biological surfaces.

Online derivatization of poorly ionizable analytes ESI-MS was challenged in the detection of poorly ionizable samples. One efficient solution is to use chemical derivatization to change the physical and chemical properties of the analytes to increase their ionization efficiency, and thus improved the analysis sensitivity and specificity of ambient MS systems.31,32 In these systems, derivatization reagents were commonly added into ESI buffers, and derivatization reactions were performed together with sample extraction. However, such a design was limited if the derivatization reagent was not compatible with ESI buffer or sample surfaces. For example, ESI buffers containing methanol and water cannot be used with 10


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betaine aldehyde.33 In this study, we provide an approach to mitigate this problem by performing derivatization reactions after samples were extracted. We integrated a mixing tee junction between the channels of the sampling probe and electrospray probe, as shown in Figure 4a. No dead volume existed in the on-chip mixer and thus significant dilution of the sample plug in the mixer as in externally integrated TEE devices was avoided. Derivatization reagent was delivered to the mixer with a pump. The mixing ratio of sample and reagent can be simply adjusted by changing the relative flow rates of the ESI solvent pump and reagent pump. Figure 4b shows a typical mixing process after a plug was sampled into the microchip. The mixing ratio of sample and reagent agreed well with their flow rates. The mixing efficiency was mainly determined by mixing time and molecular diffusion coefficients, because the flows were operated at laminar region. In the present design, the mixing time was calculated to be ca. 2 s with the total flow rate of 600 nL/min, and the mixing channel length of 1 cm. The required diffusion time to allow glucose molecule to migrate across half of the channel (40 μm) is ca. 2.3 s. Thus, sufficiently efficient mixing could be achieved in the present chip. In addition, the BA derivatization is a fast reaction, and thus satisfactory analytical performance was obtained. Despite this, enhanced mixing with chaotic mixer or longer mixing channels should be considered in some situations such as low reaction speed, and high M.W. samples with low diffusion coefficients. As a demonstration, glucose and fructose were directly sampled and then reacted with betaine aldehyde (BA) in the online mixer to generate highly charged hemiacetal compounds (Figure 4c). BA can react rapidly and selectively with hydroxyl groups of the alcohols by nucleophilic addition, which produced a MS signal at m/z [M+102]+.33 Without BA derivatization, both glucose and fructose have very low ionization efficiency and thus did not generate observable MS signals (Figure 4d1 and 4e1). In contrast, significant MS signals were observed at m/z 282.33 ([M+102]+) for both samples when online BA derivatization was incorporated (Figure 4d2 and 4e2). Because the presence of water and methanol in the ESI solvent, we also observed their reaction products with MS signals at m/z 134 and m/z 120, respectively. Although the high percentages of water and methanol were used, their signal intensities were much weaker than those of glucose and fructose, which can be ascribed to the short reaction time in the mixer. The present results demonstrate that chemical derivatization after in-situ sampling process was feasible with the integrated online 11



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mixer, which would be difficult to achieve with capillary-based extraction devices. Thus, optimal ESI buffers could be retained to achieve high ESI performance and sample extraction efficiency. U-shaped channel probe for nanoliter droplet analysis The combination of ambient MS technique with miniaturized reaction systems could provide a cost-efficient tool for rapidly screening of reaction conditions. We have developed a droplet robot system, which could perform chemical and biological reactions and assays in nanoliter droplet array.11,24 The droplets were covered by a layer of immiscible oil phase to prevent evaporation during long term incubation. Compared with other droplet-based microfluidic systems, a major advantage of droplet array system is the capabilities to generate and dispense multiple different samples and reagents for screening applications. The semi-open structure of the droplet array also allowed direct access to the droplet contents through the cover oil using a probe. We have demonstrated its versatility in various research fields including enzyme inhibition assay,24 protein crystallization screening,34 digital PCR assay,35 and single-cell gene expression analysis.36 In the present work, our initial attempts to couple a droplet array with the parallel-channel probe failed, as we observed oil plugs were frequently introduced into the sampling channel when the probe was immersed into the oil layer, resulting in the interruption of electrospray. The parallel-channel probe was improved by introducing a section of internally connected channel in the probe end, forming the U-shaped channel probe. We observed this type of probe could efficiently prevent the leakage of oil into the sampling channel. The system setup of the U-shaped channel probe for droplet analysis is shown in Figure 5a. Before the probe contacts the droplet, a continuous ESI solvent flow is formed inside the probe channel, which could efficiently prevent the leakage of oil into the sampling channel (Figure 5b1). Unlike the pull-push mode in the above dry-spot sampling process, we employed the spontaneous injection method for introducing droplet samples into the probe.11 On one hand, the high flow rate of the ESI solvent set for spontaneous injection helped to prevent oil leakage. On the other hand, the variation in sampling volume could be minimized, because the spontaneous injection is independent of the pull flow of ESI process and sampling time. To achieve spontaneous injection, the probe was allowed to contact with droplet and rapidly removed (contact time < 1 s) (Figure 5b3 and 5b4). The total sampling time was less than 6 s for each droplet, which is valuable for high throughput analysis of large number of samples in screening applications. In addition, the oil 12


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layer in the droplet array system also help to minimize cross-contamination by preventing the generation of sample residuals on sampling probe, as indicated in Figure 5b5 and 5b6. The performance of the U-shaped channel probe microchip-based ESI-MS system in the analysis of nanoliter-scale droplets was evaluated using a model sample, reserpine. It was used to sample and analyze an array of reserpine droplets with concentrations from 100 nM to 5 μM. As shown in Figure 5c, the MS signal intensities linearly increased with concentrations with a linear correlation coefficient (R2) of 0.99 (Figure S2). The concentration detection limit was calculated as 41 nM, which is comparable to or even higher than those of previously-reported droplet analysis systems.27,37,38 Similar to the sampling of dry analytes on surface, the high sensitivity can be ascribed to the relatively low sample dispersion and dilution before it arrived at the electrospray probe. Next, we assessed the long-term stability and repeatability of the microchip-based ESI-MS system by continuously analyzing an array of 63 reserpine droplets with a concentration of 5 µM. As shown in Figure 5d, all the droplets were successfully sampled and detected within 14 min, corresponding to a throughput of ~13 s/droplet. The relative standard deviation (RSD) of peak areas calculated from the 63 droplets was 15.6%, demonstrating the high reliability of the present system for high throughput ESI-MS analysis. Although high analytical performance was obtained in the present device, evident peak broadening was still observed as shown in Figure 5c, which can be attributed to the dispersion of the sample plug in the sampling channel. Thus, the sensitivity and throughput could be further improved by minimizing the “dead volume” in the sampling channel, for example, reducing its total length and the diameter. Rapid evaluation of nanoliter-scale Ugi-type reactions Multicomponent reactions play a key role in combinatorial chemistry because of their ability to synthesize large amounts of small drug-like molecules with high diversity.39-42 They greatly accelerated the drug discovery process by rapidly providing lead compounds for biological evaluations. Determination of the reaction yield and purity of the target products was essential for quality control of the screening process. However, commonly-used liquid chromatography (LC)-MS methods have suffered from low throughput, high cost, and large sample consumption. We investigated the feasibility of combining the U-shaped channel probe microchip-based ESI-MS system with the nanoliter droplet array system to rapidly evaluate the quality of 13



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multicomponent reactions. We chose Ugi-type three-component reactions as a model system (Figure S3a). The three-component reaction contained one isocyanide (Compound 1), two amines (Compound 2 and 3), and four aldehydes (Compound 4−7), forming total 8 (2 × 4) different reaction conditions (Figure S3b). Each condition was tested in triplicate and thus a total 24 droplet reactions were performed. The results are summarized in Table 1. We observed evident MS signals of the expected products in 6 out of 8 conditions. Two conditions showed stronger product signals than those of starting materials (Figure S3c2 and S3c3). To validate these results, large volume (0.15 mL) reactions were performed and analyzed with direct infusion-based ESI-MS system for comparison, and similar results were obtained (Figure S4). Although only qualitative information was provided with the present droplet-based reactions and the MS analysis platform, it still showed many attractive features for multicomponent reactions. The reaction and analysis volumes were reduced by over 1000 times, resulting in ultra-low cost and negligible organic waste. The assay throughput was hundreds of times higher than those of conventional LC-MS analysis systems. Therefore, the present platform could be used as a prescreening tool to optimize the design of multicomponent reactions.

CONCLUSIONS We have demonstrated the versatility of the integrated dual-probe microchip for ESI-MS microsampling and analysis. The capability to integrate a pull-push sampling probe, an ESI emitter probe, and a mixer for online chemical derivatization on a single microchip substantially enhanced the analytical performance of liquid extraction-based ambient MS methods. Because of the monolithic design, the system setup was simplified compared with capillary assembly methods. We envision it could find broad applications in various fields requiring in-situ surface sampling, such as tissue imaging, explosive detection, and single cell analysis. In addition to dry-spot samples, we also demonstrated the applications of ambient MS technique can be extended to analyze nanoliter-scale liquid phase reactions in an array of droplets, which is highly valuable for high throughput screening of chemical and biological reactions. The scope of the present microchip-based ambient MS platform can be further broaden by integrating other functional modules including sample cleanup with solid phase extraction, chemical separation with capillary electrophoresis or liquid chromatography, and enzymatic digestions for protein samples. 14


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In this work, the integrated probes were fabricated on glass microchips with multi-step grinding procedures. Although reproducible performance was obtained, the manual fabrication procedures could be a potential rate-limiting factor if large number of microchips are needed. The procedures can be automatized with microCNC (Computer Numerical Control) techniques, which have been successfully demonstrated to directly produce nanospray emitter on glass microchips.43,44 Recently, the Kennedy’s group has also developed a series of deep reactive-ion etching (DRIE) procedures to fabricate pull-push sampling probes on silicon chips in batch.45,46 If these methods were employed to fabricate the integrated probes, we anticipated both the fabrication throughput and analytical performance could be improved.

ACKNOWLEDGMENT Financial supports from Natural Science Foundation of China (Grants 21475117, 21435004, and 21227007), and Natural Science Foundation of Zhejiang Province (Grant LY14B050001) are gratefully acknowledged.

SUPPORTING INFORMATION The Supporting Information including the linear regression curves of thiabendazole standard samples and reserpine droplet samples, and mass spectra of Ugi-type reactions is available free of charge on the ACS Publications website.

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FIGURE CAPTIONS Figure1. (a) Schematic diagram of the integrated dual-probe microchip-based ESI-MS system. The microchip contains a sampling probe and an ESI emitter probe. (b–c) Magnified view of two types of sampling probes including (b) a parallel-channel probe for dry samples on surface and (c) a U-shaped channel probe for liquid-phase samples in droplets. (d) The setup of the dual-probe microchip in front of the MS inlet for sampling from a nanowell array chip. Figure 2. (a) Fluorescence images showing the sampling process of dried sodium fluorescein spots using the parallel-channel probe. (b) Extracted ion chromatogram (m/z 609-610) of reserpine dried-spot samples ranging from 3 pg to 300 pg, and (c) the corresponding linear response curves. Each spot was prepared on a hydrophobic slide by dispensing 5 nL of reserpine solutions with concentration ranging from 1 μM to 100 μM. Each sample amount was measured in triplicate. ESI solvent containing 80% methanol and 0.1% formic acid was delivered at 500 nL/min. Figure 3. Application of the microchip-based ESI-MS platform to evaluate pesticide residue on apple surfaces with different cleaning methods. Each condition was tested 5 times. Figure 4. Integration of post-sampling derivatization function with an on-chip TEE mixer. (a) Schematic diagram of the system setup of the dual-probe microchip integrated with a TEE mixer. (b) Fluorescence imaging of mixing process in the TEE channel. (c) Reaction equation of betaine aldehyde cation and the hydroxyl group of alcohol. (d) Typical mass spectra of glucose (d1) without and (d2) with online BA derivatization. (e) Typical mass spectra of fructose (e1) without and (e2) with online BA derivatization. The ESI solvent was 70% methanol containing 0.1% formic acid and operated at 400 nL/min. BA reagent was prepared in acetonitrile/chloroform (1:2, v/v) at a concentration of 196 μM, and was operated at 200 nL/min. Figure 5. Nanoliter-scale droplet analysis with the microchip-based ESI-MS system with U-shaped channel sampling probe. (a) Schematic diagram of the system setup for droplet analysis with ESI-MS. (b) Fluorescence images showing the spontaneous injection-based sampling process with the U-shaped channel probe. (c) Extracted ion trace (m/z 609-610) showing the continuous analysis of reserpine droplets with concentrations from 100 nM to 5 μM. Droplet volume is 100 nL. (d) Analysis of 63 reserpine droplets with a concentration of 5 μM to show the long-term stability and reproducibility of the present system. The flow rate of ESI solvent (80% methanol containing 0.1% formic acid) was 800 nL/min. 19



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Figure 1

20


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Figure 2

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Figure 3

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Figure 4

23



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Figure 5

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Table 1. MS analysis of Ugi-type three-component reactions in nanoliter droplets with U-shaped channel probe microchip.

Aldehyde

Amine

4

5

6

7

2

533.44

468.46

468.80

594.44

3

479.89

/

/

541.95

25



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F For TOC only

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Direct Surface and Droplet Microsampling for ... - ACS Publications

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