Compact Nanowire Sensors Probe Microdroplets - Nano Letters (ACS

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Compact nanowire sensors probe microdroplets Julian Schuett, Bergoi Ibarlucea, Rico Illing, Felix Zoergiebel, Sebastian Pregl, Daijiro Nozaki, Walter M. Weber, Thomas Mikolajick, Larysa Baraban, and Gianaurelio Cuniberti Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b01707 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 18, 2016

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Compact nanowire sensors probe microdroplets Julian Schütt1, Bergoi Ibarlucea1,2, Rico Illing,1,2 Felix Zörgiebel1,2, Sebastian Preg1l,2, Daijiro Nozaki1, Walter Weber2,3, Thomas Mikolajick2,3, Larysa Baraban1,2*, and Gianaurelio Cuniberti1,2 1

Max Bergmann Center of Biomaterials and Institute for Materials Science, Dresden University of Technology, Budapesterstrasse 27, 01069 Dresden, Germany 2

Center for Advancing Electronics Dresden, 01062 Dresden, Germany 3

4

Namlab GmbH, Nöthnitzerstraße 64, 01187 Dresden, Germany

Institute for Semiconductors and Microsystems, TU Dresden, 01062 Dresden

ABSTRACT: Conjunction of miniature nanosensors and droplet-based microfluidic systems conceptually

opens a new route towards sensitive, optics-less analysis of biochemical processes with high throughput, where single device can be employed for probing of thousands independent reactors. Here we combine droplet microfluidics with the compact silicon nanowire based field effect transistor (SiNW FET) for inflow electrical detection of aqueous droplets one by one. We chemically probe the content of numerous (~104) droplets as independent events, and resolve the pH values and ionic strengths of the encapsulated solution, resulting in a change of the source-drain current ISD through the nanowires. Further, we discuss the specificities of emulsion sensing using ion sensitive FETs and study the effect of droplet sizes with respect to the sensor area, as well as its role on the ability to sense the interior of the aqueous reservoir. Finally, we demonstrate the capability of the novel droplets based nanowire platform for a bioassay applications, and carry out a glucose oxidase (GOx) enzymatic test for glucose detection, providing also the reference readout with an integrated parallel optical detector. 1

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KEYWORDS: silicon nanowires FET, nanosensor, droplets based microfluidics, point-of care diagnostics, glucose assay

The fast evolution of high performance automated laboratory tests1, point of care (POC) approaches for medical diagnostics2, and smart routes towards new logic for chemical information processing is to great extent driven by the smart combination of microfluidics and nanotechnology. Droplet-based microfluidics (or digital microfluidics)– a versatile approach for operating a large amount of same size reactors in parallel3– is of particular interest. With each single droplet being an independent miniaturized laboratory for bio-chemical experiments, this technology allows extremely parallelized and fine-tuned measurements, surpassing the precision of conventional assays (Figure 1 a)3–14. Although making an important contribution to classical detection and characterization laboratory assays10,15,16, the application of droplet microfluidics in the spirit of miniature and light weight devices for e.g. POC applications remains a work in progress. The main impeding factor is related to the need for development and integration of novel miniaturized optics-less detection principle, outperforming or being complementary to the conventional approaches

17–19

. In contrast, new devices make the

measurement processes independent on the limitations of optical microscopy, i.e. dynamic range or use of molecular labels. Another shortcoming is related to the ability to probe the interior of each droplet with sensitive and nondestructive sensors for monitoring the biochemical processes inside. The latter will gain crucial importance for e.g. compact medical diagnostics platform, where conventional microscopy is not an option. In these regards, recently proposed integrated nanosensors, i.e. ion sensitive field effect transistors (ISFETs), offer real-time and ultra-sensitive detection of disease markers or pathogens20–26 as well as monitoring of multiple physiological parameters, like glucose level27 or pH28. Although the ISFET principle goes back to the 1970s29,30, it founds a new fascinating realization in the last decade with 2


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utilizing silicon nanowires (SiNW)20–24,31–34 (Figure 1 b) or carbon nanotubes, etc. as sensor building blocks. In comparison with planar semiconducting elements, the performance of nanowire devices, namely the sensitivity21,24,32,35,36 is significantly improved by reducing their size and increasing surface to volume ratio37. Silicon nanowire-based FETs reveal great electrical characteristics with high output current and low power dissipation.20,38,39 and are CMOS compatible40,41. While being an excellent candidate for integration as transducers for pH or biomarkers detection in single liquid phase conditions21,40, to our knowledge SiNW FETs have not been combined with the multiphase dropletsbased approach so far. Due to the inherent amplification the current of an ISFET is a sensitive measure for surface potential changes34,42,43 that suggests the potential excellent compatibility of this approach with water-in-oil emulsion. Namely, digitalized flow of the water droplets separated by the oil phase, results in significant signal changes in the sensor elements. This facilitates reliable one-by-one droplet detection and counting. Furthermore, the relatively thin oil film separating droplet contents from the sensor surface (typically a few nanometers thick), makes it possible to probe the interior of the aqueous droplets and to get an information about e.g. ionic strength and charged constituents, pH value, or biomolecules. The generic functionalization is an oxide surface, which allows for pH detection, but can also serve for detection of the concentration of other ions in the liquid. Here, we realize for the first time the integration of droplet-based microfluidics with silicon nanowire field effect transistor (SiNW FET) and demonstrate in-flow electrical detection of aqueous emulsion droplets one-by-one (Figure 1 c). For this miniaturized microdevices, integrating both the silicon nanowire transistors and microfluidics network on a single chip (see Figure 1 c), are fabricated. We chemically probe the content of every single droplet in a row as independent events, and investigate the effect of the pH values and ionic strength of solution the source drain current ISD of the device at constant gate voltage. Further, we discuss the underlying physical processes, which take place once the droplet and the microscopic reference electrode (liquid gate) are in close contact. 3


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We believe that even ordinary pH and ionic concentration sensing in droplets opens a huge range of applications, since the majority of chemical processes performed in liquid environment are associated with the change of ionic composition or pH value in a medium. While ionic strength of the solution drives the synthesis and catalysis of diverse chemical species44,45, it also tremendously influences biological processes and responses46,47. Cytosolic pH changes48, chromatin aggregation49, antimicrobial peptide activity50 and controlled DNA (dis)assembly into supramolecular systems51, as a response to changing ionic and pH surroundings are only some of the many prominent examples. On the other hand, the ionic composition and pH level serve as indicator helping to monitor the actual state of the ongoing chemical process, such as microorganisms growth52 or enzymatic activities

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. In order to support

aforementioned statements we finally demonstrate the capability of the novel nanowire platform for a bioassay applications, and carry out a glucose oxidase (GOx) enzymatic test for glucose detection and measure related pH change in every single droplet.

Results and discussions The key component of the developed droplets detection platform is a large area SiNW FET, revealing high output currents and low standby power consumption34,54. The fabrication of ISFET devices is briefly described below (see Figure 2 a, stages I-IV). Firstly, a dense array of vertically aligned intrinsic SiNW with a length distribution from 2 to 20 µm and a mean diameter of about 20 nm are grown on a silicon wafer using vapor-liquid-solid (VLS) mechanism55,56. The nanowires are then transferred onto the rigid receiver Si substrate with an isolating SiO2 top layer of 100 nm (panel I. in Figure 2 a). This procedure results in a large scale and dense array of horizontally aligned nanowires, which are further integrated into a conventional FET electrode geometry. Parallel arrays cover 100 x 400µm, leading to up to 103 nanowires per ISFET device (areal density 0.03 nanowires/µm2). The connection of multiple nanowires in parallel helps to deliver macroscopic current outputs20,21,33,57 which greatly outperforms 4


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single nanowire FETs (generating current outputs of about 1 µA)57. Furthermore, devices connected by NW arrays reveal broader dynamic range and higher on/off ratio, which is beneficial for the sensing devices. Interdigitated microelectrodes (source-drain) are formed via sputtering of a 50 nm nickel (Ni) film on top of the substrate, patterned by means of optical lithography (see Methods). Thermal annealing of the sample at 450°C was performed in order to reduce the contact resistance between Si nanowire and Ni electrode. This process causes the axial diffusion of Ni into the NW body20,55 and decreases the contact resistance by approximately factor of 234. Metal-oxide-semiconductor (MOS) geometry was realized by atomic layer deposition (ALD). At last, the chip was equipped with Ag/AgCl top gate electrodes on top of its FET structures (see Methods) (Figure 2 a, panel II and d). The transfer and output characteristics of the fabricated transistors are displayed in Figure 2 e and f, demonstrate the excellent ambipolar behavior, reaching the high 106 on/off current ratio and a subthreshold swing of about 205mV/dec. Due to residual negative charged traps in the interface oxide between Si and Al2O3 and the electron-hole asymmetry in barriers to the connecting electrodes in real devices, the current minimum of SiNW FETs as a function of the gate voltage is slightly shifted towards positive gate voltages (see Figure 2 e).33,34 Finally, we assembled a microfluidic channel system with a width of 300µm and a height of 15 µm onto a chip with 20 integrated SiNW FET devices (see Figure 2, panel IV) and Methods). In order to make the surfaces of the chip (PDMS and Al2O3) homogeneously hydrophobic for water droplets propagation, channels were functionalized with (3-Aminopropyl)triethoxysilane (APTES) (see Figure 2 a, panel III and Methods). Mineral oil (M5904, Sigma Aldrich) with adding nonionic 2% Span 80 (S6760, Sigma Aldrich) was used for preparing water-in-oil emulsion. The flow-focusing geometry for droplets formation58 results in a chain of uniform aqueous reservoirs directly guided over the sensing area of multiple FET structures (Figure 2 c, insets I and II). Droplets were produced with an oil flow rate 2 µL/min (PHD2000, Harvard Apparatus)). This value allowed for an exact observation of every 5


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single droplet with a bright field microscope and to provide a sufficient number of samples per droplet. The ratio of water and oil flow rates was adapted to modulate the size and rate of the droplets (Figure 2S in Supporting information). Note, we realize low rate of droplets production and detection for better understanding the time dependent response of the ISFET sensor during recording of the nonpolar/polar solvents interphases. Droplet volumes between 1 nl and 10 nl could be produced at rates between 1 Hz and 12 Hz. Considering the channel width of 300 µm the length of droplets could be adjusted to be longer or shorter than a single ISFET device. The detection of the droplets passing the nanowire FETs is realized via real time recording of the source drain current at fixed liquid gate potential.

In order to gain an understanding of the device sensitivity and its dynamic ranges, we measured transfer characteristics of SiNW ISFET devices in aqueous conditions (exemplary solutions) as well as in organic solvents (that resemble the oil properties), as shown in Figure 3a and b, respectively (Figure 3S in Supporting information). Measured characteristics clearly demonstrate that the ability of the gate voltage VG to change source drain currents ISD, so-called gate coupling, is directly related to the conductivity (Table 1S in Supporting information) of the liquid medium. While the deionized water (DI) water and ethanol, having electrical conductivities above 10-12 A/V lead to strong gate coupling and thus, easy gating of FET devices, the non-conductive substances, e.g. toluene and hexane lead to the similarly weak gate coupling. Evaluation of the results in Figure 3 a-b suggests that the water-in-oil droplets can be detected using SiNW FET in the subthreshold regime, applying gate voltage in the range between 0 and 1 V (Figure 3 a-d). We therefore probe the p-type branch of transistor characteristics. In the following we investigate the aspects crucial for probing the interior of every single droplet, i.e. interaction of the droplets interface with the liquid reference (gate) electrode, and size of droplet-reactor with respect to the sensor dimension. Figure 3 c demonstrates ISD modulation in a time domain upon 6


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arrival of a single DI water in mineral oil. The red dashed line in the figure insets marks its front appearing directly on top of FET device. Synchronization the videos of droplets flow at the sensors surfaces (see Supporting Video S1)) with the FET signals, reveals that the ISD is higher for the water than for the oil phase. In the absence of the aqueous phase ISD is stable at the value of about 0.24 µA (red datapoints in Figure 3c); once the oil-water interface approaches the FET sensing area, current increases abruptly by more than 10% up to 0.265 µA; this increase is followed by a gradual decay of the signal to a plateau at about 0.255 µA (blue area in Figure 3 c), corresponding to the case when the aqueous phase covers the FET completely; crossing the sensor area by the droplet’s tale leads to the source drain current decrease and further signal stabilization in an oil phase (blue data points in Figure 3 c). We assume that the signal overshoot, taking place during the detection of the oil-water interfaces, is caused by the polarization of oil- dispersed DI water droplet59,60 in an external electric field of the gate electrode. Furthermore, such signal change can also be attributed to the strong disturbance of the surface potential of the liquid gate electrode, once a water droplet interface is passing the sensing area. The further relaxation of the ISD peak into the plateau is probably due to a stabilization of surface potential at the nanowires after the passage of the charged interface. Figure 3 d shows the typical electrical signal readouts recorded for multiple water–in-oil emulsion droplets (note the possibility of scanning the thousands of droplets in one run, see Figure 7S in Supporting information). The measurements are shown for two different volumes of 2.5 nL (flow ratio

Rwater/oil

= 0.15, top panel) and

of 9 nL (Rwater/oil = 1, bottom panel), which correspond to 200 µm and 900 µm long droplets, respectively. Assuming that the sensor area, exposed to the liquid environment, is around 100 x 300 µm (see Figure 3 c), the droplets detected in Figure 3 d have the lengths below and above the size of single sensor device. Interestingly, the plateau in the time dependent ISD curve was formed only for droplets that were larger than the sensor dimensions (see Figure 3 d, bottom panel, and Figure 4 a, bottom panel). It has to be stressed that the established plateau in the ISD current profile indicates the stabilization of the 7


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surface potential, which is convenient for probing the interior of the aqueous phase. Possibility to detect droplets at different frequencies i.e. 3Hz, 5Hz and 10Hz, is summarized in Figure 8S in Supporting Information. In contrast, the ISD current profile for small droplets measured by NW FET in Figure 4 a (top panel) resembles a sharp peak with the heights, comparable to those detected for the oil-water interface in larger droplet (see the red area in Figure 3 c). This statement is investigated in details by analyzing the ratio of source drain currents corresponding to peak and plateau values of droplets with different sizes. Figure 4 b suggest that three different regimes in droplet detection can be considered, depending on their length: smallest droplets are characterized by the highest peak-to-plateau ratio (assuming that the background oil signal serves as a reference, see Figure 4 a top panel); the middle droplet size regime is characterized by the evolution of plateau for droplets comparable or slightly larger than the sensor size (Figure 4 a, middle panel); droplets larger than the sensor area are characterized by a stabilized signal of peak-to-plateau ratio (Figure 4 a, bottom panel). Although the presence of the peaks makes a strong influence onto SD current during detection of DI water droplets, it can be substantially reduced by increasing ionic strength of the solution. In particular, higher concentrations of ions lead to the increased charge screening at the nanowire surface and at the water-oil interfaces inside droplet, better gate coupling and more efficient stabilization of the surface potential, which finally lowers peak values, caused by droplets interfaces. This statement is supported by detecting the droplets of phosphate buffered saline (PBS, 10 mM of PO43−) which raises the ionic strength of the solution. The blue data-points and a curve in Figure 4 b show that the peak-to-plateau ratio stays practically unchanged for PBS droplets of different sizes. The observations above enable us to conclude that the droplets shorter than the NW sensor linear dimension are less suitable for probing their inner environment (due to the strong influence of the oilwater interfaces) and e.g. using them for monitoring the biochemical processes inside. However, this configuration might be of higher interest for monitoring the reactions happening at the water-oil 8


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interface, i.e. structural reorganization of proteins61 and liposomes formation62. Thus, further experiments for probing the pH and ionic concentrations inside single reactors are realized employing the droplet-plugs with lengths of about 400 µm and Rwater/oil = 0.25 that slightly exceeds the sensor size. In the following, the ratio of ISD currents for the plateau values and the oil signals as a background, are used for the data analysis (Figure 6S in Supporting information). Sensing the pH and ionic strength in droplets Finally, we perform the detection of pH values and ionic strengths of solution inside of the aqueous droplets using SiNW FETs. Figure 5 summarizes the obtained results, represented by recording source drain currents versus time upon altering pH and ionic concentrations (in Figure 5 a and c), and the corresponding quantitative analysis of pH and ionic sensitivities, respectively (in Figure 5 b and d). For pH analysis the prepared PBS solutions (isoosmotic solution for multiple biochemical species, i.e. bacteria, cells) were mixed with 1M HCl and 0.1 M NaOH to tune pH value from acidic pH= 4 to alkaline one pH= 8 (for details see Methods and Figure 7S in Supporting Information). Figure 5 a and b show the increase of the source drain current once the pH values in droplets become higher. Whereas the raw data depicting the readout of every single droplet is represented in panel a, the averaged current change versus pH (average over 30 measurements per data point (Figure 6S in Supporting information)) is represented in panel b, respectively. Such trend can be briefly explained in terms of energy band diagram shift, summarized below. Since the applied gate voltage is slight positive, the current is driven by holes through valence band, which is clearly seen from the left subthreshold regime at low VG (see Figure 3 b). This results in the horizontal shift of the transfer characteristics due to the inherent surface charges. The lower pH protonates the APTES surface, thus it works as local positive gate suppressing the hole current (see inset of Figure 5 a). Note that this tendency is in agreement with the results demonstrating the pH sensing with SiNW FETs in a single phase aqueous medium, previously reported by multiple groups21,63,64. In addition to the analysis of 9


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absolute current values, we calculated the ratio of ISD for each droplet, via separate determination of current levels for both phases (water and oil). This thereby eliminates the influence of potential current drifts due to i.e. temperature instabilities or electric field fluctuations at the reference electrode (see Figure 5 b). Further, we proceed with altering the ionic strength of the aqueous medium, realized via gradual addition of DI water to the aliquots of PBS stem solution. The results for 4 different concentrations of PBS, spanning from 10-3X to 1X (10-2 to 10mM PO43−), is depicted in Figure 5 c and d. In contrast to the tendency, observed for pH analysis, we observe a significant ISD decrease as a response on higher salt concentrations (Figure 5 c, d). This is due to the screening of local gate field from surface charge by accumulated ions (Figure 5 c, inset). Overall, the dependence of the source drain current versus ionic strength of the solution is in qualitative agreement with investigations of i.e. ionic concentration detection using Si-NW FETs65. Although pH values and ionic strength were detected in droplets with high signal-to-noise ratio, the demonstrated nominal sensitivity of the measurements is lower, compared to similar technology applied in aqueous medium. Such sensitivities of absolute current (about 1.4x10-3, and 0.03 decades per decade of concentration change of H+ and PO43− ions66, respectively) could be partially caused by arbitrary device fluctuations, i.e. not proper operating the FET in the sub-threshold regime but in a different controlled range in the trans-conductance regime. Generally, observed aqueous buffer-to-oil ratio unveils the weak gate coupling of the mineral oil phase (see Figure 5 a, c). Therefore, in order to optimize the sensing of emulsions and to avoid the situation of thermodynamically undefined surface potential at the nanowires, the better gate coupling for oil phase has to be realized in the future platforms, by e.g. using conductive oils or organic solvents, enriched by metal salts. The proof-ofconcept validation for this our assumption is introduced in the inset of Figure 5 b. We used dichlorobenzene with diluted tetraethylammonium tetrafluoroborate (TBABF4, 0.1%) for making large 10


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plug-like aqueous droplets with different pH values (pH from 4-8) and their further detection using SiNW FET sensor. The measured profile reveals better stabilization of the continuous phase signal, compared to the mineral oil signal. Water-in-dichlorobenzene emulsion could be formed, but its stability should be improved for controllable formation of monodisperse droplets in the channel. Thus, research on the stabilizing of the aforementioned emulsion using suitable interfacial agents, as well as looking for an alternative biocompatible solvents, and suitable stabilizing agents, is ongoing (see Figure 9S in Supporting information). Among other reasons leading to the lowering of the device sensitivity, we name the formation of the thin lubricating oil film between the sensor surface and the droplets, which can cover the sensor either partially or completely (due to the hydrophilic nature of isolating oxides) and the weak gate coupling of the oil phase. Further improvements of the system are necessary, in order to use its full power for performing and monitoring the bio-chemical reactions in time. For example, tuning emulsion composition to involve the conductive oils or organic solvents, the use of high-quality threshold voltage followers, and implementing the selective chemical functionalization of the channels, enabling the hydrophilic surface at the sensing area, are the crucial steps to do. Finally we verify the capability of the novel droplets based nanowire platform for a bioassay applications, and carry out a glucose oxidase (GOx) enzymatic test for glucose detection. The GOx enzyme catalyzes the oxidation of glucose into gluconolactone, which spontaneously hydrolyzes to Dgluconic acid by releasing a proton and giving rise to an acidification of the buffer (see Figure 6 panel a), measured in time70. This time dependent pH decrease should be detected by the FET71. For this, the first enzymatic reaction was linked to a second one involving horseradish peroxidase (HRP). This second enzyme reduced the hydrogen peroxide (H2O2) produced from the initial reaction by concomitantly oxidizing a chromogenic substrate, e.g. 3,3’,5,5’-Tetramethylbenzidine (TMB), into TMBH2. The last oxidation step resulted in a color change from clear to blue. Thus, the success of 11


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enzymatic reaction could be confirmed by the parallel readout of the droplets using integrated optical detection setup72 (see Figure 6 b, c). In detail, 2 mM glucose in DI water was mixed in a 1:1 ratio with 2 mM TMB in 0.1 M phosphate citrate buffer (pH 7) which contained 3.3% GOx and 1.6% HRP. After mixing the solution, it was immediately guided to the microfluidic geometry thereby creating droplets in a continuous oil phase with a final concentration of 1 mM glucose and 1 mM TMB in each droplet. Here, each droplet, passing the nanowire FET structure, allows to monitor precisely every stage of the reaction. In order to determine the change of the pH value during the reaction, the ratio of the droplet signal (droplet) to the baseline (oil) (Rdroplet/oil) passing the FET was firstly calculated for each reservoir. Furthermore, ∆Rdroplet/oil was derived via subtraction the instantaneous Rdroplet/oil from the mean value of droplet-to-oil ratios before the reaction (R0) (see Figure 6 d). Here, R0 (R0=1.085, at pH of about 6) is exclusively valid for the observed glucose oxidation reaction and comprises negative values for acidification and positive values for alkalization processes, as depicted by pink region in Figure 6 d. By comparison of the arising ratio ∆Rdroplet/oil value with reference droplets (2mM glucose: 0.1M phosphate citrate buffer 1:1) R0 (see Figure 6 panel d) we were able to determine the pH changing dynamics during the reaction. Thus, Figure 6 b shows an acidification process during the initial phase of the reaction with the FET. This leads to a lowering of the droplets signal (pink region). After completion of the glucose decomposition process, the FET signal was recovered. We attribute this to the pH equilibrating character of the phosphate citrate buffer, as it was later confirmed by using a pH meter to follow the dynamics of the reaction. Here, a decrease of 2 pH units was measured for the first minute, which returned later to the original value. For the optical measurement we used a LED with a cold white spectrum. The light was focused onto the transparent FEP tubing with the emulsion inside. A fiber coupled spectrometer was used as a detector on the other side of the tubing. Here the integrated signal of the spectrometer was used for the 12


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measurement of the absorbance. When the liquid is changing its color from clear to blue it is acting as a filter blocking the yellow wavelengths of the LED which is causing the decrease of the intensity as shown in Figure 6 c. Finally we realized GOx assay to demonstrate the decomposition of different concentrations of glucose in the range between 0mM and 1mM (C(GOx) is constant) (see Figure 6 e). Lowering of the glucose concentrations inside of the droplets, leads to the weaker pH change during the reaction and results in a decrease of ∆Rdroplet/oil, as shown in Figure 6 e.

Conclusions In conclusion, we presented a new optics-less method for high throughput detection and analysis of emulsion droplets in the microfluidic channel using compact integrated nanowires based ISFET. In particular, we investigate the effect of the droplet size with respect to the sensor area and its role on the ability to sense the inner environment of the droplet. Namely, we report that the small drops (shorter than the sensor linear dimensions) are not suitable for probing their inner environment with ISFET, due to the strong influence of the oil-water interfaces on the liquid gate potential, but can rather be used to detect the interfacial charges. Note, we chemically probe the content of every single droplet in a row as independent events, and resolve the pH values (H+ sensitivity) and ionic strength of the solution, resulted in a change of a source-drain current ISD through the nanowires. Taking into account that the sensor is driven in electron conduction mode by a positive gate voltage, we achieve the increase of the source-drain current upon increase of the pH value in solution; the ISD decrease, once the concentration of PBS in the solution becomes higher, is detected. Finally, on a proof of concept level we demonstrate the GOx assay in droplets, measured by SiNW FET sensor and integrated optical spectrometer in parallel. Such realization of dual detection (SiNW sensor and optical luminescence) is demonstrated for the first time to the best of the authors’ knowledge. 13


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We envision the high potential impact of the described technology for the early detection of pathogens in in vitro assays, microorganisms and drug screening or establishing the low-cost enrichment of nucleic acids67 in miniaturized format, where the change of pH or ionic strength can serve as an indicator of the ongoing biochemical process68. In these regards, the advantage of the proposed platform is in the possibility of high throughput analysis and automatization of complex assays, where single nanowire sensor device can be employed for probing of thousands independent reactors. Finally, the discrete and pronounced signal change between oil and water phases allows for a stable counting of numerous droplets (Figure 7S in Supporting information), allowing a repeated checking of the signal of every individual aqueous reservoir. This feature opens the plethora of applications in e.g. chemical computing or information processing, where a single drop, detected by an integrated nanodevice can serve as an information bit (so-called droplet logic14,69 concept), able to trigger an avalanche of the programmed chemical processes. Methods Fabrication of Schottky Barrier Si-NW FETs: The sensing device fabrication is described elsewhere in detail[20,59] and is shown in figure 2. Briefly, the chip consists of a parallel array of bottom-up silicon nanowires transferred to the chip substrate by contact printing technique. Following the electrode design, consisting of nickel, is deposited on the substrate (I. in Figure 2a). The FET geometry consists of an interdigitated electrode pair connected via silicon nanowires. By a silicidation process an intruded atom-sharp NiSi2-Si Schottky junction is formed within the intrinsic Si-nanowires. Exhibiting a Schottky-barrier height of 0.45eV for holes and 0.66eV for electrons, the Schottky-FET shows a preferred p-type polarity[20,59]. After the silicidation, the device was passivated with a 20nm of Al2O3. The concentration of parallel nanowires can reach up to 103 per device, having a diameter of 22nm ± 5 nm. Additionally, the chip was equipped with Ag/AgCl liquid gate electrodes at its FET structures (II. in Figure 2a). The chip was spincoated (Karl Süss RC8, Garching, Germany) with the image reversal 14


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photoresist (AZ 5214E, Micro-Chemicals GmbH, Ulm, Germany) at 4000rpm for 50s. The electrode pattern was realized by a double UV-illumination (MJB4, Süss Microtec, Garching, Germany) including a heating step for 2min at 120°C. Now, 100nm pure silver with a 5nm chrome adhesion layer were evaporated on the chip and residual resist removed in acetone (VWR International GmbH, Darmstadt, Germany) for 15min. For the electrochemical reaction to AgCl, one electrode of the FET and a covering 0.1M KCl droplet were connected to a sourcemeter (2604B SourceMeter, Keithley Instruments, Germering, Germany) at 1 µA for 2min. The Al2O3 passivation layer was additionally treated with (3Aminopropyl)triethoxysilane (APTES, Sigma Aldrich, USA) (III. in Figure 2a). After surface activation in O2 plasma (Plasma Prep II, SPI supplies, USA) for 20s, the chip was immersed into APTES solution (Ethanol:DI Water:APTES =100:5:2) for 1h in saturated environment. After a cleaning step the chip was finally heated for 10min at 100°C. We excluded the possible influence of APTES on the Ag/AgCl electrode by showing experimentally that APTES is not able to bind to silver with the followed protocol (Figure 1S in Supporting information). UV lithography: Channel master structures (15µm high and 300µm wide) were fabricated using a negative photoresist (SU-8 2010, Microchemicals, Westborough, USA) on 3’’ silicon wafer (Silicon Wafer , Active Business Company GmbH, Germany). After substrate cleaning, an adhesion promoter (TI-Prime, Microchemicals GmbH, Germany) was coated at 4000rpm for 50s. Now, 15µm SU-8 were coated, pre-baked at 95°C for 3min and exposed to UV light (Karl Süss MJB4, Garching, Germany). After postbaking at 95°C for 6 min, the structure was developed (mr600 dev, Micro-resist Technology GmbH, Germany), cleaned and finally baked at 120°C for 30min. PDMS-based microfluidic integration: Silicone elastomer and its curing agent (Sylgard 184 & Sylgard 184 silicone elastomer curing agent, Dow Corning, USA) were mixed well in a 1:6 ratio and vented in the vacuum oven (Vacutherm VT6025, Heraeus, Hanau, Germany) for 10min. After curing at 100°C for 15min over the master structure, PDMS was treated in O2 plasma and gently brought in 15


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contact using a little weight and incubated at 100°C in the oven and left overnight at room temperature. Finally, Teflon tubes were connected to the channel openings and additionally sealed with PDMS (IV. in Figure 2a) Measurement setup for droplet detection: For droplet formation, either DI water or PBS aliquots with different pHs and molar concentrations were applied. The continuous phase consisted of a mixture of mineral oil (M5904, Sigma Aldrich, USA) with 2% non-ionic surfactant (Span80, Sigma Aldrich, USA). Control of liquid flow rates was given by a low pressure microfluidic pump system (NemeSYS, Cetoni GmbH, Germany). By connecting of the electrode pads of a single Si-NW FET to the integrated conductive needles of a tip probe station (Karl Süss, Garching, Germany) to the sourcemeter allowed measuring and manipulating the conductivity of the respective SiNW FET and detection of droplets. For real-time observation the chip was placed under the built-in microscope with an integrated camera (Olympus SZX-ZB12, Hamburg, Germany). For a time dependent droplet detection the SiNW FET was set at constant gate voltage with continuously recording of the ISD. Solutions for pH and ionic measurements: Phosphate buffered saline (PBS) tablets (Sigma Aldrich, USA) were dissolved in 200ml DI water resulting in 1X PBS solution, pH 7.4 (containing 10mM PO43-). In order to manipulate the pH, aliquots from the stem solution were manipulated with 1M HCl and 0.1M NaOH to alter the pH from 4 to 8 using a pH-meter (InoLab, Germany). PBS Concentration gradients (10mM to 10µM) were created by diluting aliquots of PBS with DI water in the desired quantity.

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FIGURES

Figure 1. Conceptual figure describing the integration of (a) Multiphase microfluidics, in which two immiscible liquids are combined to create identical isolated droplets with (b) Si-NW FETs. Left side: The presence of the charges result in a change of the conductance of the channel. Right side: Microscopic appearance of the FET structures (top) and close-up SEM picture of the SiNW connecting source and drain electrodes (bottom). (c) Concept and real-life illustrations of the integration of a SiNW FET into a droplet microfluidic system. Droplets with different pH will give rise to a different conductance change.

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Figure 2. (a) SiNW ISFET fabrication process and integration with the microfluidic channel. (b) Topview of a single ISFET, showing source (S), drain (D), and the Ag/AgCl reference electrode (G) on silicon wafer. (c) Layout of an array consisting of 21 electrode sets, aligned with the microfluidic channel (blue) design, including (I) a picture of the created water-in-oil emulsions and (II) a close-up of an individual electrode set. (d) Cross-section of an individual ISFET after surface modification with APTES. (e, f) Electrical characteristics (transfer and output) of the fabricated SiNW FET.

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Figure 3. Comparison of transfer characteristics in DI Water and different solvents: (a) Hexane and toluene, (b) Chloroform, ethanol and DI water. (c) ISD modulation through time as a single droplet passes the SiNW FET. After a short current peak when the droplet is reaching the sensing element, the signal equilibrates in a plateau signal representing the specific sensor response of the droplet. (d) Example of typical electrical signal readouts for small (top) and large (bottom) droplets. Note that the plateau signal is only able to fully establish if the droplet is large enough to cover the SiNW FET for more than 300ms (for DI water). Black and red arrows indicate the peak and the established plateau, respectively.

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Figure 4. (a) Current profile through time when droplets of different volumes pass the ISFET: 1.9nl (top), 3.7nl (middle) and 9nl (bottom). Blue arrow indicates peak signal, red arrow indicates plateau signal. Note that the plateau signal becomes more and more for increasing droplet volumes. (b) Comparison of water droplets in a microfluidic channel (300µm width, 15µm height) with their corresponding volumes and schematic width of the sensing SiNW FET (yellow box). Starting from ~3.7nl, the droplet is able to fully cover the FET. (c) Peak to plateau current ratio for different droplet sizes. PBS (pH7, 10mM) and water droplets were recorded in a wide spectrum of volumes (and corresponding lengths) including shorter droplet lengths than the FET width (yellow box) and higher. The ratio is able to equilibrate if the droplet volume exceeds approximately 9nl for DI water and is stable for every measured droplet volume for PBS.

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Figure 5: (a) Raw data presentation of ISD values upon altering of pH values ranging from 4 to 8. For decreasing pH values, the absolute signal is decreasing due to protonation of the amino groups of APTES. The mean value of different pH values for PBS peak signals (red stars) and baseline values (yellow stars) are shown in (b). (b) Averaged absolute signal is increasing for increasing pH values (red). Inset: water-in-dichlorobenzene emulsion droplets with different pH values (from 8 to 4), measured by FET. (c) Raw data appearance for decreasing ionic concentration of PBS droplets. The absolute signal is decreasing for increasing molar concentrations of PBS. (d) Mean value of raw data signal of various ionic concentrations (green).

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Figure 6. (a) Realization of label free GOx assay in multiple droplets, using SiNW FET for signal readout; (b) time dependent change of pH in droplets, measured by SiNW sensor, reflected in a change of water to oil ratio in droplets. Enzymatic decomposition of glucose leads to a lowering of pH (pink region), followed by the restoring of pH in citric buffer (light pink region); (c) Optical absorbance readout measured for comparison purposes. (d) Calibration measurements, depicting the droplet to oil ratio as a function of pH (black datapoints); determination of differential ratio of water to oil, with

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respect to the baseline (water to oil ratio before the reaction R0) (blue datapoints); (e) GOx assays in droplets with different concentration of glucose.

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ASSOCIATED CONTENT Supplementary information on APTES functionalization of the chip substrate, droplet size analysis, conductivities of the liquids, gate coupling for different liquids, real-time measurement data of PBS droplets with pH values ranging from 4 to 8, real-time measurement data of PBS droplets with decreasing ionic concentrations, raw data ratio analysis for various pH values and ionic concentrations and long-time high throughput measurement data.

Author Contributions J.S., F.Z., B.I. R.I. and S.P. conducted experiments under supervision of L.B. Simulation were realized by D.N. L.B. W.W., L.B., G.C. and T.M. wrote the proposals and oversaw the research in their groups. The manuscript was written by L.B. and J.S. with input from G.C, W.W., T.M. All authors co-wrote the paper and agree to its contents.

ACKNOWLEDGMENT This work is financially supported in part by the German Excellence Initiative via the Cluster of Excellence EXC 1056 Center for Advancing Electronics Dresden (cfAED). We further acknowledge the supported from the Initiative and Networking Fund of the Helmholtz Association of German Research Centers through the International Helmholtz Research School for Nanoelectronic Networks, IHRS NANONET (VH-KO-606). We thank Dr. Alexey Popov from Leibniz Institute IFW Dresden for fruitful discussions about physico-chemical aspects of the system.

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Graphical table of contents Conjunction of miniature silicon nanowire sensors and droplet-based microfluidic systems for label free probing of thousands independent reactors.

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