Ultrasensitive Single-Molecule Enzyme Detection and Analysis Using

Feb 9, 2018 - Using a simple and robust loading method to introduce the reaction solution, individual enzyme molecules are trapped in picoliter microw...
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Ultra-sensitive single molecule enzyme detection and analysis using a polymer microarray Barrett K. Duan, Peter E Cavanagh, Xiang Li, and David R. Walt Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03980 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 10, 2018

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Analytical Chemistry

Ultra-sensitive single molecule enzyme detection and analysis using a polymer microarray Barrett K. Duan, Peter E. Cavanagh, Xiang Li and David R. Walt

Department of Pathology Harvard Medical School Brigham and Women’s Hospital Wyss Institute for Biologically Inspired Engineering Building for Transformative Medicine, 60 Fenwood Road, Boston, MA 02115, U.S.

Corresponding Author: David R. Walt Office: 1-857-307-1112 Email: [email protected]

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Abstract

This report describes a novel method for isolating and detecting individual enzyme molecules using polymer arrays of picoliter microwells. A fluidic flow-cell device containing an array of microwells is fabricated in cyclic olefin polymer (COP). The use of COP microwell arrays simplifies experiments by eliminating extensive device preparation and surface functionalization that are common in other microwell array formats. Using a simple and robust loading method to introduce the reaction solution, individual enzyme molecules are trapped in picoliter microwells and subsequently isolated and sealed by fluorinated oil. The sealing is stable for hours in the COP device. The picoliter microwell device can measure enzyme concentrations in the low femtomolar range by counting the number of active wells using a digital read-out. These picoliter microwell arrays can also easily be regenerated and reused.

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Introduction Recent advances in microscopy and microfabrication techniques allow research scientists to perform highly sensitive measurements, drastically improving detection limits.1-6 These techniques enable scientists to measure and observe individual molecules, providing insight that cannot be gleaned from traditional ensemble

measurements.

Traditional

biochemical

measurements,

such

as

immunoassays and enzyme activity assays, only yield information about the average behavior of a large number of molecules. Such ensemble measurements mask the significant variation between individual molecules revealed in recent studies involving enzymology,7-11 protein transport,12,13 molecular interactions and conformational changes,1,14,15 and redox reactions.2,16,17 Droplet-based microfluidics are an emerging tool for single molecule analysis. Biomolecules, such as enzymes, are encapsulated in femtoliter to nanoliter-sized water-in-oil droplets.18-21 Droplets can be generated and analyzed in high throughput21,22 but the stability of the droplets requires surfactants to prevent droplets from coalescing and exchanging their contents.20,22 However, surfactants may also interfere with some biological assays.22 The surface of the microfluidic device in which the droplets are prepared often requires modification to minimize interactions with the droplets.21 Microwell arrays are an alternative to droplets and provide a versatile platform for confining individual molecules in small volumes. Similar to dropletbased microfluidics, microwell arrays allow a large number of reactions to be

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performed in parallel and observed simultaneously at the single-molecule or singlecell level. Microwell arrays provide a physical compartment to capture and isolate individual molecules. They have been used to detect and study biomolecules and biological

systems

with

high

sensitivity,

including

RNA

and

DNA,1,23-25

proteins8,10,11,13 and cells.26-32 Our group has previously developed a reliable single molecule technique using an optical fiber glass bundle to generate femtoliter-sized microwell arrays to detect proteins and nucleic acids,33-35 to enable the study of enzyme and nanocatalyst kinetics8,10,11,36,37 and to detect protein biomarkers.38-40 Experiments using this optical fiber array employ Poisson statistics to describe the distribution of molecules into wells at very dilute concentrations. For example, filling an array that contains 10,000 (3.5pL) wells with a 5fM concentration of protein results in about 1% (or 100) of the wells containing an individually trapped protein molecule. Therefore, an array with a large number of wells can capture, isolate and study a very small number of individual molecules even at low concentrations. In this paper, we describe the design of a high-density array that uses easily accessible materials, making it a good alternative when compared to many other microwell array formats. The use of this array simplifies experimental protocols, allows for design flexibility, and decreases detection limits when compared to the optical fiber glass femtolitersized well arrays. Cyclic olefin polymer (COP) is an emerging polymeric material used for labon-a-chip applications and has been demonstrated for microfluidic devices.41-43 COP possesses a number of properties that make it a desirable material for microarray

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Analytical Chemistry

fabrication. COP has low water absorption and is highly resistant to chemicals including acids, alkaline reagents, polar solvents (acetone, ethanol, methanol), and reactive

chemicals

(hydrogen

peroxide,

ethylene

oxide).44,45

Similar

to

polydimethylsiloxane (PDMS) and fused silica, COP has a high optical transparency from the near UV to the near-IR regions and has low autofluorescence.46 COP is also biocompatible and has been used with various biological materials.46-50 Significantly, COP devices can be fabricated by injection molding, providing low cost and high volume production of precise microstructures.51,52 Taking advantage of the properties of COP, Kan and coworkers fabricated a COP femtoliter well array that was capable of detecting individual protein molecules captured on antibody-coated paramagnetic beads.53 The beads were loaded and isolated in 4.5μm diameter wells. The beads that contained a single enzyme-labeled immunocomplex generated fluorescent signals in the microwells, which were monitored with a fluorescence microscope. Thus, the COP femtoliter well array was used to perform digital ELISA read-outs of single protein molecules; however, their results show that less than 50% of the wells contained a bead. The combination of small femtoliter microwell sizes and the hydrophobic surface of the device creates a challenge for loading aqueous solutions. Here, we present the advantages of using a COP array with picoliter microwells as compared to using optical fiber arrays or femtoliter microwells. The larger microwells provide a number of experimental advantages. First, an array of large microwells is easier to focus and image for fluorescence analysis because larger wells project onto more pixels on the detector. More pixels for each well can

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minimize the effect of pixel-to-pixel noise variations and the non-uniform pixel response in the camera.54,55 Larger microwells can also eliminate the use of more expensive high-powered objectives. Second, the surface-to-volume ratio of larger microwells is lower, which minimizes the interaction between the well surface and the target molecule.56 Third, according to Poisson statistics, a microwell array with larger wells can analyze a lower concentration of the target molecule than a microwell array with smaller wells when both arrays have the same array area and their center-to-center spacing : well diameter ratio is equal.36,57 This aspect allows us to improve the detection limit. In the work reported here, we exploit these advantages and demonstrate that we can eliminate surface functionalization and simplify experimental protocols using the large-well COP microarray for single molecule experiments. Briefly, a simple loading method was used to introduce freein-solution single enzyme molecules into individual microwells.58 By using a large number of picoliter wells combined with oil sealing to isolate individual enzyme molecules, a lower enzyme concentration can be monitored and quantified compared to previous methods.

Results and Discussions

Device design The design of the COP microarray chip is similar to the bead-based digital ELISA read-out platform.53 In both microarray chips, the area of the microwell array, the height and width of the channel, and the size of the inlet and outlet are identical.

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Analytical Chemistry

The microarray chip is composed of two bonded polymeric compartments: one contains the microarray and the other contains the fluidic channel. The microarray chip used in this report is designed for use in an upright microscope as illustrated in Figure 1. The bottom microarray compartment contains ~9% carbon black, and the top compartment that contains the fluidic channel is composed of clear, colorless polymer. A photograph of the COP femtoliter well array is shown in Figure 1 (a). The black microarray is composed of about three times more carbon black than the microarray chips used in the bead-based digital ELISA COP femtoliter well array. The clear polymer allows light to pass through the fluidic channel and excite the microarray. The black polymer minimizes background and prevents fluorescence crosstalk between wells. The microwell array chip contains about 15,200 microwells, which are 16.5μm in diameter, 16.5μm in depth and have 30μm center-to-center spacing, as shown in the scanning electron microscope image in Figure 1. Each well has a volume of about 3.5pL. The 16.5μm diameter of these microwells is about four times larger than the microwells of the COP femtoliter well array.

Device preparation In this work, individual molecules in solution are trapped directly; functionalized surfaces and paramagnetic beads are not needed for capturing and loading. To overcome the hydrophobic surface of the device, the microarray devices are first pre-filled with water prior to loading with the molecules of interest. A simple, surface modification-free approach is used to achieve an even filling of solution across the microarray that results in no bubble formation in either the

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fluidic channel or the microarray. Wells of PDMS devices can be easily filled by immersing the entire device in an aqueous solution and applying vacuum.58 To mimic this approach, the COP device is first taped to a standard microscope slide. Then the slide is immersed in the reaction buffer or water at an angle in a beaker. The beaker is then placed under a vacuum of approximately 10kPa. After 10 minutes, air bubbles trapped in the channel and in the wells expand due to the vacuum and are replaced by the aqueous solution. Intermittently releasing and re-applying the vacuum can speed up the filling process. Once the channel and array are filled, the microscope slide is placed on a standard stage of an upright fluorescence microscope, as shown in Figure 1 (b); this configuration eliminates the need for a custom holder and sealing stage. The filled device can also be stored in water up to 24 hours.

Sealing and sealing efficiency Figure 2 illustrates the experimental steps of loading, sealing, and imaging single molecules in the microwell array. It is important for each picoliter microwell to have the same volume of aqueous solution and to be isolated from one another. A non-uniform filling will give each microwell a different probability of containing a single enzyme molecule. In addition, if the sealing technique is poor or inconsistent, fluorescent product can diffuse into adjacent microwells and give false positive signals. To test whether the volume of solution is equal in all the wells and whether the oil seal is leak-proof, 100μL of 10μM resorufin standard dye is pulled into the inlet by vacuum into a pre-filled microarray. After the dye solution has completely

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displaced the pre-filling buffer (or water), the dye molecules are allowed to diffuse for an additional 30 sec. Afterwards, 100μL of fluorocarbon oil is pulled through the inlet to displace the dye solution in the channel and to seal the dye solution that has filled the microwells. The fluorescent wells are then imaged; a portion of a microwell array image is shown in Figure 3 (a). The fluorescence intensities of the picoliter wells are uniform across the array. This result demonstrates the vacuum filling method is able to fill the entire array with aqueous solution evenly, and therefore each well has the same probability of containing a single molecule. To determine that the wells are isolated and sealed, an octagonal-shaped aperture in the microscope is used to expose a small area of the array to a high intensity excitation light for 15 min, resulting in partial photobleaching.57 The octagonal-shaped aperture is then removed and an image is immediately taken to show the photobleached area, as shown in Figure 3 (b). Consecutive images are taken every 30 min, and the fluorescence intensity of the photobleached area is compared to the first photobleached image. The fluorescence trajectories of four photobleached wells are plotted in Figure 3 (d). The individual fluorescence intensities of the photobleached wells remain the same after 120 min, indicating the wells are effectively isolated and sealed with fluorocarbon oil. The seal remains stable and effective for at least 18 hours, which is the longest experimental time that has been tested. Sealing stability of the COP microarray was further tested by heating the array. The array withstood 50oC for 1 hour without losing the seal stability (data not shown).

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The increased sealing efficiency and stability of the COP microarray as compared to the optical fiber array is due to a number of contributing factors. Since the surface of the COP picoliter array is hydrophobic, the hydrophobic fluorocarbon oil effectively seals and isolates individual wells, eliminating the need to functionalize the surface. Optical fiber arrays require the use of a hydrophobic silane to selectively modify the glass surface to aid the oil sealing technique.37,59 Furthermore, the seal stability of the COP microarray is aided by the location of the wells. In the present format, the microwell array is located on the bottom and the sealing oil is on top of the array. In contrast, the wells of an optical fiber array point downward, leading to sealing complications as gravity pulls on both the sealing oil and reaction solution. Overall, the oil seal in the COP microwell array device is stable for long periods of time and is able to withstand increased temperature. With the ability to vary experimental temperatures, COP large-well microarrays facilitate single molecule analyses of a wide variety of enzyme reactions.8,10,11,29,60

Single molecule experiments After pre-filling a COP microarray, 100μL of reaction solution containing femtomolar concentrations of β-galactosidase (β–gal) and 100μM resorufin-β-Dgalactopyranoside (RDG) substrate in the same buffer as the pre-fill are pulled into the inlet by vacuum pressure. Solution displacement, diffusion, and the introduction of fluorocarbon oil are performed as described above. Previously, single enzyme molecules have been isolated and their fluorogenic products detected using femtoliter reaction vessels, such as optical fiber arrays.57,59

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Analytical Chemistry

An optical fiber array containing 50,000 46fL microwells can give digital read-outs for enzyme concentrations ranging from 72fM to 1.8pM. To compare the quantification and detection limits of the COP picoliter microarray to the optical fiber femtoliter array, we have used the same β-gal mediated, fluorogenic RDG substrate reaction system in the COP microarray experiments as was used in previously reported optical fiber arrays. The picoliter-sized wells allow for the capture of larger volumes of low concentration analyte solution while still maintaining singlemolecule sensitivity. Based on Poisson statistics, picoliter-sized wells should further lower the detection limit compared to femtoliter microwell arrays.57 Here, we load solutions of 0fM to 20fM β-gal enzyme and 100μM RDG into COP microarrays comprised of approximately 15,200 wells. This concentration range corresponds to a 0:24 to 1:24 ratio of β-gal enzymes to wells. Therefore, at the highest tested concentration of 20fM, the probability that a well contains one enzyme is 4.2%. Of the 15,200 wells, the fluorescence intensities of approximately 9,200 wells can be monitored simultaneously using a 5x objective lens (NA=0.15). A trapped β-gal enzyme molecule catalyzes the production of many fluorescent resorufin product molecules, which accumulate in the well. Since the bottom component of the device is designed to minimize background fluorescence and increase the signal to noise ratio, the fluorescence intensity from the confined resorufin is quickly detected and can easily be distinguished from empty wells. Figure 4 (a)-(c) shows typical fluorescence images of the COP microarray over a 10 min time frame. A small number of wells (e.g. #1, #2 and #3) show fluorescence intensity increases while the majority of the wells (e.g. #4 and #5) show no increase

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in fluorescence intensity. To determine whether the wells contain a single enzyme molecule, three negative control COP microarrays containing no enzyme were also run. The intensity changes of the control wells over the same time period were compared to the single enzyme experiments, as shown in Figure 4 (d). When the intensity from resorufin production exceeds the intensity threshold of the control wells, a well is considered to be active. In this way, the active wells and background wells can be distinguished as a simple binary readout. Figure 4 (e) shows the percentage of active wells at different enzyme concentrations as well as the predicted percentage based on Poisson statistics. The experimental results (n≥3) are in good agreement with the Poisson calculations and have a well-fitted linear relationship with increasing concentrations of β-gal. This observation is consistent with previous optical fiber experiments, demonstrating the potential of the COP microarray to provide high-quality quantitative single molecule measurements.33,59 The good agreement between the experimental results and Poisson calculations also shows that the loading time is sufficient for β-gal and the dye solution to diffuse into the wells. At the lowest tested concentration of 2fM β-gal enzyme, an average of about 60 wells were identified as active. This detection capacity is ~36 times lower than the detection limit of the femtoliter optical fiber array, due to the COP microarray’s larger-sized wells, enabling a larger total sample volume to be confined in the microwell array.57,59 Notably, the dynamic range of 2fM to 20fM in this COP microarray is not as broad as the optical fiber array because of the reduced number of wells. However, the dynamic range can be expanded if more microwells were

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Analytical Chemistry

imaged, provided that there are a statistically significant number of active wells at low concentration to maintain accuracy and precision. For example, if only 0.1fM βgal (20X less than 2fM) was loaded into the 15000 microwell array and imaged with the same 5X (N.A.=0.15) objective lens, the Poisson equation predicts that only three wells would be detected as active. The shot noise error for such a small number of wells is large, making it difficult to quantify very low concentrations. Consequently, one would need to use both wide field imaging and a larger microwell array to expand the dynamic range. The great benefit of using microwell arrays is the ability to measure individual molecule activities in parallel, such as the kinetics of many individual enzyme molecules. The change in the fluorescence intensity can be converted to the enzyme turnover rate by imaging at a fast rate and using a calibration curve to determine the concentration of fluorescent product in each well at a given time point. The average turnover rate of the β-gal enzyme is measured to be 296±108 s-1 in the COP microwell array device. This value matches results from previous optical fiber array experiments that used either mechanical sealing or oil sealing techniques.36,59 Thus, the COP microarray with oil sealing is able to reliably measure single enzyme kinetics. Factors that may affect the average turnover rate include threshold selections that incorrectly mislabel non-active wells,37 batch-to-batch variation of enzyme,61,62 enzyme interactions with the surface63,64 and the lag time of the experiments. Many materials, including PDMS and optical fiber glass bundles, require surface passivation to minimize protein adsorption and interaction with the

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surface.36,65-67 Our result indicates that surface interaction of β-gal is minimal in the COP microarray and surface passivation is unnecessary. Finally, the COP device was tested for re-usability. The device was pre-filled with PBS reaction buffer, loaded with a reaction solution containing 100μM RDG and 20fM β-gal enzyme, and oil sealed. Following a 10 min imaging sequence, the microarray was flushed with 100μL of reaction buffer, followed by 100μL of acetone, and then 100μL of methanol. The device preparation and experiment were repeated a total of three times, and the fluorescence images are shown in Figure 5. The percent of active wells remained constant, but the active wells appeared randomly in different locations across the three experiments, indicating the enzyme molecules were completely removed upon washing. (Supporting Fig. 1). This result demonstrates the reusability of the COP microarray. By comparison, the use of many organic solvents in PDMS-based fluidic devices can cause the PDMS-based devices to swell68 and the swelling can affect the sealing and microchannel structures.69 Therefore, the regeneration of COP microarray devices potentially reduces the experimental time and cost compared to optical fiber bundle and PDMS-based devices.

Conclusions COP has low autofluorescence and is highly transparent over a wide range of wavelengths. The material offers superior chemical resistance and low water absorption. The results here demonstrate the use of a larger diameter picoliter COP microwell array that uses a simple and effective oil sealing technique to perform

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Analytical Chemistry

single molecule experiments. Using COP devices simplifies experimental protocols by eliminating surface modifications and the need for sophisticated instruments and specialized microscope setups. We have shown that individual β-gal enzyme molecules captured in the picoliter wells and detected based on their fluorogenic products. The picoliter microarray has a low femtomolar detection limit but a relatively small dynamic range. The stability and reproducibility of the oil sealing technique allow us to observe molecules over a prolonged period of time. The COP picoliter array offers some unique capabilities, including long-term single molecule monitoring of many molecules simultaneously, good stability at elevated temperature, and a larger volume that can potentially accommodate more complex chemical and biological systems than femtoliter microwells, all of which can be accomplished in a simple and robust manner.

Experimental

Materials β-galactosidase from Escherichia coli was obtained from Sigma-Aldrich (Gravde VIII, lyophilized powder) and dissolved in β-gal reaction buffer (pH 7.4) containing 10mM phosphate buffered saline (PBS), 1mM MgCl2, and 20% glycerol to a stock concentration

of

1μM,

aliquoted

and

stored

in

-80oC.

Resorufin-β-D-

galactopyranoside was obtained from Invitrogen, dissolved in anhydrous dimethyl sulfoxide (DMSO) (Invitrogen), and was aliquoted at 100mM and stored at -20 oC. The β-gal reaction buffer (pH 7.4) containing 10mM PBS and 1mM MgCl2 was filtered

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with 0.2μm polyethersulfone filters twice before used. Fluorocarbon oil (Fluorinert FC-70) was purchased from Sigma-Aldrich.

COP picoliter array devices were

customized and purchased from STRATEC Consumables GmbH (Anif, Austria). All other reagents were used as received.

Imaging system All experiments were observed under a 5x objective (Olympus, NA=0.15) using an Olympus BX-51 upright epifluorescence microscope. A 100mW short arc mercury lamp was used for excitation and filtered through a filter cube (λex.=572-582nm, λem.=590-650nm, dichroic 585LP, Chroma Technology Corp, Bellows Falls, VT). All images were acquired by a CMOS camera (ORCA-Flash 4.0 LT, Hamamatsu, Bridgewater, NJ) using cellSens Software (Olympus, Center Valley, PA) at an exposure time of 1.5 s.

Experimental Setup and Oil sealing The picoliter microwell array devices were first fixed to a glass slide using double sided tape.

The devices were then completely submerged in a container with

deionized water or β-gal reaction buffer at a 45-degree angle. The container was then placed in a chamber and the chamber was evacuated to a vacuum of 10 kPa. After ten minutes, the pressure was slowly returned to atmospheric pressure. This was repeated two to three times until the microarray and the channels were completely free of bubbles. The microarray area would look visibly dark after filling with aqueous solution. The microarray devices were stored in the same container

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Analytical Chemistry

submerged in aqueous solution and were used within the same day. The filled devices were placed underneath a fluorescent microscope. A metal tubing insert was then plugged into the outlet of the microarray device, and silicone tubing connected the metal tubing to a syringe pump. A clamp was placed on the tubing between the device and the syringe pump to completely block flow when needed. Solution was pipetted in increments of 20μL into the inlet port, with care to avoid air bubbles that could enter the device. First, 100μL of β-gal reaction buffer (1xPBS, 1mM MgCl2) was pulled into the device at a rate of 40μL/min. Next, 100μL of the reaction buffer containing 100μM RDG and femto-molar concentration of β-gal was pulled into the device at a rate of 40μL/min. The fluidic flow was then paused for an additional 30 seconds to allow the solution and enzyme to diffuse evenly across the array of microwells. Finally, 80μL of Fluorinert FC-70 was pulled at a rate of 40 μL/min into the device. The syringe pump was then stopped and the tubing clamp was fastened to completely stop the flow of oil before imaging. To determine the β-gal concentration, images were taken at 0 min and after 20 min. For the kinetic studies of β-gal, images were taken every 20 seconds for 10 minutes with a 1.5 second exposure time.

Data Analysis x-y drift of the images was corrected for using ImageJ. A custom MATLAB code analyzed the images as briefly outlined here. A mask was created to identify the wells and obtain the mean intensity of each well. The fluorescence intensity percentage change between the first and last frames was calculated for each well. Based on the

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Gaussian distribution of the control experiments, a threshold of 6% was determined. The intensity percentage change of a well that exceeded the 6% threshold was considered to be active. The fluorescence intensities from the images were converted to the number of resorufin molecules using a calibration curve of fluorescence intensity versus resorufin product concentration. Curve fitting and plots were determined and created by WaveMetrics Igor Pro software.

Acknowledgements The authors thank Dr. Pratyusha Mogalisetti for her assistance with the MATLAB code. This work was supported by the Defense Advanced Research Projects Agency (N66001-15-2-4-23).

Supporting information: Fluorescence images showing a section of a microwell array during an experiment and after washing to remove the enzymes.

References (1) Bouilly, D.; Hon, J.; Daly, N. S.; Trocchia, S.; Vernick, S.; Yu, J.; Warren, S.; Wu, Y.; Gonzalez, R. L.; Shepard, K. L.; Nuckolls, C. Nano Letters 2016, 16, 4679-4685. (2) Zaino, L. P.; Grismer, D. A.; Han, D.; Crouch, G. M.; Bohn, P. W. Faraday Discussions 2015, 184, 101-115. (3) Klein, T.; Proppert, S.; Sauer, M. Histochemistry and Cell Biology 2014, 141, 561575. (4) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Science 2006, 313, 16421645. (5) Rust, M. J.; Bates, M.; Zhuang, X. Nature methods 2006, 3, 793-795. (6) Wang, C.; Nam, S.-W.; Cotte, J. M.; Jahnes, C. V.; Colgan, E. G.; Bruce, R. L.; Brink, M.; Lofaro, M. F.; Patel, J. V.; Gignac, L. M.; Joseph, E. A.; Rao, S. P.; Stolovitzky, G.; Polonsky, S.; Lin, Q. Nature Communications 2017, 8, 14243.

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Figure 1. Diagram of the COP picoliter microwell array device and setup. (a) Image of a COP picoliter microwell array device. (b) The buffer-filled fluidic device is placed under an upright epi-fluorescence microscope. The aqueous solution is pulled by vacuum through the outlet. The inset is a scanning electron microscope image showing a portion of a picoliter microwell array.

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Figure 2. Schematic diagram of the loading, sealing, and imaging of single molecules in a picoliter microwell array. (a) The fluidic device is pre-wet and filled with aqueous solution. (b) A buffer solution containing the enzyme and substrate molecule is loaded into the channel and a fraction of the enzymes are trapped in the picoliter microwells. (c) Fluorinated oil is pulled into the channel to displace the buffer solution and to seal the microwells. (d) Fluorescent products (purple wells) are generated only in the microwells where an individual enzyme molecule is present. (e) The non-fluorescent RDG substrate is catalyzed by β-gal enzyme to generate a fluorescent product.

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Figure 3 The stability of oil-sealing a picoliter microwell array. Fluorescence images are taken (a) after a 10uM resorufin standard dye is loaded and oil-sealed, (b) immediately after and (c) 120 min after a fraction of the microwell array is partially photobleached for 15 min with high intensity light. The fluorescence trajectories of the selected wells in the photobleached microwells are plotted in (d).

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Figure 4 Single molecule experiments. The fluorescence products catalyzed by individual β-gal enzymes are monitored over time in the experiments shown. Fluoresence images are taken (a) immediately (b) 5 min, and (c) 10 min after the microwells are oil-sealed. The fluorescence intensity increases for microwells containing a single enzyme (#1-3) but the intensity remains low for microwells containing no enzyme (#4-5) (d) Histogram of the fluorescence intensity change from all wells that do not contain an enzyme (combined data from n=3). The blue line shows the threshold for a well to be considered active. (e) All microwells measured above the threshold are plotted as the percentage of active microwells versus the concentration of β-gal enzyme. The experimental plot is well fit by linear regression (red line, y=0.23+0.19x, r2=0.99). The values calculated by the Poisson distribution are also shown. Error bars represent the standard deviation of multiple experiments.

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Figure 5 Fluorescence images of the same microarray device after three single molecule experiments. Images are taken at the end-point of the experiments. The circle denotes a surface defect specific to this microarray device.

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