Single Molecule Ionic Current Sensing in Segmented Flow Microfluidics

Jan 7, 2014 - Single Molecule Ionic Current Sensing in Segmented Flow. Microfluidics. Thomas R. Gibb, Aleksandar P. Ivanov, Joshua B. Edel,* and Tim ...
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Single Molecule Ionic Current Sensing in Segmented Flow Microfluidics Thomas R. Gibb, Aleksandar P. Ivanov, Joshua B. Edel,* and Tim Albrecht* Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ, United Kingdom W Web-Enhanced Feature * S Supporting Information *

ABSTRACT: Herein, we describe the integration of two glass nanopores into a segmented flow microfluidic device with a view on enhancing the functionality of label free, single molecule nanopore sensors. Within a robust and mechanically stable platform, individual droplet compositions are distinguished before single molecule translocations from the droplet are detected electrochemically via the Coulter principle. This result is highly significant, combining the sensitivity of single molecule methods and their ability to overcome the clouding of the ensemble average with the “isolated microreactor” benefits of droplet microfluidics. Furthermore, devices as presented here provide the platform for the development of systems where the injection and extraction of single molecules allow droplet composition to be controlled at the molecular level. “label-free” experiments become possible. Importantly, the absence of a label ensures that the molecular properties being measured are not influenced by its effect on analyte behavior. In addition, artifacts arising from the labels, such as photobleaching or quenching, are completely eliminated. The operating principle of a nanopore sensor is simple: by application of an electric field, single molecules are driven (or “translocated”) through a nanoscale aperture, similar to the operating principles of a Coulter counter.10 The change in ionic current during the translocation process is dependent on several factors which include the molecule’s charge and shape and the ionic strength of the solution. Therefore, this mechanism can be used to quantify and classify single molecules among a larger population.11 Optical and electronic methods for detecting single molecules are powerful analytical tools, but their optimum operation relies on their successful incorporation into a suitable platform. One strategy that is often explored with fluorescence based detection, but not electrical detection, is to downsize the sample volumes sufficiency to maximize the detection throughput and efficiency. With this in mind, micro- or even nanofluidics offer an attractive solution as sample volumes can often be comparable to what can be realistically probed. Other advantages that are often touted include portability, short analysis times, and the potential for parallel analysis. On the other hand, downsizing can introduce significant challenges in the form of nonspecific adsorption to the channel walls. Problems of absorption and interaction can be overcome

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he ability to detect, analyze, and monitor single molecules in solution remains one of the ultimate aims of analytical chemistry. Engaging in such studies enables the measurement of fundamental variations in individual molecules which are often considered identical when looking at an ensemble averaged measurement. Removal or even a reduction in ensemble averaging is hugely advantageous when investigating small but significant differences in a subset of a larger population, such as the detection of point mutations,1 protein folding,2 or exonuclease binding3 to name but a few examples. While much work has already been done to develop single molecule manipulation and sensing techniques,4,5 further work is required to extend these abilities across the entire spectrum of analytical chemistry. Given the many benefits of single molecule detection (SMD), it is perhaps unsurprising that many methodologies have been developed for these studies, one of the most popular being fluorescence. However, most single molecule fluorescence strategies are based on the use of a confocal microscope which results in the requirement of working concentrations to be less than 5 nM. This can become problematic when studying biomolecular interactions where dissociation constants are often in the micromolar range. To compensate for this limitation, techniques based around plasmonic nanostructures and nanofluidic confinement have been developed to minimize detection volumes and increase analyte concentrations while still achieving single molecule resolution.6−9 For single molecule studies in these higher concentrations, an alternative approach is the use of electrochemical methods, such as nanopore sensing. As well as single molecule detection at higher concentrations, due to the possibility of only a single molecule entering a correctly sized pore at any one time, key advantages include the speed of acquisition and the fact that © 2014 American Chemical Society

Received: November 29, 2013 Accepted: January 7, 2014 Published: January 7, 2014 1864

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demonstrated before and, more recently, glass nanopores have been used to study translocations from microfluidic flows in more detail.34 In this report, the microfluidic integration of nanopores is taken and developed to produce a platform that demonstrates the first reported translocations of single molecules from a segmented microfluidic flow. Glass nanopores are used to probe the gross and molecular contents of individual droplets without permanent deformation of the droplets themselves. As droplets remain intact throughout, their status as isolated microreactors is preserved resulting in no loss of functionality compared to previous analysis techniques. The translocation of single molecules from within microfluidic droplets generated at rates up to 15 Hz is a key result, improving upon fluorescent detection methods due to its label free nature and lack of a high concentration limit. Detection efficiency is found to compare favorably to that of a glass nanopore in bulk solution with further improvement possible to produce an efficient nanopore sensor. In addition, the translocation of a small number of molecules per droplet is of significance as it offers potential for changes in droplet composition via molecular extraction and injection and forms the basis for the extension of single molecule techniques across a wide range of experimental systems, allowing analysis beyond the ensemble average.

with the use of segmented microfluidics, a subset of microfluidic technology where immiscible fluids, most commonly an oil and aqueous phase, are introduced into a device in such a way that droplets of one of the phases are formed. For biological studies, aqueous droplets in an oil carrier phase are most common. Although the ability to continuously measure the analyte of interest is lost, interactions and absorption are removed as obstacles in such a device as the oil carrier fluid coats the walls of the microfluidic channel, preventing any interactions between the droplet and its external environment. Thus, each droplet can be considered as an isolated microreactor, 12 a major advantage for many applications, leading to a rapid increase in the use of segmented flow, microdroplet devices in a wide range of fields such as kinetics,13 synthesis,14 crystallization,15 and single molecule encapsulation.16 The analysis of droplet contents is thus an important analytical challenge and, like single molecule detection, most commonly undertaken by optical means17,18 in particular with experiments involving fluorophores.19,20 In droplets, fluorescent studies have been used to detect single molecules of DNA,21 detect cells,22 and quantitatively describe protein expression23 in a high throughput setting. In such studies, droplets confine the analyte to ultrasmall volumes of a femtolitre and below, advantageous as detection efficiency is greatly increased. Reports of similar studies, or any other requiring molecular sensitivity, using an electrochemical means of detection in a segmented flow microfluidic device remain unreported in the literature. While single molecule studies remain elusive due to the speed of acquisition, sensitivity, and difficulty of fabrication associated with such devices, electrochemical methods have been used to measure the physical properties of droplets including their length, frequency, and velocity.24−26 In addition, the bulk content of individual microdroplets has proven amenable to study using such methods.25 An obvious candidate for the electrochemical detection of single molecules in a multiphase microfluidic device is the integration of proven nanopore devices with a microfluidic channel. In doing so, significant issues must be overcome such as electrode incorporation, droplet piercing, and the minimization of disturbance to the flow profile. Suitable nanopore technologies include planar, wafer based systems,27−31 biological pores,32 or glass nanopores33,34 fabricated by pipet pulling, each method with its own advantages and disadvantages. Glass nanopores are the ideal candidate for the electrochemical interrogation of droplets; the sharp, nanometer scale pipet tips readily pierce droplets as they pass. The low cost, ease of fabrication, and chemical and mechanical stability35,36 of glass nanopores are also key benefits of such a platform. Away from microfluidics, glass nanopores have proved to offer a versatile platform for single molecule translocation experiments. Single molecule detection of both DNA36 and proteins37 has been described as well as the translocation of functionalized nanoparticles.38 Furthermore, glass nanopore technologies have been used to probe the detailed structure of translocated molecules, for example, the folded state of DNA.39 Additionally, the sensitivity of glass nanopores can be increased with insertion of DNA origami structures into the nanoscale pipet opening.40 The integration of nanopores with microfluidics has, to date, resulted in little discussion in the literature. Single molecule detection via biological41 and inorganic42 nanopores integrated into a microfluidic device has been

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RESULTS AND DISCUSSION Glass nanopores as used in this study were pulled from quartz capillaries with a commercially available laser pipet puller, producing pores with a diameter of 24 ± 1 nm as imaged in Figure 1c. Additional detail on all fabrication steps is provided in the Supporting Information. Conductance measurements were also used to characterize the glass nanopores with the conductance of individual nanopipettes measured at 54 ± 3 nS in 1 M KCl, from which a diameter of 20 ± 1 nm was calculated,43 in excellent agreement with the diameter as measured by scanning electron microscopy and comparable to pipettes elsewhere in the literature.33,37,38 All microfluidic experiments were carried out in devices fabricated in PDMS and glass. Droplets were generated using a T-Junction geometry shown to be previously successful21 with channel widths of 50 μm in the area of droplet generation. In areas of the channel other than those used for droplet generation, channel widths were 100 μm to ease the integration of the glass nanopores into the main microfluidic channel, facilitated by two access shafts downstream of the droplet generating geometry as seen in Figure 1a,b. In contrast to previous work, two nanopores are integrated into the microfluidic channel. One nanopipette is used in the traditional sense, to detect translocation events, while the second nanopipette is used to ensure a second electrode can pierce the droplet, achieving an electrical connection. In all experiments, glass nanopores were prefilled with 1 M KCl before insertion by hand such that their tips were in the center of the microfluidic channel. Access shafts were subsequently sealed with a two-part silicone, cured rapidly on a hot plate to prevent blockage of the droplet channel. The experimental device geometry is included in the Supporting Information (Figure S1). Upon completion of this fabrication process, around 25% of devices were found to be viable for experimentation. Devices were most commonly rendered unusable by nanopipettes breaking or becoming blocked with PDMS on integration. Additionally, the flow of silicone into the main microfluidic 1865

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Figure 1. (a) Schematic of the microfluidic device. (b) Optical image of two glass nanopores inserted into the main microfluidic channel via access shafts. (c) SEM image of the nanopipettes in the microfluidic channel. (d) SEM image showing the taper of the glass nanopipette. Inset: 600k× magnification of the glass nanopore. All scale bars are 100 μm, apart from inset, scale bar of 20 nm.

Figure 2. (a) Typical optical trace taken from an image sequence. Inset: Fourier transform of the droplet signal showing an initial peak at 11.9 s−1. (b) Characteristic square wave trace as seen during the electrical detection of droplets, here using 1 M KCl. Inset: Fourier transform of the droplet signal showing an initial peak at 12.2 s−1, in excellent agreement with the optically recorded data. For both (a) and (b), data was collected with a flow rate of 5 mm s−1.

channel upon access shaft sealing also reduced the percentage of viable devices. Successfully fabricated devices had a measured conductance of 30 ± 4 nS with a microfluidic channel filled with 1 M KCl, as measured with a patch clamp amplifier (Axopatch 200B, Molecular Devices, USA) connected to Ag/ AgCl electrodes inserted into both pipettes, one containing the working electrode and the other the reference electrode. When considering the conductance of the device as a whole, it should be noted that by far the most significant contribution to the resistance of the device is the glass nanopores themselves. Hence, to a first approximation, the devices would be expected to have twice the resistance of a single glass nanopore, containing as they do two glass nanopores in series. The measured conductance for these devices is thus within expectations, 55% of the single glass nanopore conductance, confirming the successful integration of both glass nanopores into the microfluidic channel. Devices showed no rectification when the applied voltage bias was swept between +0.5 and −0.5 V and the microfluidic channel was filled with an electrolyte of 1 M KCl, in agreement with single nanopipette configurations using solutions of the same concentration.44 To form the segmented flows, a continuous phase of Fluorinert FC-40 (3M, USA) (containing 2.5% by weight surfactant45) was used with KCl solutions of varying molarity (0.05 to 2 M) to form the segmented phase. The segmented

and continuous phases were introduced into the device in a 1:1 ratio to produce flow velocities up to 30 mm s−1, well within the range where droplet microfluidics have previously been demonstrated (see Supporting Information, Figure S-2).13,20,22 Successful operation of the device requires all droplets to remain intact during their passage past the glass nanopores, at odds with the introduction of a sharp object, in this case the glass nanopores themselves, into the microfluidic channel. In devices as fabricated, there was no permanent deformation of the droplet during its passage; no splitting was observed, and no volume was lost, as indicated by the droplet size, while passing through the glass nanopore area (see Supporting Information, Figure S-3). This lack of leakage from the droplet is due to the lowered surface tension of the droplet with the presence of the surfactant, enabling the facile piercing of the droplet and preserving its isolated microreactor status. The only observed change in droplet behavior across the double nanopore structure is a slight increase in velocity due to the channel volume excluded by the pipettes themselves (see Supporting Information, Figure S-4). 1866

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Figure 3. (a) Typical droplet length distribution as measured optically at 10 mm s−1, fitted with a Gaussian curve to obtain the most probable droplet length and standard deviation, here 0.442 ± 0.007 mm. (b) Typical droplet length distribution as measured electrically at 10 mm s−1 with a Gaussian fit giving an average droplet length of 0.442 ± 0.006 mm. (c) Optically measured droplet length between flow rates of 3 and 30 mm s−1. (d) Droplet length as measured with the glass nanopores at flow rates between 3 and 30 mm s−1. Errors presented are the standard deviation in droplet length for each device.

excellent agreement with each other at 11.9 and 12.2 s−1, respectively, indicating consistent droplet detection between the two platforms. Data as presented in Figure 2 was obtained for flow velocities between 3 and 30 mm s−1 and used to determine the droplet length distributions for each flow rate. Histograms illustrating the distribution in droplet lengths were produced from the data collected with examples, measured at a flow rate of 10 mm s−1, shown in Figure 3a,b. These histograms exhibited a Gaussian distribution in droplet lengths from which fitting parameters were extracted to obtain the droplet length and standard deviation, in these examples, 0.442 ± 0.007 and 0.442 ± 0.006 mm, respectively. Figure 3c,d illustrates these parameters across the full range of experimental flow rates, showing that droplet length remained consistent across flow rates and between devices whether measured electrically or optically, as expected.47 The agreement between the two methods of measurement is best illustrated by comparing the mean droplet length across all measured droplets for both electrical and optical experiments, found to be within error at 0.43 ± 0.03 and 0.45 ± 0.03 mm, respectively. A polydispersity index, α, was calculated for the entire data set via the formula, α = (δ/lav) × 100%, with δ being the standard deviation and lav being the average droplet length. Droplets as produced had a polydispersity index of 6.9%, around three to four times as large as values previously achieved in microfluidic droplet devices.48 This increase in the droplet polydispersity can likely be accounted for by backpressure in the access shafts causing a small amount of instability in the microfluidic flow. This instability is enhanced by the presence of small, residual bubbles of air that frequently remain around the glass nanopores in the access channels after device filling, their easily compressible nature increasing any pressure variations occurring in the device during operation. Such

Initial experiments with the fabricated devices centered on the electrical measurement of droplet length and the comparison of the collected data with optically collected control data sets. Electrical measurements of droplet length were facilitated by changes in the measured current during droplet passage. On application of a 500 mV bias across the glass nanopores, the high resistivity of the fluorinated oil continuous phase resulted in a current of around 20 pA with a root mean squared (RMS) noise of ∼15 pA across the device. When both glass nanopores pierced a droplet containing 1 M KCl, a current increase to around 15 nA with an RMS noise of ∼30 pA was typically seen due to the increased conductivity of the KCl segmented phase. The overall effect was the production of a characteristic “square-wave” fluctuation in the current against time during droplet production as seen in Figure 2. Such a square-wave pattern on droplet passage has been observed previously in fluorescence experiments, in these cases due to the background fluorescence of the droplets themselves.21 Fluorescent monitoring in droplets has been demonstrated down to a time scale of 1 μs,20 comparing favorably with the 50 kHz sampling rate used here, although such rates of electrical measurement are possible.46 Fabricated devices exhibited levels of noise that were large compared to other glass nanopore experiments37 with the syringe pumps providing the microfluidic flow proving a source of noise despite being located outside the Faraday cage and the filtering of all acquired data at 10 kHz. Optical control experiments used videos of the devices taken with a high speed camera with droplet length subsequently analyzed using Image-J. Figure 2a shows a typical processed experimental trace from an optical control, and Figure 2b shows the raw data from an electrical experiment. Data from Figure 2a,b were analyzed with a Fourier transform, inset into the main graphs, showing major peaks in 1867

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bubbles to escape and preventing any variability in backpressure affecting droplet formation. As a first step in probing the contents of the droplets themselves, devices were used to distinguish different KCl concentrations within droplets. KCl was introduced to devices as the segmented phase with molarities between 0.05 and 2 M and a voltage of 500 mV applied across the device. The change in the measured current associated with these differing concentrations was then used to distinguish droplets of different molarities. Glass nanopores were filled with 1 M KCl throughout. For each molarity, data was obtained for 2 min, and the mean peak droplet current recorded and plotted. After the current was measured for each KCl concentration, the device was flushed with the subsequent KCl solution and carrier oil for 2 min to remove any residual KCl droplets from the device and ensure accurate measurement. Figure 4a,b shows typical current traces for KCl of 1 and 0.1 M with peak droplet currents of 15.84 ± 0.04 and 1.62 ± 0.07 nA, respectively. The measured current associated with the passage of droplets was seen to decrease linearly with a slope of 14.56 ± 0.24 nA M−1 across the range of concentrations studied, as seen in Figure 4c. This linear decrease in conductance is in agreement with a model of the glass nanopores and the droplet as three resistors in series. In many, but not all devices, a transient spike in current was seen upon droplets being pierced by the glass nanopores; see Figure 5aii for an example. Such spikes could be up to 500 pA in size and 10 ms long and are likely due to the capacitive charging of the interface between the oil and salt solution and the initial wetting of the surface of the nanopore by the droplet. Such a hypothesis is supported by the time scale of droplet piercing observed in some experiments (see Video 1 and Video 2, available in the HTML version of the paper). Here, nanopipettes are seen to be in contact with the droplets before piercing for a period similar to the observed current peak (see Supporting Information, Figure S-5). The size of these transient peaks, while highly consistent for each device, varied between devices preventing the reliable determination of KCl concentration below 0.05 M. In addition, precision at low KCl concentrations could be limited by changes in the droplet composition due to fluid out-flow from the nanopipettes.49 While the analysis of bulk droplet composition is a valuable tool, single molecule detection is required to fully exploit the ability of the microfluidic platform to perform analysis on small volumes without the clouding of the ensemble average. As such, translocation studies involving the passage of a DNA molecule from within the droplet into the glass nanopores were undertaken as a proof of principle experiment. Experiments used 10 kbp double stranded DNA diluted to a concentration of 0.5 pM in an electrolyte of 1 M KCl, 10 mM Tris, and 1 mM EDTA. This DNA suspension was then used as the segmented phase as droplets were created at a flow velocity of 5 mm s−1 using the previously described oil carrier phase. At this concentration, each droplet was calculated to contain ∼1300 molecules of DNA. Experiments were carried out at five (applied) voltages: 600, 500, 400, 300, and 200 mV. Figure 5a,b shows a series of current time traces for a droplet containing a control solution of the buffered electrolyte and the DNA solution, respectively, the latter showing several translocation events seen as characteristic drops in the measured current. At applied voltages of 600 and 500 mV, the majority of droplets contained only a single translocation event with many droplets containing no events at all. These event free droplets

Figure 4. (a) Electrical measurement of droplets containing 1 M KCl at a flow rate of 5 mm s−1. (b) Electrical measurement of droplets containing 0.1 M KCl at a flow rate of 5 mm s−1, showing a drop in peak droplet current compared to the 1 M case presented in (a). (c) Measured peak droplet currents across KCl concentrations from 0.05 to 2 M. Measured current showed a linear decrease with KCl concentration across the range studied. Errors as presented are the standard deviation of the mean measured peak droplet current at each KCl concentration across five devices.

problems can be removed with the addition of “drainage” channels in the access shafts, allowing any trapped micro1868

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Figure 5. (ai) Sequence of three droplets containing a control solution of 1 M KCl, 10 mM Tris, and 1 mM EDTA at a flow velocity of 5 mm s−1 with an applied potential of 500 mV . Expanded to a single droplet in (aii), (a.iii) compares 8 differing droplet traces, none of which show translocations. (bi) Sequence of three droplets containing a 0.5 pM 10 kbp DNA solution at a flow velocity of 5 mm s−1 and an applied potential of 500 mV. (bii) Expansion to a single droplet during which two DNA translocations can be seen. (biii) Eight droplet traces from using the DNA solution, six of which show translocations.

Figure 6. (ai and aii), (bi and bii), and (ci and cii): Histograms of translocation time and charge for 400, 500, and 600 mV, respectively. Gaussian fits of the histograms give mean dwell times of 0.43 ± 0.09 ms at 600 mV, 0.55 ± 0.07 ms at 500 mV, and 0.77 ± 0.12 ms at 400 mV as presented in (d). Data is linear when when plotted as expected.55 Gaussian fits of the integrated charge histograms give mean values of 46.94 ± 4.56, 53.62 ± 4.96, and 52.54 ± 3.79 fAs at 600, 500, and 400 mV, respectively (e).

became the majority at 400 mV, and at lower applied voltages, no translocation events were observed due to the decreased driving force. Even at the highest applied voltages, this frequency of DNA translocation represents a low DNA sampling efficiency of ∼0.1% given the large number of molecules (∼1300) per droplet. While this efficiency seems low at first glance, it should be noted that it compares favorably to

typical bulk nanopore experiments. Bulk translocation measurements were undertaken with glass nanopores fabricated using the same protocol as those in the device. With a 1 mL sample volume, a detection efficiency of ∼0.0003% was achieved after measuring the current across the pore for 1 min, over three hundred times lower than the microfluidic device (see Supporting Information, Figure S-6). 1869

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the device as presented does not achieve the detection efficiency possible with fluorescence,53 it is still ∼300 times greater than that of a bulk nanopore measurement and thus offers potential as a device for high throughput analysis. Furthermore, the single molecule detection offered enables the interrogation of large populations and the reduction or even removal of the ensemble average, a key advantage in a wide range of studies. The robust nature of the fabricated devices and the low cost and optical transparency of their constituent materials offer benefits for larger scale production and the potential for the integration of other, secondary, detection processes. In addition, the isolated microreactor status of the droplets in this device enables individual droplet composition to be measured accurately even if it is changing rapidly such as in droplet dilution devices.54 Finally, with careful control of the time of droplet passage and translocation frequency, such glass nanopores devices have the potential to control droplet composition at the single molecule level via removal, or even injection, of single molecules.

Distributions of dwell time and integrated charge for translocation events at 400, 500, and 600 mV are shown in Figure 6a−c. Events at 600 and 500 mV were analyzed at a 5 σ level of significance whereas data for 400 mV was analyzed at 4 σ. At all three voltages, translocation times exhibited a Gaussian distribution, indicating a high friction translocation regime.50 Between 400 and 600 mV, translocation time decreased with increasing voltage as expected with average dwell times of 0.43 ± 0.09 ms at 600 mV, 0.55 ± 0.07 ms at 500 mV, and 0.77 ± 0.12 ms at 400 mV as shown in Figure 6e. With a contour length of 3.4 μm for 10 kbp DNA, this represents a translocation speed of 6.2 mm s−1 or at 500 mV, in good agreement with reported literature values: 10.3 mm s−1 by Steinbock et al.,39 10 mm s−1 by Li et al.,51 and 8.2 mm s−1 by Gong et al.34 In addition, the excluded ionic charge per translocation event, measured as the integrated current area per translocation, is expected to be constant for molecules of the same size.39 Figure 6e shows the integrated charge for the 10 kbp DNA: 46.94 ± 4.56, 53.62 ± 4.96, and 52.54 ± 3.79 fAs at 600, 500, and 400 mV, respectively. The values of integrated charge for translocations at 500 and 400 mV are in excellent agreement with each other, and while the value for 600 mV differs slightly, it is still within experimental error. These values are also of the same order of magnitude as those previously reported in the literature.34,39 The device presented in this work could be used in two conceptually different ways. At low analyte concentrations and short analysis times, as shown here, individual or a known small number of analyte molecules could be injected into (or extracted from) each droplet. The focus is then on sample manipulation and/or reaction monitoring at the singlemolecule level. On the other hand, at higher analyte concentrations and sufficiently long analysis times, the same structure could also be employed for analyzing the content of individual droplets, i.e., with the pipettes as conventional nanopore sensors. This is provided given that a sufficiently large number of translocation events can be recorded during the residence time of the droplet, in order to obtain a sufficiently robust statistical basis. Calculations, presented in the Supporting Information, estimate that a 200-fold increase of the translocation frequency could be expected on the basis of the concentration alone, or a factor of ∼1600 combining all other pertinent factors. In principle, this would allow for nanopore based analysis of individual droplets, even though further work is clearly needed to substantiate the above estimates. Other possibilities for the total analysis of droplet contents include the passive trapping of droplets52,53 or, with improvements in pipet integration, a multiplexed cascade of nanopipettes along the microfluidic channel.40

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ASSOCIATED CONTENT

S Supporting Information *

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

Videos of the time scale of droplet piercing are available in the HTML version of the paper.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.B.E. acknowledges the receipt of an ERC starting investigator award and an EPSRC basic technology grant.

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CONCLUSION We have presented the first successful demonstration of label free, single molecule detection from a segmented microfluidic flow. In addition, we show that glass nanopores can be used to reliably probe the physical dimensions and contents of a microfluidic droplet without its permanent deformation, opening the door to high throughput electrical analysis with all the analytical benefits of droplet microfluidics. Upon the introduction of a 10 kbp DNA analyte population to the droplets, the recorded current signal upon the application of a bias shows current drops consistent with translocation data previously reported in the literature. While 1870

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dx.doi.org/10.1021/ac403921m | Anal. Chem. 2014, 86, 1864−1871