Manipulating Femtoliter to Picoliter Droplets by Pins for Single Cell

Apr 12, 2018 - Manipulating Femtoliter to Picoliter Droplets by Pins for Single Cell Analysis ... Institute of Microanalytical Systems, Department of ...
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Manipulating Femtoliter to Picoliter Droplets by Pins for Single Cell Analysis and Quantitative Biological Assay Xiao-Li Guo, Yan Wei, Qi Lou, Ying Zhu, and Qun Fang* Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, 310058, China S Supporting Information *

ABSTRACT: Herein, we developed an automated and flexible system for performing miniaturized liquid−liquid reactions and assays in the femtoliter to picoliter range, by combining the contact printing and the droplet-based microfluidics techniques. The system mainly consisted of solid pins and an oil-covered hydrophilic micropillar array chip fixed on an automated x-y-z translation stage. A novel droplet manipulation mode called “dipping-depositing-moving” (DDM) was proposed, which was based on the programmable combination of three basic operations, dipping liquids and depositing liquids with the solid pins and moving the two-dimensional oil-covered hydrophilic pillar microchip. With the DDM mode, flexible generation and manipulation of small droplets with volumes down to 179 fL could be achieved. For overcoming the scale phenomenon specially appeared in picoliter-scale droplets, we used a design of water moat to protect the femtoliter to picoliter droplets from volume loss through the cover oil during the droplet generation, manipulation, reaction and assay processes. Moreover, we also developed a precise quantitative method, quantitative droplet dilution method, to accurately measure the volumes of femtoliter to picoliter droplets. To demonstrate its feasibility and adaptability, we applied the present system in the determination of kinetics parameter for matrix metalloproteinases (MMP-9) in 1.81 pL reactors and the measurement the activity of β-galactosidase in single cells (HepG2 cells) in picoliter droplet array. The ultrasmall volumes of the droplet reactors avoided the excessive dilution to the reaction solutions and enabled the highly sensitive measurement of enzyme activity in the single cell level. from 50 to 300 μm, corresponding to spot volumes ranging from picoliters to nanoliters. The nanotip printing method commonly uses an atomic force microscopy (AFM) probe7 or other nanotips8 as the printing devices, known as dip-pen lithography (DPN).7 The DPN method is capable of fabricating microspots with the sizes from 15 nm to 10 μm, 9 corresponding to spot volumes in the range from subfemtoliters to picoliters. The microstamping method10 usually is adopted to form a microspot array of one reagent in parallel using an elastomeric stamp array as the printing device. According to the standard protocol used in most of microarray chip-based assays, the reagent microspots are first predeposited on the chip surface by the printing devices under a controlled ambient humidity condition and allowed to be dried. Then the sample solution is loaded on the chip to allow the analyte to react with the reagent ligands (such as oligonucleotides or antibodies) immobilized in the microspots on the chip surface to complete the biological assays.11 However, such a liquid−solid reaction

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iniaturization of assay reactors continues to be a major trend in the field of analytical and bioanalytical chemistry.1 With the development of various miniaturization technologies, the volumes of microreactors have been decreased from conventional microliter scale to picoliter− nanoliter scale,2 or even to femtoliter scale.3 The decrease of microreactor volumes can not only significantly reduce the consumption of samples and reagents, but also depress the dilution of samples to achieve highly sensitive assays, such as single cell analysis or single molecular analysis. In addition, the reaction time could also be shortened from hours to seconds or less due to the short diffusion distances and high surface to volume ratios within the microreactors.4 Currently, two major techniques, microarray chips and microfluidic chips, are frequently used in the generation and manipulation of microreactors. Among the different microarray chip techniques, contact printing technique5,6 is the most widely used one by which pins,5 nanotips,7−9 or microstamps10 are commonly used as printing devices to print microspots (microreactors) of reagents on the chip surface, mainly based on the physical contact. The contact pin printing method5 usually can form a microspot array with the spot sizes ranging © XXXX American Chemical Society

Received: January 22, 2018 Accepted: April 9, 2018

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DOI: 10.1021/acs.analchem.8b00343 Anal. Chem. XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION Setup of the System. The system is composed of three major parts: solid pins for generating and manipulating droplets, a glass microchip with hydrophilic pillar array for loading droplets of reagents and samples (Figure 1), and an

mode may limit its application in liquid−liquid reaction systems, which are frequently adopted in many biological assays. Some researchers have tried to complete biological assays under the liquid−liquid reaction mode on the biochip by using a low volatile liquid, glycerol, as the solvent for reagents and samples to minimize the evaporation of microspot reactors.12 While the use of highly viscous glycerol also resulted in the application limit in most biological reactions and assays. Microfluidic techniques have evolved as an efficient way for manipulating microreactors ranging from nanoliters to femtoliters by using various microchips with micropumps, microvalves, microwells, or other control modules. Among these, droplet-based microfluidics using small droplets in immiscible liquid as microreactors is one of the promising techniques to miniaturize the microreactor volume for chemical and biological assays.13 Various droplet-based microfluidic techniques have been developed to manipulate the droplets ranging from femtoliters to nanoliters, and these techniques mainly include continuous flow-based droplet systems, inkjet printing-based droplet systems, and two dimentional (2D) static droplet array systems. In the continuous flow-based systems, femtoliter to nanoliter scale droplets can be generated by merging an aqueous stream with immiscible streams with a T-junction14,15 or a flow-focusing microchannel.16,17 The sample and reagent are premixed before droplet formation or postmixed after droplets formation with additional droplet fusion inducing devices.18 The inkjet printing-based systems usually use piezoelectric or thermal inkjet printing methods19,20 to continuously form droplets in the range of femtoliters to picoliters. In 2D droplet array systems, microchips with microwell21 and micropatterning22−24 array are commonly used to generate small droplets in the femtoliter to picoliter range by mainly utilizing the microwell volume or the hydrophilic pattern size to control droplet volumes. These 2D droplet array systems have been successfully applied in single cell analysis and single molecular assay, while adding reagents into the generated droplets still presents challenges in these systems. In 2013, the authors’ group developed a sequential operation droplet array (SODA) system,25 which used the programmable combination of the capillary-based liquid aspirating−depositing operation and the moving of the oil-covered 2D droplet array to manipulate picoliter to nanoliter-scale droplets, including droplet generation, assembling, transferring, splitting, and fusion. This technique has been applied in enzyme reaction, protein crystallization,26,27 gene analysis,28 and single cell analysis.29 However, due to the limitation of the syringe pump precision, the volumes of droplets capable of being manipulated by these SODA systems were in the picoliter to nanoliter range with a smallest droplet size of 60 pL. Herein, combining the contact printing and the dropletbased microfluidics techniques, we established a solid pin-based droplet system for performing automated and flexible miniaturized liquid−liquid reactions and assays in the range of femtoliters to nanoliters. A novel droplet manipulating approach with femtoliter-scale precision was developed using the programmable combination of the solid pin-based liquid dipping-depositing operation and the moving of the oil-covered 2D droplet array. To demonstrate the potential and versatility of this droplet system in biological assays, we utilized it in the measurement of kinetic parameters for matrix metalloproteinase-9 in picoliter droplets and the enzymatic activity assay in single cells.

Figure 1. Setup of the solid pin-based droplet system. (a) Schematic diagram of the system setup. (b) Photographs of a micropillar chip (left) and a droplet array with different dye droplets (right) formed on hydrophilic pillars with a pillar diameter of 50 μm, surrounded by a water moat.

automated x-y-z translation stage (Zolix, 3 μm repositioning accuracy, Beijing, China) on which the microchip was fixed. The x-y-z translation stage was controlled by a LabVIEW program (LabVIEW 8.0, National Instruments, TX). A stereomicroscope (SMZ 850T, Touptek, Hangzhou, China) was used to assist in observing droplet generation and manipulation. An inverted fluorescence microscope (ECLIPSE TE-2000-S, Nikon, Tokyo, Japan) coupled with a CCD camera (SPOT RT-SE6, Diagnostic Instruments, Sterling Heights, MI) was used in the observation of single cells and measurement of fluorescent intensities of droplets. For the fabrication of the solid pins, first, commercial acupuncture needles made of stainless steel (Beijing Keyuanda Co., Beijing, China) were treated with a commercial agent (Aquapel, PPG Industries, Inc. PA) to obtain hydrophobic surface for whole needles. Then the tips of the needles were perpendicularly polished on a sandpaper (7000#) to obtain appropriate pin tip diameters in the range of 5 to 500 μm, with a hydrophilic cross section on the pin tip and hydrophobic lateral surface for each pin. A digital microscope (Aigo GE-5, Huaqi Co., Nanjing, China) was used to meter the tip diameters of pins. The microchips with hydrophilic pillar array were fabricated using standard photolithography and wet etching technique based on glass substrates with chromium film (SG2506WC, Shaoguang Co., Changsha, China) as described previously.30 First, a pillar array chip with chromium film remained on the top of each pillar was produced. The sizes of pillars for loading assay droplets were 3 μm in height and 50 to 250 μm in B

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Analytical Chemistry diameter, while 3 μm in height and 1000 μm in diameter for loading stock droplets of samples and reagents. Then, the whole surface of the microchip was silanized with 1% OTCS in isooctane (v/v) to obtain hydrophobic surface. After the chromium films on the pillars were removed, the microchip with hydrophilic top pillar surface and hydrophobic surface for the other part of the chip was obtained. Finally, a 2 mm thick glass frame was glued on the microchip surrounding the hydrophilic pillar array for containing the cover oil and coupling with the cover glass plate. Procedures. Droplet Generation and Manipulation. Before experiment, the whole microchip was covered with 200 μL of mineral oil presaturated with water, then 500 nL of sample and reagent solutions were pipetted onto the corresponding hydrophilic pillars (1000 μm in diameter) as sample and reagent stock droplets, respectively. When the pillar diameters smaller than 50 μm were used, 10 μL of water or buffer solution was required to be loaded on the hydrophilic region surrounding the hydrophilic pillar array. The sample droplet for assay was first generated by moving the x-y-z stage to allow the pin tip to be dipped into the preloaded sample stock droplet and then removed from it to form a droplet on the pin tip end by its surface hydrophilic effect (Figure 2a). The sample droplet on the pin tip could be used to further generate assay droplets with smaller volumes on the chip hydrophilic pillars, by moving the x-y-z stage to make the droplet on the pin contact with the hydrophilic pillar and then remove it (Figure 2b), leaving part of the droplet deposited on the pillar to form a smaller sample droplet. To add reagent into the sample droplet on the hydrophilic pillar, a pin with smaller tip diameter was first dipped into the reagent stock droplet and remove it and then inserted into the sample droplet by using the movement of the x-y-z stage (Figure 2c). After adding the reagent solution, the pin tip was washed by immersing it into a 1 μL droplet of washing solution before handling a new sample droplet. During the whole liquid handling process, the droplet on the pin tip was immersed in the oil phase to avoid droplet evaporation. Measurement of Droplet Volume. To accurately measure the volumes of droplets formed in the present system with the range from femtoliter to picoliter, sodium fluorescein (1 mM) was used as model sample and sodium borate solution (50 mM) as buffer. The quantitative dilution method was used in the droplet volume measurement, by which the sample droplet on the pin tip end or the hydrophilic pillar was first mixed with a much larger buffer droplet (10 nL) generated by using a capillary probe connected with a picoliter-scale precision syringe pump (PHD 2000, Harvard Apparatus, Holliston, MA). The fluorescence intensities of the mixed droplet as well as a series of 10 nL standard droplets with different fluorescein concentrations in the range of 10−8 M to 10−3 M were measured with the fluorescence microscope. Thus, the concentrations of fluorescein in the mixed droplets could be calculated using a calibration curve obtained from the serial concentration droplets. According to the dilution law, the accurate volume of sample droplet could be calculated with eq 1. Vs =

Cm × Vm C0

Figure 2. Working principles of droplet generation on a solid pin (a) and a hydrophilic pillar (b), and reagent addition (c). Both the schematic diagrams (a1), (b1), and (c1) and the actual microscope images (a2), (b2), and (c2) are used to show the working processes of the droplet generation and reagent addition.

droplet volume Vs in the range of 100 fL to 500 pL was much smaller than the 10 nL buffer droplet, its influence to the mixed droplet volume Vm could be omitted, that is, Vm was equal to 10 nL. When the sample droplet volume was larger than 1 nL, 50 nL or more than 50 nL of buffer droplet was used. The Cm value was obtained with the calibration curve of in-droplet fluorescein concentration vs corresponding fluorescence intensity. Then, the value of Vs could be calculated by eq 1. Determination of In-Droplet Enzyme Kinetic Parameters. Matrix metalloproteinase-9 (MMP-9) and MMP substrate III (QXLTM520, Pro-Leu-Gly-Cys-(Me)-His-Ala-D-Arg-Lys-(5carboxyfluorescein)NH2) were used in the measurement of the in-droplet kinetic parameter of MMP-9. MMP-9 droplets

(1)

where C0 and Vs are the concentration and volume of the sample droplet, and Cm and Vm are the concentration and volume of the mixed droplet, respectively. As the sample C

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fluorescein droplets on hydrophilic pillars with a diameter of 50 μm decreased to 10% after a 20 min incubation period at 37 °C (Figure 3a), even with the protection of oil saturated by water

were first generated on hydrophilic pillars with a diameter of 50 μm by a pin with a tip diameter of 150 μm, then MMP substrate III were added into the MMP-9 droplets by a pin with a tip diameter of 40 μm. During this process, the microchip was kept at 15 °C to depress the enzyme reaction before incubation. After the droplet generation was completed, the droplet array was incubated at 37 °C to trigger the enzyme reaction for 15 min and detected in real-time with the fluorescence microscope. Single Cell Enzymatic Activity Assay. The activity of βgalactosidase in single human hepatocellular carcinoma cell (HepG2) was measured using fluorescein di-β-D-galactopyranoside (FDG) as substrate. Cells were dispersed in PBS with the density of 1 × 107 cells/mL, and FDG was diluted with PBS, which was premixed into Triton X-100. Droplets of 1 × 107 cells/mL cell suspension was first generated on a pin tip with a diameter of 150 μm, then part of the cell suspension droplet was deposited on a hydrophilic pillar with a diameter of 50 μm to form a single cell droplet on the pillar. Next, the substrate solution with 0.5% Triton X-100 as cell lysis agent was added into the single cell droplet by a pin with a tip diameter of 40 μm to trigger the single cell enzyme reaction. Then, the single cell droplet array was incubated at 37 °C for 30 min and detected in real-time with the fluorescence microscope.

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RESULTS AND DISCUSSION Design of the System. The main objective of this work was to develop a flexible and automated platform for precise manipulation of ultrasmall droplet in the picoliter to femtoliter range to perform the microscale biological assays. In our previous work, we developed the SODA system which can reliably and flexibly manipulate the droplets ranging from picoliter to nanoliter by combining the capillary-based liquid aspirating-depositing operation and the moving of the oilcovered 2D droplet array under the aspirating-depositingmoving (ADM) mode.25 Differing from the SODA systems, we used a solid pin instead of the capillary-syringe pump module to handle the droplets, based on the programmable combination of the solid pin-based liquid dipping-depositing operation and the moving of the oil-covered 2D hydrophilic pillar-based droplet array, namely, “dipping-depositing-moving” (DDM) mode. The volume of the droplets generated under the DDM mode was mainly depended on the hydrophilic cross sectional area of the pin tip or the pillar, so femtoliter to nanoliter droplets could be readily generated by adjusting the sizes of the pin tip or the hydrophilic pillar in the range of 3 to 500 μm. However, in the SODA systems, the precision in liquid handling was in the picoliter scale due to the precision limit of the currently used commercial syringe pumps. It should be noted that the droplets generated under the DDM mode is different from the contact pin printing mode5,6 despite the similar liquid contact method is used in both modes, as the droplets in our system was stored in the oil phase during the whole droplet formation and manipulation process, which enables the possibility of achieving miniaturized liquid− liquid reactions and biological assays with volumes as low as femtoliter scale. During the experiments, we observed evident scale effect that when the volumes of droplets were reduced to femtoliter scale, the droplets exhibited obvious volume decrease during the incubation period under the observation of the microscope in the bright field mode. When quantitatively observing under the fluorescence mode, the fluorescence intensities of 1 mM

Figure 3. Droplet preservation experiments without additional measurement (a), with one side water moat (b) and surrounded water moat (c). Conditions: droplet volume, 6 pL; diameter of hydrophilic pillar, 50 μm; gap between pillars, 200 μm; incubation temperature, 37 °C. The mineral oil saturated with water and coverglass were used to protect the droplet array in all experiments. (a1), (b1), and (c1) show the schematic diagrams of the droplet arrays and typical fluorescence images of a droplet formed on a hydrophilic pillar in different incubation times. (a2) and (c2) show the relationships of the integrated fluorescence intensity of 25 droplets in the droplet array and the incubation time. (b2) shows the relationships of the integrated fluorescence intensity of 5 droplets in one column in the droplet array with different distance to the water moat and the incubation time.

and an additional glass cover plate. This phenomenon could be attributed to the evaporation and dissolution of the water in the microdroplets through and in the relatively large-volume oil. The water loss in the droplets led to the excessive increase of the in-droplet fluorescein concentrations, resulting in the decrease of droplet fluorescence intensities. To further verify this presumption, we first tried to use an additional one-sided water zone to protect these small droplets. The results showed that the fluorescence intensity of the droplets closer to the onesided water zone decreased less than the other droplets under the same incubation conditions (Figure 3b). Inspired by this phenomenon, we designed a water moat closely surrounding the array of small-volume droplets to protect the droplets (Figure 1b), and the results are shown in Figure 3c. The variations in droplets fluorescence intensities were only 3% in 4 h at 37 °C, using a water moat with an inner size of 1 mm × 1 mm. These droplets could be stably preserved over 48 h, which demonstrated the effectiveness of the water moat in microdroplet protection. D

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Figure 4. Relationships of the droplet volume and the solid pin diameter (a), and the droplet volume and the hydrophilic pillar diameter (b) in droplet generation. Conditions: removal velocity of solid pin, 0.5 mm/s; diameter of solid pin used to measure the droplet volume on the hydrophilic pillars, 350 μm.

speed (Figure S1a). Thus, a relative low removing speed is favorable for obtaining small droplet volume. With a fixed removing speed (e.g., 0.5 mm/s), the diameter of the solid pin tip became the main influencing factor on the droplet volume. As shown in Figure 4a, with the pin tip diameters ranging from 3 to 500 μm, the droplet volume increased with the pin tip diameter. The smallest droplet of 179 fL was obtained with a 3 μm tip diameter, and the largest one of 7.94 nL with 500 μm tip diameter, spanning 5 orders of magnitude of droplet volume from femtoliter to nanoliter range. The repeatability of droplet formation by the pin tips with tip diameters ranging from 55 to 500 μm were in the range of 1.5% to 8.0% (relative standard deviation, RSD, n = 12), while RSDs ranging from 15.7% to 19.4% (n = 12) were obtained when the pin tip diameters were less than 10 μm. In the present work, on the basis of the primary droplet formed on a pin tip, we also used another droplet formation method to generate secondary droplet by depositing part of the primary droplet on the pin tip on a hydrophilic pillar on the chip. We studied the effects of removing speed of the pin tip from the hydrophilic pillar on the volume of the secondary droplet. The results showed that the volumes of secondary droplets on the hydrophilic pillars decreased with the removing speed (Figure S1b). A removing speed of 0.5 mm/s was adopted in the subsequent experiments. The relationship between the hydrophilic pillar diameter and sample droplet volume on the pillar exhibited similar trend as the pin tip-based method (Figure 4b). When the hydrophilic pillars with diameters of 50, 100, 150, 200, and 250 μm were used, the corresponding droplet volumes were 5.76 pL, 68.0 pL, 250 pL, 649 pL, and 1.18 nL, respectively, with RSDs of droplet volumes in the range of 2.1−7.2% (n = 12). We further tried to use double logarithmic curves to fit the above data. Good linear relationship between the logarithms of

Another major challenge in the present droplet system lied in the accurate measurement of the volumes of femtoliter to picoliter droplets, as the microscope imaging method currently used in many droplet systems could not be used to accurately estimate the volume of such small droplets. For accurate measurement of these droplets with extremely small volumes, we developed the precise quantitative droplet dilution method, with the aid of the ability of the SODA system in forming droplets with accurate volumes in the tens to hundreds nanoliters25 using the picoliter-scale syringe pump. This method overcomes the difficulty in direct and precise volume measurement of small droplets in the range of femtoliter to picoliter, by first quantitatively diluting the small droplet in a larger nanoliter-scale droplet with known volume, and then measuring the fluorescence intensity (proportional to the fluorescein concentration) of the large droplet by microscopic imaging to calculate the volume of the small droplet. In the fluorescence measurement, we added an antioxidant, propyl gallate, into the sodium fluorescein solution with a final concentration of 0.5% to avoid the photobleaching. The validity of this method was demonstrated by the result that, with the irradiation time of excitation light of 10 s, the decreases of the fluorescence intensities of 10 nL sodium fluorescein droplets (10−6 M) were 19.4% and 1.0% without and with the addition of propyl gallate, respectively. As shown in Figure 4, the quantitative droplet dilution method could measure droplet volumes as small as 179 fL, which provides an effective method for the measurement of small droplet volumes in the femtoliter to picoliter range. Generation and Manipulation of Droplets. We first studied the effects of removing speed of the solid pin from the sample solution on the droplet volume using sodium fluorescein as model sample. The results showed that the volume of droplet on the solid pin increased with the removing E

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Figure 5. Determination of kinetic parameter for enzyme MMP-9. (a) Standard curve of the droplet fluorescent intensity vs FAM concentration. (b) Typical images of the droplet microreactors with a fixed MMP-9 concentration of 50 nM and different concentrations of MMP substrate III in different reaction times. (c) Variations of fluorescence intensity of droplet reactors with different concentrations of MMP substrate III during the enzymatic reaction; (d) Lineweaver−Burk plots of MMP-9 obtained in the measurement. Conditions: concentrations of MMP substrate III, 10, 20, 40, and 50 μM; diameter of the solid pin for generating MMP-9 droplets: 150 μm; diameter of the solid pin for adding MMP substrate III into the generated MMP-9 droplets: 40 μm; removing speed of the solid pin: 0.5 mm/s; incubation temperature: 37 °C.

maximal activity) value of MMP-9 was 42.08 ± 6.33 μM (n = 3), which agrees with that reported previously.31 Single-Cell Enzymatic Assay. We applied the DDM method to form droplets containing single cells and determine the enzymatic activity of β-galactosidase in single HepG2 cells. For forming single cell droplets, a cell suspension with the density of 1 × 107 cells/mL was used to first generate a cell suspension droplet on the tip of a solid pin with a tip diameter of 150 μm. The cells in the droplet would rapidly sink to the bottom of the droplet, which significantly increased the cell density at the droplet bottom. When this cell suspension droplet first contacted with a hydrophilic pillar with a diameter of 50 μm and then removed from the pillar, approximately 6 pL of cell suspension at the bottom of droplet remained on the pillar, forming a droplet with a single cell capture probability of about 60%. The relatively higher capture probability for single cells than other methods based on Possion distribution32 could be attributed to the relatively high cell density at the bottom of the droplet which increased the probability of the cells contacting with the hydrophilic pillar, as well as the use of the hydrophilic pillars with a diameter (50 μm) close to the cell sizes (20−30 μm) which facilitated the capture of single cells. After the single cell droplets were formed on hydrophilic pillars, FDG solution with Triton X-100 was added into each single cell droplet to trigger the reaction, meanwhile the fluorescence intensities of the droplets were recorded by a microscope at a 5 min interval. The typical results of droplets containing zero, one, and two cells, as well as the recordings of the droplet fluorescence intensity during the enzyme-catalyzed reaction process, are as shown in Figure 6a,b. The fluorescence

droplet volume (lg V) and pin or pillar diameter (lg D) were observed for both the pin tip-based and the pillar-based methods with linear correlation coefficients of 0.989 and 0.997, respectively. Determination of Kinetic Parameters in Picoliter Droplets. We applied the present droplet system in determination of kinetic parameters for MMP-9 in 1.81 pL droplets to demonstrate its ability in performing enzyme catalytic assay. First, the droplet volumes of MMP-9 and MMP substrate III on 50 μm hydrophilic pillars were accurately determined by using the droplet dilution method and the enzyme reaction buffer, and the obtained droplet volume of the enzyme and substrate were 1.81 ± 0.10 pL and 0.75 ± 0.08 pL (n = 6), respectively. Then, for calibrating the velocity characterization of the enzymatic reactions of MMP-9 with MMP substrate III, a series of droplets with different concentrations of 5-carboxyfluorescein (FAM, product of enzymatic reaction) on the 50 μm hydrophilic pillars were formed and measured to obtain a standard curve (Figure 5a). To determine the kinetic parameters for MMP-9, we varied the MMP substrate III concentrations as 10, 20, 40, and 50 μM by keeping the enzyme concentration fixed at 50 nM. The fluorescent intensities of the enzymatic reaction droplets were continuously detected with an interval of 2 min for a period of 10 min (Figure 5b,c). The initial velocity of the reaction in each droplet could be calculated from the results. To determine the kinetic parameters, the initial velocities (1/V) and substrate concentrations (1/C) were plotted as a Lineweaver−Burk plot (Figure 5d), then the Y-intercept (1/Vmax) and X-intercept (1/ KM) values were calculated by linear fitting. The KM (halfF

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the DDM mode could eliminate the limitation of picoliter-scale precision by the currently used capillary-syringe pump modules25 and achieve flexible droplet manipulation with femtoliter precision. In addition, we also used the water moat design to protect the femtoliter to nanoliter droplets from volume loss through the cover oil during the processes of droplet generation and manipulation as well as the prolonged in-droplet liquid−liquid reactions, which belongs to a special scale phenomenon that only obviously appeared when droplet volumes decreased to several picoliters. In the present work, we also developed an accurate quantitative method, quantitative droplet dilution method, for the measurement of volumes of femtoliter to picoliter droplets. Such a method could be adopted in the volume measurement of other types of small droplets when imaging methods cannot achieve the accurate measurement. We applied the present system in the determination of kinetics parameter for MMP-9 and the high sensitive measurement of enzyme activity in single cells, which demonstrated its feasibility and adaptability to different types of assays. The picoliter-scale reactor volumes and flexible operation make it particularly suitable for high sensitive assays in the single cell and single molecule level or for mimicking incell biological microreactions. In the present system, a single solid pin was used to handle the droplets, the throughput of droplet generation and manipulation may be limited. If higher throughput or larger assay number are required, an array of multiple solid pins instead of a single pin, such as 8 and 12 pins, or even 96 pins, could be adopted to handle multiple droplets in parallel.

Figure 6. Assay for enzymatic activity of β-galactosidase in single HepG2 cells. (a) Typical microscope images of droplets formed on hydrophilic pillars with a diameter of 50 μm encapsulating one single cell, two cells, and zero cell. (b) Recordings of fluorescence intensities of droplets encapsulating one single cell and two cells during the enzymatic reaction process. Conditions: cell suspension density, 1 × 107 cells/mL; diameter of the solid pin for capturing cells, 150 μm; diameter of the solid pin for adding FDG (200 μM, premixed with 0.5% Triton X-100) into the generated single cell droplet, 40 μm; other conditions are the same as in Figure 5.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00343. Chemicals and materials and supporting figures (PDF).

intensities of the single cell droplets increased quickly with the time, while the blank droplets showed no obvious fluorescence signal increase. It was noted that the intracellular βgalactosidase activity of single cells exhibited a large degree of heterogeneity due to the individual difference of cells. For comparison, we also performed the enzymatic activity assay for β-galactosidase in single cell level using droplet microreactors with volume of 4 nL which is 660 times larger than the present picoliter-scale reactors. The fluorescence intensities of these 4 nL droplets containing single cells showed no obvious change in 3 h, demonstrated that the small volume droplet reactors could effectively avoid the excessive dilution to the reaction solution and enable the high sensitive measurement of enzyme activity in single cell level.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Qun Fang: 0000-0002-6250-252X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate the financial supports from Natural Science Foundation of China (Grants 21435004 and 21227007).

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CONCLUSIONS In summary, we coupled the contact printing technique with the droplet-based microfluidic system to establish an automated and flexible platform for the precise manipulation of droplets in the picoliter to femotoliter range to perform microscale liquid− liquid reactions and biological assays. In this system, solid pins and a movable oil-covered 2D hydrophilic pillar microchip were used to handle droplets, which provides an effective and flexible droplet manipulation mode, dipping-depositing-moving (DDM), for small droplets with volumes down to femtoliter scale. Since the droplet volumes were mainly determined by the hydrophilic cross sectional areas of the pin tips or the pillars,

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REFERENCES

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DOI: 10.1021/acs.analchem.8b00343 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.8b00343 Anal. Chem. XXXX, XXX, XXX−XXX