Swan Probe: A Nanoliter-Scale and High-Throughput Sampling

Oct 10, 2014 - *Phone: +86-571-88206771. ... The Swan probe is fabricated using a single capillary with quite low cost, and it consists of a U-shaped ...
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Swan Probe: A Nanoliter-Scale and High-Throughput Sampling Interface for Coupling Electrospray Ionization Mass Spectrometry with Microfluidic Droplet Array and Multiwell Plate Di-Qiong Jin,†,§ Ying Zhu,†,§ and Qun Fang*,†,‡ †

Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310058, China Key Laboratory for Biomedical Engineering of Ministry of Education of China, Zhejiang University, Hangzhou 310027, China

‡

S Supporting Information *

ABSTRACT: Mass spectrometry provides a versatile detection method for high-throughput drug screening because it permits the use of native biological substrates and the direct quantification of unlabeled reaction products. This paper describes the design and application of a Swanshaped probe for high-throughput and nanoliter-scale analysis of biological samples in both a microfluidic droplet array and a multiwell plate with electrospray ionization mass spectrometry (ESI-MS). The Swan probe is fabricated using a single capillary with quite low cost, and it consists of a U-shaped section with a micrometer-sized hole for sampling and a tapered tip for sample electrospray ionization. Continuous sample introduction was carried out under both sampling modes of push−pull and spontaneous injection by sequentially dipping the probe in the sample solutions and then removing them. Highthroughput and reliable ESI-MS analysis was achieved in analyzing 256 droplets within 90 min with a peak height RSD of 12.6% (n = 256). To validate its potential in drug discovery, the present system was applied in the screening of inhibitors of acetylcholinesterase (AchE) and the measurement of the IC50 values of identified inhibitors.

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mainly on the basis of autosamplers with multiwell plate (MWP) and microfluidic devices with parallel ESI emitters or droplets. Autosampler systems are commonly operated under flow injection analysis mode, that is, loading samples into injection valves using robotics and then delivering them to ESI sources (Figure 1a). High-speed3 or parallel4 liquid chromatography-based separation systems and automated solid phase extraction systems6 are often incorporated prior to ESI-MS to remove nonvolatile salts and additives in sample solutions for enhancing ESI efficiency. In these systems, sample volumes in the range of 1−10 μL are usually required. To reduce the sample consumption, Felten et al.10 developed a direct infusion ESI-MS system (Figure 1b) that is capable of sampling and ionizing nanoliter-scale samples from MWP using a subatmospheric ESI source and a tapered capillary. Most recently, the Zenobi group12 reported a high-throughput ESI-MS system in which a nanoliter-size sample droplet was transferred to a capillary gap sampler and delivered to an ESI emitter. The microfluidic technique has emerged as a powerful platform for high-throughput ESI-MS analysis with low sample/reagent consumption. Microchip-based parallel ESI

he development of novel analytical techniques with high throughput and high efficiency is in great demand,1 especially in current drug discovery processes.2 Most of the current protocols for high-throughput screening are based on optical detections, in which specially designed fluorescent or chromogenic substrates must be employed to generate detectable signals. The use of these synthetic substrates leads to increasing challenges of assay development, high research cost, and high risks of false positives/negatives, which can be attributed to their changed biological activities compared with native substrates.3,4 Mass spectrometry (MS) is a label-free detection technique that allows the use of native biological substrates and direct quantification of reaction products without the need for fluorescent or chromogenic labeling. However, the assay throughput of MS is evidently lower than that of optical detection. Thus, more and more efforts have been focused on the study of high-throughput MS systems, including the invention of ionization methods,5 the improvement of sample preparation procedures,4,6−8 and the development of sample introduction approaches.9,10 As an online analytical technique, electrospray ionization mass spectrometry (ESI-MS) has many intrinsic advantages for high-throughput screening, including compatibility with continuous-flow systems, operation in atmospheric environment, high sensitivity, and low sample consumption.11 Current ESIMS-based high-throughput analytical techniques are built © 2014 American Chemical Society

Received: July 29, 2014 Accepted: October 10, 2014 Published: October 10, 2014 10796

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coupled with ESI-MS. Compared with droplet systems with microchannels, the droplet array system is naturally suitable for droplet generation of different samples and subsequent multistep droplet handling using external robots, which is of importance for high-throughput screening.28,29 A capillary with an “L” shape was used as a sampling probe as well as an ESI emitter, aspirating sample solutions from droplets with one of its ends and then ionizing them with the other; however, the sampling end of the capillary must be alternately inserted into blank droplets and sample droplets to reduce cross-contamination and sample consumption, which limited the analysis throughput. In this study, we developed the Swan probe (Figure 1e), an integrated capillary device capable of performing continuous sampling, delivery, and ESI-MS analysis of nanoliter-scale samples with high throughput and high reliability. The Swan probe was inexpensively produced using a single capillary with a U-shaped section with a micrometer-sized hole for sample introduction and a tapered tip for sample electrospraying. With the Swan probe, continuous nanoliter-scale sample introduction and direct MS analysis of samples preloaded in both MWP and droplet array systems was achieved by simply dipping the probe into sample solutions and then leaving them to allow nanoliterscale sample plugs to be introduced. Two sample injection modespush−pull sampling and spontaneous injectionwere undergone during the sample introduction process. The former sampling mode is frequently used for in vivo sampling and measurement of neurotransmitters in the brain30 and also adopted in other MS techniques for sampling and identifying analytes on solid surfaces, such as liquid microjunction surfacesampling probe/electrospray ionization31 and nanospray desorption electrospray ionization (NanoDESI),32 although the latter is commonly used in capillary electrophoresis33 and is reported in ESI-MS for the first time. We applied the Swan probe in label-free screening of enzyme inhibitors of acetylcholinesterase (AchE) to preliminarily demonstrate its potential in high-throughput drug screening.

Figure 1. Schematic diagrams of five typical sample introduction approaches for high-throughput electrospray ionization mass spectrometry analysis. (a) Flow injection analysis. (b) Direct injection analysis. (c) Microchip-based parallel ESI emitters. (d) Direct droplet electrospray. (e) Swan probe-droplet array. C, carrier buffer; S1, sample 1; S2, sample 2; S3, sample 3; O, oil.

emitters (Figure 1c) can be produced on glass, silicon, and polymer substrates with photolithography and microfabrication procedures.9,13−15 Each of the emitters in the chip is used for one individual sample via an infusion microchannel, and multiple samples are analyzed in a serial fashion without a rinsing step and cross-contamination. Liu et al.9 described a 96channel microdevice with an array of 96 ESI tips, which was capable of analyzing 96 samples in 480 s. The Nanomate is a commercially available high-throughput ESI analysis system14 that is composed of a silicon microchip with an array of monolithic nanoESI emitters and an automated sampler with disposable pipet tips. In addition to multiemitters-based parallel analysis, dropletbased microfluidic systems provide an alternative high-speed serial analysis method for high-throughput analysis. In these systems, picoliter to nanoliter-volume droplets are generated, merged, diluted, split, and sorted with various microfluidic techniques.16−18 Droplets are isolated with immiscible oil phases, which effectively eliminate droplet evaporation, diffusion, and cross-contamination between adjacent droplets. Each droplet can serve as a microreactor undergoing a screening reaction/assay independently. The outstanding advantage of droplet technique for the screening application is the significantly reduced consumption of sample and reagent compared with those of conventional MWP and parallel microfluidic emitter-based techniques. To achieve the ESI-MS analysis of these small droplets, special microfluidic designs are commonly employed for extracting droplets into continuous aqueous streams to remove the interference of oil phase on electrospray process.19−21 However, droplet dispersion and dilution usually occur in the continuous aqueous streams, which may limit the analysis sensitivity and throughput of these methods. To address this , the Kennedy group22,23 developed a simple but efficient approach for dilution-free droplet analysis by directly coupling a droplet cartridge to a commercial nanospray emitter (Figure 1d). This approach was applied to label-free screening of enzyme inhibitors24 and offline coupling of ESIMS with an LC system.25,26 Recently, the authors’ group27 described a semiclosed 2-dimentional droplet array chip

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EXPERIMENTAL SECTION Fabrication of the Swan Probe. Figure 2 shows the fabrication process of the Swan probe. First, an 18-cm-long polyimide-coated, fused-silica capillary (360 μm o.d., 100 μm i.d., Refined Chromatography Co., Yongnian, China) was heated at its middle region (2 mm length) with a butane lighter to a softening state and then pulled to form two 9 cm-long tapered capillaries with a tip inner diameter of ∼50 μm (Figure 2a). Second, the tapered capillary was heated and bent to form the initial Swan probe with a U-shaped section for sample introduction and a tapered tip for ESI-MS detection (Figure 2b). The Swan probe was then fixed on a glass slide with epoxy to make it more durable and to avoid its breakage in the following fabrication process (Figure 2c). The outer surfaces of the U-shaped section and the tapered emitter were treated with 1% (v/v) octadecyltrichlorosilan (OTCS) in isooctane for hydrophobic modification.21 Third, the bottom of the U-shaped section was ground on a 2000-grit sandpaper until an elliptical hole ∼200 μm in length and 100 μm in width was formed on the capillary wall (Figure 2d), which served as the sampling inlet of the Swan probe. During the grinding process, an annular-shaped hydrophilic surface surrounding the sampling hole was also generated by removing the hydrophobic coating, as indicated in Figure 2e−g. The grinding process was continuously monitored with a stereomicroscope (ST60-

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Figure 3. (a) Schematic configuration of the setup of the Swan probeESI-MS system coupled with a microchip with high-density droplet array. The inset shows an image of the Swan probe during the sampling introduction process for a fluorescein sample. (b) Image of a prototype microchip with 256 nanowells. (c) A close view of the Swan probe and the nanowell chip installed in front of the mass spectrometer.

Hamilton, Reno, NV) for continuously infusing ESI carrier into the Swan probe; (3) an oil-covered microchip27,28,34 (see details of the fabrication in the SI) with 2D nanowell array for containing droplet reactors28,35 (see details of the droplet array generation in the SI); (4) an automated x−y−z translational stage (PSA series, Zolix, Beijing, China) for rapidly switching droplets to the sampling inlet of the Swan probe; and (5) an ion trap mass spectrometer (LCQ DECA XP, Thermo-Fisher, Waltham, MA) for sample detection. The Swan probe was fixed on a stage with a distance of 2 mm between its emitter tip end and the MS inlet orifice. High voltage for ESI was applied between the metal needle of the syringe and the MS inlet. The droplet array chip was mounted on the x−y−z translational stage. A program written with LabView (Version 8.0, National Instruments, TX) was used to control the translational stage. Unless mentioned otherwise, the translational stage moved with an initial velocity of 8 mm/s, an acceleration of 20 mm/s2, and a uniform velocity of 20 mm/s. Two cameras (DH-SV1401/ FM, Daheng Image, Beijing, China) equipped with magnifying lenses (MLM-3XMP, Computar, Tokyo, Japan) were used to monitor the sample introduction and electrospray process, respectively. Procedures. Enzyme Inhibition Assay in Droplet Array. The assay procedures mainly from a previously published method23 were adopted for the inhibitor screening of acetylcholinesterase (AchE) in a chemical library. To perform the enzyme assay in a droplet array, 50 nL of each test compound was first deposited into nanowell and then mixed with 50 nL of 100 μg/mL acetylcholinesterase (AchE) solution (pH 7.8). After an incubation time of 30 min at room temperature, 50 nL of 200 μM acetylcholine iodide solution was added into each droplet to trigger the enzyme reaction.

Figure 2. Fabrication process of the Swan probe. (a) A capillary is heated with a torch and pulled to form a tip. (b) The tapered capillary is heated and bent to form a Swan-shaped probe with a U-shaped section and a horizontal emitter tip. (c) The Swan-shaped probe is fixed on a glass slide with epoxy. A salinizing reagent, 1% OTCS solution, is used to treat the outer surfaces of its tapered tip and the Ushaped section. (d) The probe is vertically ground on a sandpaper to produce a sampling inlet hole on the bottom of the U-shaped section and an annular-shaped hydrophilic surface surrounding the hole (e, f, g). (h) The final appearance of the Swan probe.

24T2, Ningbo Sunny Instruments Co., China) equipped with a CCD camera (UMD200, Superimage Digital Technology Exploitation Co., Hangzhou, China), which ensured the size variation of the sampling holes was lower than 15%. The final appearance of a typical Swan probe is shown in Figure 2h. A movie clip (ac503014k_si_002.avi) of the fabrication process can be found in the Supporting Information (SI). Setup of Swan Probe ESI-MS System. The structure of the Swan probe ESI-MS system coupled with a droplet array is schematically illustrated in Figure 3. Five main components were included: (1) a Swan probe served as sampling probe and ESI emitter; (2) a syringe pump (PHD 2000, Harvard Apparatus, Holliston, MA) with a 500 μL syringe (1700 series, 10798

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sequential operation droplet array (SODA) system28 was employed to assemble a high-density and nanoliter-scale 2D droplet array for enzyme reactions and assays. With the SODA system, sample/reagent consumption can be significantly reduced by 30−1000 fold compared with those of conventional multiwell plate-based screening systems. The SODA system could flexibly achieve multistep droplet manipulation required in enzyme inhibition assay, including generation of different compound droplets, addition of an enzyme solution, long-term incubation, and addition of a substrate solution for triggering an enzyme reaction. More importantly, the oil-covered 2D droplet array system had a semiopen structure, and thus, the Swan probe could pass through the oil to sequentially contact with different droplets for sample introduction. Sample Introduction of Swan Probe. The sampling process of the Swan probe was studied using sodium fluorescein solution (1 mM) as a model sample and a homebuilt fluorescent microscope for real-time monitoring. A movie clip (ac503014k_si_003.avi) is provided in the SI, showing the sampling process with a sampling time of 5 s and with carrier flow rates of 600 nL/min and 1000 nL/min, respectively. Two different sampling modespush−pull and spontaneous injection modescould be observed during the sample introduction process, as shown in Figure 4 and SI movie file ac503014k_si_003.avi. Once the probe sampling inlet contacted with the sample droplet, a continuous sample injection occurred in the capillary channel to the emitter tip under the push−pull mode at carrier flow rates lower than 800 nL/min

After a reaction time of 25 min in room temperature, 150 nL of aqueous solution containing 60% methanol and 2% acetic acid was rapidly added into each droplet to terminate the enzyme reaction and improve its compatibility with MS detection. Automated Sampling and ESI-MS Analysis. Before the experiment, the droplet array chip or 96-well plate loaded with multiple samples was mounted on the translational stage. Sample introduction was achieved by moving the translational stage to allow the sampling inlet of the Swan probe to be sequentially immersed into the sample solutions in the droplets or microwells and aspirate nanoliter-volume samples into the probe. The aspirated sample plugs were then delivered to the ESI emitter for MS detection by the continuous ESI carrier from the syringe pump (Figure 3).

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RESULTS AND DISCUSSION Design of Swan Probe. We aim to develop a simple and reliable sample introduction approach for high-throughput ESIMS analysis of large numbers of samples preloaded in both conventional multiwell plates and microfluidic droplet arrays. In our previous study, we developed an integrated microchipbased system for high-throughput droplet analysis with ESI-MS detection.21 A droplet extraction interface with K-shaped channel was adopted in the microchip, which was composed of three modules: a V-shaped hydrophilic channel36 with an emitter for sample delivery and MS detection; a straight hydrophobic channel for droplet transportation; and a hydrophilic tongue structure at the junction of the two channels for droplet extraction. The hydrophilic tongue could allow the flowing droplet contact with the ESI buffer through a liquid bridge and reliably be extracted into the V-shaped channel without oil leakage. However, the application of this microchip-based system in screening of multiple different samples was limited because droplets were generated under the continuous-flow mode, with which sample changing cannot be conveniently performed. Here, on the basis of the previous system, we designed the Swan probe, which also consisted of a U-shaped hydrophilic channel with an ESI emitter for sample delivery and MS detection and a sampling inlet surrounded by a hydrophilic surface for contacting with aqueous droplets (See Figure 2f, g), while it did not integrate the straight hydrophobic channel for segmented flow used in the microchip system in the probe. This endows the Swan probe with an open structure and allows it to directly access different samples loaded in highdensity wells or on planar surfaces. This Swan probe design also allow us to fabricate the sampling probe using a capillary instead of a microchip which requires expensive microfabrication equipment and complicated procedures. The key step of the fabrication process lies in the grinding fabrication of the sampling inlet at the U-shaped section of the capillary. We observed that fixing the two upper ends of the U-shaped capillary on a glass slide with epoxy could dramatically enhance the strength the capillary, which made it tolerate the grinding operation of the U-shaped section of the capillary rather than breaking by a yield of 90%. This technical breakthrough in capillary fabrication enables a T-shaped channel interface to be produced in a single capillary using a simple grinding method, with which the probe can be fabricated using simple devices and materials with quite low cost. The total time for fabricating a Swan probe was ∼70 min. The other objective of this work was to apply the Swan probe in label-free screening of enzyme inhibitors with high throughput and using low sample/reagent consumptions. A

Figure 4. Study of the sampling process with fluorescent imaging and MS detection. (a, b) Images recording two sampling processes at carrier flow rate of 600 nL/min (a1−a5) and 1000 nL/min (b1−b5), respectively, with a sampling time of 5 s. (c) The relationships between sampling time and MS signal at carrier flow rates of 600 nL/ min (c1) and 1000 nL/min (c2), respectively. (d) The relationship of MS signal intensity and carrier flow rate in the range of 200−1600 nL/ min. 10799

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Figure 5. Analytical performance of the Swan probe ESI-MS system. (a) Extracted ion trace (m/z 609) shows the effect of droplet volume on the corresponding MS signal of a model sample (reserpine, 1 μM). (b) Extracted ion trace (m/z 609) for reserpine with concentrations from 100 to 500 nM in 300 nL droplets. (c) High-throughput analysis of a 16 × 16 droplet array to show long-term stability and reproducibility of the present system.

dead time of the translational stage. As shown in Figure 4d, the MS signals are quite stable at a flow-rate range from 500 to 1200 nL/min, indicating the sampling volumes are almost the same at these flow rates. Further increasing the push flow rate would lead to leakage of the carrier buffer from the probe into the sample droplet and dilute the sample solution (Figure 4d). On the basis of the above results, the spontaneous injection mode was adopted in the present work for achieving highthroughput ESI-MS analysis with a pushing flow rate of 1000 nL/min and a sampling time of 1 s for the Swan probe (ac503014k_si_004.avi). Compared with the push−pull mode, the spontaneous injection mode can avoid the sampling-volume variation induced by the dynamic fluctuation of pulling flow rate in the electrospray process. In addition to increasing analysis throughput, the use of a high pushing flow rate of 1000 nL/min can also effectively eliminate the leakage of gas bubbles and oil into the probe channel during the sampling process and thus can guarantee highly stable electrospray performance in a prolonged working period. The good robustness of the spontaneous injection mode was further demonstrated in a long-term analysis for large number of 256 samples . Analytical Performance. The performance of the Swan probe ESI-MS system in the analysis of oil-covered nanoliterscale droplets was evaluated using a model sample, reserpine. The flow rate of carrier buffer was set as 1000 nL/min, and the sample droplets were introduced under the spontaneous injection mode with a sampling time of 1 s. To test the ability of the system for droplets with variable volumes, an array of droplets with volumes of 50, 80, 100, 200, 300, and 400 nL were generated and analyzed. As shown in Figure 5a, the MS signal of reserpine increases with droplet volume in the droplet volume range of 80−300 nL and tends to be stable above 300 nL. The repeatability of peak height tended to be worse when the droplet volume was lower than 80 nL, which may be attributed to the relatively large size of the probe sampling inlet. Further reducing the droplet volume could be achieved by reducing the sampling hole size with improved capillary fabrication technique or replacing the capillary probe with microchip-based probe with more accurate structure. Next, the sensitivity of the system was evaluated by sequentially sampling an array of 300 nL reserpine droplets with concentrations of

(Figure 4a). The driving force for this continuous sample injection came from the flow-rate difference between the pull flow generated by the self-aspiration force of the electrospray and the push flow produced by the syringe pump in the channel of the Swan probe. The injected sample plug was delivered by the continuous carrier flow to the ESI emitter for detection, which could significantly reduce the cross contamination between different samples. With the push−pull injection mode, the sampling volume is dependent on sampling time (Figure 4c1). The sample injection volumes with sampling times of 1, 6, 11, and 21 s were estimated as 6, 11, 15, and 24 nL, respectively. When the sampling inlet of the Swan probe was removed from the droplet, the other sample injection, spontaneous injection, could be observed (Figure 4a4, b4). During the removing process, a small sample droplet remained on the sampling inlet end of the Swan probe because of the hydrophilic property of the surface surrounding the probe inlet, and then it was sucked into the probe channel rapidly and spontaneously by its surface tension. The spontaneous injection process and its MS signal could be observed more obviously with higher carrier flow rates than 1000 nL/min, at which almost no sample was injected into the probe by the push−pull mode because of the larger push flow rate (i.e., carrier flow rate) relative to the pull flow rate (Figure 4b). The spontaneous injection phenomenon was first reported by Zare’s group33 and used in capillary electrophoresis to introduce nanoliter-scale37 and picoliter-scale38−40 samples into capillaries for achieving high-speed separation. We observed this phenomenon in the sampling process for MS analysis for the first time. Because the spontaneous injection process occurred with the removal of the probe from the sample droplet, its sampling volume (∼6 nL) and MS signal are independent of sampling time (Figure 4c2). The additional small peaks at the sampling time of 6, 11, and 21 s in Figure 4c2 could be attributed to the sample injection when the probe just contacted the droplet, whereas the injection volumes were quite smaller than those of the spontaneous injection. With the spontaneous injection mode, we further tested the effect of the pushing flow rate on the sampling volume by measuring the corresponding MS signals of 1 μM of reserpine with a minimal sampling time of ∼1 s, which is the mechanical 10800

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100, 250, and 500 nM. A relatively small sample injection volume of ∼3.7 nL was obtained among oil-covered droplets, which was ∼60% of those of droplets without oil layer. This was probably due to the relatively lower interfacial tension between the interface of the oil phase and aqueous droplets than that of air/water interface. Because the distance between the sampling hole and the emitter tip of the Swan probe was very short (∼1 cm), the dispersion of the injected analyte plug in the probe channel was quite small. We observed the intensities of the MS peak signals of reserpine droplets were almost at the same level as that of continuous perfusion experiment with the Swan probe. The lowest concentration that could be detected was 100 nM (Figure 5b), indicating the detection sensitivity of the Swan probe-based system is comparable to or even better than that of previously reported droplet analysis system.19−21 As a high-throughput analysis system, an essential prerequisite is the good stability in long-term operation of the device. Thus, we assessed the long-term stability and repeatability of the Swan probe by continuously analyzing a 16 × 16 droplet array generated with the same sample (1 μM reserpine). As shown in Figure 5c, all of the 256 droplets were successfully sampled and detected within 90 min, corresponding to an analysis time of 21.1 s per droplet. The relative standard deviation (RSD) of peak height obtained from the extracted ion trace (m/z 609) was 12.6%, demonstrating the high reliability of the present system for high-throughput ESI-MS analysis. In the continuous analysis of 256 droplet samples, no oil leakage into the probe channel was observed, which can be attributed to the high pushing flow rate and the surface tension effect at the hydrophilic surface surrounding the sampling hole. By reducing the sampling interval, we achieved the shortest analysis time of 9.8 s per sample (SI Figure S1). Further increase of the analysis speed led to evident cross talking between adjacently injected sample plugs. Considering the single capillary probe was used to handle different droplets, we also tested the cross contamination of the Swan probe by alternately sampling reserpine droplets (1 μM) and blank droplets (buffer) (SI Figure S2). No detectable reserpine signal could be observed in the extracted ion trace of reserpine (m/z 609), demonstrating the neglectable cross-talking of the Swan probe for droplet analysis. This low cross-contamination could be attributed to the hydrophobic property of the probe’s outer surface and the protection effect of cover oil, which effectively isolated aqueous samples from the probe.28 In addition, if necessary, a washing step could be carried out between the samplings of different samples, as in the section titled Swan Probe ESI-MS System Coupled with Multiwell Plate, to further decrease possible cross-contamination. AchE Inhibitor Screening. The Swan probe was used to screen AchE inhibitors to demonstrate its potential in highthroughput drug discovery. AchE plays pivotal roles in the central and peripheral nervous systems for the control of signal transmission among nerve−nerve and neuromuscular synapses.41 Inhibition of AchE activity can be possible treatments of neurodegenerative disorders, such as Alzheimer’s disease, myasthenia gravis, and related dementia. Thus, screening for new compounds with improved AchE inhibition and pharmacologic properties has gained broad research interest. The AchE assay was on the basis of enzymatic conversation reaction of acetylcholine to choline (Figure 6a). The reaction was performed in 10 mM NH4HCO3 buffer and quenched with 60% methanol aqueous solution containing 2% acetic acid,

Figure 6. Label-free inhibitor screening of acetylcholinesterase (AchE) using the Swan probe-ESI-MS system. (a) Enzymatic conversion reaction of acetylcholine to choline. (b) Typical mass spectra show the formation of choline without (b1) and with (b2) enzyme inhibition by eserine (200 μM). (c) Extracted ion trace (m/z 104.5) shows the formation of choline in 128 droplets for AchE enzyme assay. The 128 droplet samples include 120 sample droplets (30 compounds), 4 control droplets without inhibitor, and 4 blank droplets without enzyme and substrate. (d) Standard curve of choline signal intensity with concentration in the range from 200 μM to 10 mM (n = 8). Choline was dissolved in 50 μg/mL AchE solution in 300 nL droplets. (e) Quantification of choline formed in each droplet using the standard curve (d) (n = 4). (f) Dose−response curves of three AchE inhibitors (n = 4).

which was compatible with ESI-MS. Thus, the final reaction product could be directly detected without further sample preparation. Figure 6b1 shows a typical mass spectrum of unreacted substrate (acetylcholine, m/z at 146.2) and product (choline, m/z at 104.5). By adding a known AchE inhibitor of eserine (200 μM), the signal of choline significantly decreased because of its inhibition effect on the enzyme activity (Figure 6b2). A chemical library containing 3 known AchE inhibitors and 27 randomly picked compounds was used to conduct the 10801

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possible cross contaminations, we added a wash step between the sampling of different samples by immersing the probe into a blank reservoir agitated with a magnetic stirrer for 3−5 s. As shown in SI Figures S3d and S4b, no evident crosscontamination could be observed in the selected ion traces. The small additional peaks for caffeine (m/z 195.7) were produced by the impurities in the carrier buffer. It is also noteworthy that the additional washing steps did not significantly reduce the analysis speed of the system. The total analysis time for the 96 samples was 55 min, which was only 5 min longer than that for the same samples without washing steps. The peak-height RSDs for the 8 samples were 2.9%, 6.1%, 15.6%, 7.5%, 10.7%, 5.0%, 5.0%, and 6.6%, respectively, indicating the good reproducibility of the present system for multiple different samples.

enzyme inhibitor screening. To ensure the reliability of screening results, each compound was tested in quadruplicate; thus, a droplet array of 128 droplets was formed, including 120 sample droplets, 4 control droplets with enzyme and substrate but without compound, and 4 blank droplets without any reagent. Figure 6c shows the mass spectrum of extracted ion trace for choline (m/z 104.5) produced from the 128 droplets. The total analysis time was 52 min, corresponding to an analysis time of 24.4 s per sample. To quantify the choline formed in each droplet reaction, we measured a series of droplets containing choline with different concentrations and plotted a standard curve. As shown in Figure 6d, the signal intensity increases linearly with the choline concentration in the range of 200 μM to 10 mM. The regression equation between signal peak height and concentration is I = 3632 C + 1694 (R2 = 0.999). After the signal intensity was calibrated into choline concentration, as in Figure 6c, the three known AchE inhibitors were successfully identified as expected (Figure 6e). No evident enzyme inhibition effect was observed for the other 27 smallmolecule compounds. Using the screening results, we could also evaluate the repeatability and reliability of the Swan probe ESI-MS system for enzyme inhibitor screening by measuring the Z′ factor using a method described previously.23,28 A Z′factor value over 0.5 is generally considered necessary for a high-throughput screening system. The Z′ values for neostigmine, edrophonium, and eserine were 0.82, 0.69, and 0.88, respectively, demonstrating the high reliability of the present system in enzyme inhibition assay. To characterize the inhibition efficiency of the three identified inhibitors, their dose−response relationships were measured using the present system (Figure 6f). For each inhibitor, 10 different concentrations in the range of 0 nM to 10 mM were tested by incubating them with the same assay mixtures. The IC50 values for neostigmine, edrophonium, and eserine were 69 ± 19 nM, 26 ± 3.7 μM, and 61 ± 17 nM, respectively, using Sigmoidal fitting method.26 These results generally agree well with the values reported previously.23,42 Swan Probe ESI-MS System Coupled with Multiwell Plate. Because 96 or 384-well plate are still the primary workhorse in current high-throughput screening pipelines, we evaluated the capability of the present system for analyzing samples loaded in 96-well plate. First, all 96 wells of the plate were filled with the same sample of 1 μM reserpine in 30% methanol aqueous buffer containing 1% acetic acid and presented for ESI-MS analysis. The sampling time was 1s, and ∼6 nL of each sample was introduced into the probe. As shown in SI Figure S4a, the sample introduction and MS detection for 96 samples were completed in 50 min. The peakheight RSD was calculated as 11.4% (n = 96), demonstrating the high reliability of the present system in long-term and highthroughput multiwell plate-based analysis. Further improving the reproducibility could be achieved by adding an internal standard in each well to calibrate the variation of the peak height.40 Next, 8 different samples were filled into 8 rows of a 96-well plate, respectively, and then were analyzed column by column, to demonstrate the ability of the system in measuring multiple different samples (SI Figures S4b and Figure S5). We first tested the cross contamination of the probe in MWP system by alternately sampling reserpine (1 μM) and blank (buffer) solutions in the wells (Figure S3). An evident carryover to the blank solution was observed (Figure S3b) as a result of the lack of a protection effect by the oil layer to the probe. To eliminate

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CONCLUSIONS In summary, we have demonstrated the Swan probe could be a promising sampling interface for high-throughput ESI-MS analysis. It is not only compatible with conventional multiwell plate-based screening systems but also can be directly coupled to miniaturized reaction systems, such as the nanoliter droplet array in this work. High-quality screening data obtained from the AchE inhibitor screening assay proves the present system may provide an effective label-free and cost-efficient tool for the next generation of high-throughput screening (HTS) of drug candidates. In addition to HTS, we envision the Swan probe could find broad applications in the fields requiring smallamount sampling, such as single cell analysis, in vivo analysis, and studies of intercellular communications. In addition, the Swan probe could also be used for MS imaging of solid surfaces and tissue sections using a sampling principle similar to NanoDESI.32 Although the Swan probe was mainly designed to couple ESI-MS with static sample systems, such as droplet array chips and microwell plates, if combining with the eluent segmenting and collecting technique,43 it also has the possibility to be applied in the analysis of a flow system by first segmenting and depositing the eluent of the flow system on a chip to form a static droplet array and then analyzing the droplets individually. The Swan probe technique could be further developed from probe fabrication to function integration. To improve the structure and performance reproducibility of different probes, the Swan probe could be produced from microfluidic chips instead of capillaries using more automated and accurate microfabrication techniques. In this study, enzyme reaction products could be directly analyzed due to the use of MScompatible buffer and solvent. The analytical performance and scope of the Swan probe could be enhanced by integrating sample pretreatment functions before MS detection, such as liquid−liquid extraction, solid-phase extraction, preconcentration with electrostacking, capillary electrophoresis, and capillary liquid chromatography.

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-571-88206771. Fax: +86-571-88273572. E-mail: [email protected]. 10802

dx.doi.org/10.1021/ac503014k | Anal. Chem. 2014, 86, 10796−10803

Analytical Chemistry

Article

Author Contributions

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§

Di-Qiong Jin and Ying Zhu contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Natural Science Foundation of China (Grants 21105089 and 21227007) and Major National Science and Technology Programs (Grant 2013ZX09507005) are gratefully acknowledged.

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