Optically-Gated Self-Calibrating Nanosensors: Monitoring pH and

Letter pubs.acs.org/NanoLett

Optically-Gated Self-Calibrating Nanosensors: Monitoring pH and Metabolic Activity of Living Cells Hagit Peretz-Soroka,†,§ Alexander Pevzner,†,§ Guy Davidi,† Vladimir Naddaka,† Reuven Tirosh,† Eliezer Flaxer,† and Fernando Patolsky*,†,‡ †

School of Chemistry, the Raymond and Beverly Sackler Faculty of Exact Sciences and ‡The Center for Nanoscience and Nanotechnology, Tel-Aviv University, Tel Aviv 69978, Israel S Supporting Information *

ABSTRACT: Quantitative detection of biological and chemical species is critical to numerous areas of medical and life sciences. In this context, information regarding pH is of central importance in multiple areas, from chemical analysis, through biomedical basic studies and medicine, to industry. Therefore, a continuous interest exists in developing new, rapid, miniature, biocompatible and highly sensitive pH sensors for minute fluid volumes. Here, we present a new paradigm in the development of optoelectrical sensing nanodevices with built-in self-calibrating capabilities. The proposed electrical devices, modified with a photoactive switchable molecular recognition layer, can be optically switched between two chemically different states, each having different chemical binding constants and as a consequence affecting the device surface potential at different extents, thus allowing the ratiometric internal calibration of the sensing event. At each point in time, the ratio of the electrical signals measured in the ground and excited states, respectively, allows for the absolute concentration measurement of the molecular species under interest, without the need for electrical calibration of individual devices. Furthermore, we applied these devices for the realtime monitoring of cellular metabolic activity, extra- and intracellularly, as a potential future tool for the performance of basic cell biology studies and high-throughput personalized medicine-oriented research, involving single cells and tissues. This new concept can be readily expanded to the sensing of additional chemical and biological species by the use of additional photoactive switchable receptors. Moreover, this newly demonstrated coupling between surface-confined photoactive molecular species and nanosensing devices could be utilized in the near future in the development of devices of higher complexity for both the simultaneous control and monitoring of chemical and biological processes with nanoscale resolution control. KEYWORDS: Nanowire, nanotube, field effect transistors, pH measurement, biosensors, living cell, metabolism, ratiometric sensor

Q

is constantly maintained close to neutrality by potent ionexchange mechanisms, and the buffering capacity of the cytosol. Thus, measuring the distribution and fluctuations of pH with high temporal and spatial resolution outside and inside living cells and tissues is crucial for expanding our understanding of cellular biology.4 Various methods have been developed for measuring and monitoring pH, such as pH-sensitive fluorescent or radiolabeled probes,7−9 pH-selective microelectrodes (metal-based, glassbased, or liquid membrane-based),10−14 31P NMR spectroscopy,15 electrochemical probes,16 nanoparticles-based optical probes,17,18 ion-sensitive and extended-gate field-effect transistors (FETs) devices,19−22 and so on. During the past decade, great efforts have been invested in sensing applications based on one-dimensional (1D) semiconducting nanomaterials such as nanowires, nanotubes, and nanobelts.23−27 These devices, by virtue of their remarkably high surface-to-volume ratios, show an exceptional sensitivity for the real time label-free detection

uantitative detection of biological and chemical species is critical to numerous areas of medical and life sciences. The transduction of a signal associated with the specific recognition of the molecules of interest is the key to the detection mechanism. In this context, information regarding pH is of central importance in multiple areas from chemical analysis through biomedical basic studies and medicine to industry.1 Therefore, a continuous interest exists in developing new, rapid, miniature, biocompatible, and highly sensitive physiological pH sensors for minute fluid volumes. For instance, the pH of blood is regarded as an important indicator of human health.2 The acidity balance in extracellular fluids is tightly regulated by physiological buffering systems and body organs (such as the lungs and kidneys), so that the pH level of arterial blood is tightly kept within a very narrow range, between 7.3 and 7.4.2 This delicate homeostatic balance is often threatened by abnormal processes, such as respiratory and metabolic diseases. Furthermore, tight control of the intracellular pH is essential for the achievement of critical cellular processes, such as proliferation,3 proteins, DNA and RNA synthesis,1,4,5 controlling cell cycle,3 apoptosis,6 changes in the ionic membrane conductance, and more.1 The cytoplasmic pH © XXXX American Chemical Society

Received: April 1, 2013 Revised: June 6, 2013

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dx.doi.org/10.1021/nl401169k | Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. Preparation and binding of 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride to SiNWs. (A) Synthesis of the activated derivative of the photoactive molecule HPTS, 1-hydroxypyrene-1,3,6-trisulfonate. (B) Nanowires chemically modified by the activated HPTS derivative molecules. (1) SiNWs are first modified with 3-aminopropyl triethoxysilane (APTES) layer, followed by their reaction with 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride (HPTS activated derivative). (2) Neutralization of free sulfonyl chloride groups by reaction with morpholine (optional). (3) Acyl-protecting groups are reacted with sodium acetate for the exposure of the phenol function group.

of molecular species adsorbed on their surfaces, down to sensitivity of single molecules.28,29 The binding of charged molecular species modulates the surface potential and the carrier density within the electro-active nanostructure, which in turn changes the device-measured current. Particularly, silicon nanowire-based FETs, bare or chemically modified, have been extensively studied in pH sensing applications, and were shown to be very sensitive to proton concentration, the so-called pH.19,20 However, device-to-device electrical variations, sensitivity to even miniscule changes in the solution ionic strength and chemical composition, nonspecific adsorption of chemical and biological species, sensing limitations in complex physiological samples, temporal electrical drifts, electrical

deterioration of device performance, and additional factors lead to detrimental artifacts in the real time measurement of the absolute pH values of the media under test. While electrical calibration of each individual electrical device30 prior measurement, along with the use of stable reference electrodes, may partially alleviate few of these limitations, current electrical sensing schemes cannot overcome most of the abovementioned intrinsic factors leading to false measurements. Thus, and notwithstanding the great progress achieved in this area, the handicapping limitations inherent to all the abovementioned approaches, such as dimensionality and miniaturization limits, low sensitivity and selectivity, limited long-term stability, potential toxicity, dye local concentration and optical B


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Figure 2. Characterization of HPTS-modified SiNWs by fluorescence microscopy and XPS spectroscopy. (A−C) Confocal fluorescence images of chemically-modified SiNWs. The inset images show representative phase-contrast images. SiNWs were obtained by sonication of the growth substrate into a suitable solvent and collection by centrifugation (5 min, 10 000 rpm). (A) SiNWs chemically modified with APTES. Minor fluorescence is observed under visualization conditions. (B) SiNWs 1 day after their chemical modification with 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride. (C) SiNWs 1 month after their chemical modification with 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride. Scale bars for (A−C): 5 μm. (D−E) Characterization of the formation of the modification steps of HPTS-modified SiNWs. (D) N1s XPS high-resolution spectra of bare SiNWs, curve a, APTES-modified SiNWs, curve b, and HPTS-modified SiNWs, curve c. The N1s peak is indicated. (E) S1s XPS high-resolution spectra of bare SiNWs, curve a, APTES-modified SiNWs, curve b, and HPTS-modified SiNWs, curve c. The S1s peak is indicated. These spectra clearly reveal the increase in the S1s peak resulting from surface anchoring of HPTS molecules.

path length, calibration issues, lack of real-time multiplexing ability, biofouling-related artifacts, and many more, call for the need to develop new generations of pH sensors, and sensors in general, with enhanced capabilities. Here, we present a new paradigm in the development of opto-electrical sensing nanodevices with built-in self-calibrating capabilities. The proposed electrical devices, modified with a photoactive switchable molecular layer, can be optically switched between two chemically different states, the ground and excited states, each having different chemical binding constants, and as a consequence affecting the device surface potential at different extents, thus allowing the ratiometric internal calibration of the sensing event. At each point in time, the ratio of the electrical signals measured in the ground and excited states, respectively, allows for the absolute concentration measurement of the molecular species under interest, without the need for electrical calibration of individual devices, and without the influence of screening limiting factors as stated before. We thus demonstrate a model system for the real-time monitoring of absolute pH values outside and within single living cells at physiological conditions, based on the use of optically gated silicon nanowire, nanotube, and nanowire− nanotube-based optoelectrical devices, respectively. Furthermore, we applied these devices for the real-time extracellular monitoring of cellular metabolic activity, as a potential future tool for the performance of basic cell biology studies and high-

throughput personalized medicine-oriented research, involving single cells and tissues.31−33 Results and Discussion. Figure 1 schematically describes the creation of our optically gated nanowire-based selfcalibrating electrical sensors. First, a chemical derivative of the fluorophore, and photoacid molecule, HPTS (8-hydroxypyrene-1,3,6-trisulfonyl chloride) was synthesized as depicted in Figure 1A (see Experimental Methods Section for further information) . This procedure allows for the chemical activation of its sulfonate groups (some or all of them), thus allowing the further chemical anchoring of HPTS optically active molecules to the surface of Si nanowire sensing elements. HPTS is a highly water-soluble pH indicator with a pKa of ∼7.3 in aqueous solutions. HPTS exhibits a pH-dependent adsorption shift, and allows the performance of ratiometric pH measurements by using the excitation ratio of 405/455 nm. Notably, the excited state of the molecule HPTS is significantly more acidic than its ground state, with a pKa ∼0.4, and thus it is frequently used as a photoactivated source of protons in various studies.34−38 Next, amino-terminated silicon nanowires, modified with aminopropyl triethoxy silane, APTES, are reacted with the activated HPTS molecules to create the optically active SiNW elements, Figure 1B (see Experimental Methods section in Supporting Information for detailed information). The successful modification of SiNWs by HPTS molecules was verified by the use of confocal fluorescence microscopy measurements (Leica STED CW), Figures 2A−C, as well as C


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versus HPTS-modified SiNWs samples please refer to Supporting Information, Figures S1 and S2). As seen, a suspension of HPTS-modified SiNWs, Figure 3B curve c, follows a similar pH-dependent optical behavior to that of a standard HPTS solution. In contrast, and as expected, suspensions of bare SiNWs and of amino-modified-SiNWs do not show any coherent pH-dependent optical behavior, Figure 3B curve a and b, respectively. These results clearly show that surface-confined HPTS molecules retain intact fluorescence and ratiometric pH-dependent activity (protonation−deprotonation reaction at its phenolic site); a very important prerequisite for the further development of optically gated nanoFET sensing devices. Furthermore, HPTS molecules were extensively studied in the past for their interesting photoacid properties.38,40 Figure 3C schematically describes the operation principle of our optically gated SiNW FET devices (SiNW OG-FETs). Unlike other common fluorescent molecules, HPTS displays a dramatic change in its acidic characteristics when excited by the appropriate light wavelength. For instance, HPTS molecules display a pKa ∼ 7.3 in the ground state at pH ∼ 6.0 and exist mainly in their protonated phenol-like state (versus the unprotonated phenolate-like state, pH ∼ 8), Figure 3C. Under irradiation at 405 nm, HPTS molecules are brought to their excited state where they display a dramatic change in their pKa to ∼0.4, turning into superacid molecules which rapidly expel their phenolic protons, thus transforming into their negatively charged phenolate-like state. On the basis of this well-known phenomenon, we can expect that HPTS-modified SiNW FETs will experience a dramatic “gating” effect after light irradiation, as a result of the fast deprotonation of protonated HPTS species. The resulting unprotonated phenolate-like species will change the electrostatic environment of the SiNW FET devices, and result in a sharp change in their surface potential, leading so to a modulation in the measured device current. Of course, the extent of modulation cause by light irradiation, the “lightgating” effect, will strongly depend on the pH of the tested solution (at a constant light intensity), or the extent of HPTS molecules residing in their phenol-like state, before the light exposure, Figure 3C,D. In order to verify the ability to couple between the HPTS light behavior and SiNW FET devices, we performed a series of experiments on HPTS-modified p-type SiNW FETs, Figure 3D, using a home-built opto-electrical setup and software. This allows the simultaneous electrical measurement of multiple FETs, along with multiwavelength light irradiation capabilities and temperature controller, as illustrated in the Supporting Information Section, Figure S3. Clearly, HPTS-modified SiNW FETs rapidly respond to light irradiation at 405 nm, exhibiting a pH dependent sharp current modulation, as expected, Figure 3D curves a and b. A clear pH dependency in the extent of current modulation is observed at the different pH values tested. As expected at pH 6, where a large extent of the surfaceconfined HPTS molecules are protonated in their ground state, we observed the strongest current modulation as a result of light irradiation, Figure 3D curve a. At pH 8, the observed FET current response to light irradiation is considerably weaker, curve b. At this higher pH, the amount of protonated surfaceconfined HPTS molecules is significantly lower than for pH 6, thus a considerably weaker response to light would be expected. Importantly, light irradiation experiments performed on aminomodified SiNW FETs, Figure 3D curve c, do not show any

by chemical characterization by XPS spectroscopy, Figure 2D,E and Table 1. Clearly, HPTS molecules were successfully Table 1. XPS Characterization of APTES or HPTS Modified SiNWsa C O Si N S

SiNWs

SiNWs + APTES (NH2)

SiNWs + HPTS (NSO2)

12.53 21.10 54.17

21.84 37.04 39.29 1.83

28.19 35.57 33.20 1.69 1.35

a

XPS elemental analysis in atom %. The functionalization steps are ordered from left to right according to their chronological order as follows: bare SiNW sample, APTES-modified SiNWs and HPTSmodified SiNWs.

modified onto the surface of SiNW elements (with a surface density of 1.5−2.8 × 1013 molecules/cm 2), and more importantly, remained optically active after their activation and chemical anchoring to the SiNWs, Figure 1A,B. These results show that HPTS fluorescence activity is not irreversibly quenched during their chemical manipulation and surface attachment, and it remains highly fluorescent, see the Supporting Information, Figures S1 and S2. The resulting HPTS-modified SiNWs can be effectively used as building blocks for the fabrication of field-effect transistor devices, Figure 3A. Generally, HPTS-modified SiNWs are suspended in a suitable solvent and drop-deposited on a silicon wafer substrate,39 before source and drain contact electrodes are deposited using standard photolithography procedures.39 Notably, the HPTS molecular layer proved to be exceptionally stable and remained optically active after all the steps of SiNWFETs fabrication, Figure 3E−G. The chemical modification of SiNWs by HPTS molecules prior their deposition on the device substrate allows the selectively confinement of the photoactive molecules to the surface of SiNWs exclusively, unlike all past reported cases where the chemical modification of nanowires is performed after devices are fabricated on silicon base wafers.20,21,23,24 This is of great importance for the further unambiguous characterization of the resulting opto-electrical nanodevices, by optical and electrical means. Furthermore, this benefit will be of critical importance when employing photoacid- or photobase-modified SiNW nanodevices as H+ or OH− nanosources for the local activation and monitoring of chemical and biological processes. These optically activated “proton guns” may be used in the future for the simultaneous electrical monitoring and manipulation of chemical/biological basic and applied processes with a spatial resolution in the nanoscale. Clearly, the use of HPTS-modified p-type-SiNW building blocks lead to the creation of devices with electrical characteristics and gating behavior, as expected from p-type SiNW FETs, Figure 3A. Thus, the chemical modification sequence does not negatively affect on the electrical characteristics of the resulting FETs. As mentioned before, HPTS exhibits a pH-dependent adsorption shift and allows the performance of optical ratiometric pH measurements by using the excitation ratio of 450/405 nm.35 In order to verify that the surface-confined HPTS molecules retained their expected optical pH dependency, we performed a series of experiments by a commercial fluorescence scanner (TECAN Infinite M200), Figure 3B (for a more detailed comparison between optical behavior of HPTS D


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Figure 3. HPTS-modified SiNW elements preserve the photoacid molecule functionality. (A) Source-drain current (Ids) versus source-drain voltage (Vds) plots at different gate voltages (Vg) for a typical p-type SiNW chemically modified with HPTS molecules. The inset graph shows Ids versus Vg (Vg = water gate) for a p-type HPTS-modified SiNW FET at a constant Vds of −0.1 V. (B) Calibration curve of pH titration (x-axis) as a function of the fluorescence intensity ratio (Ex.405 nm, Em.517 nm/Ex.455 nm, Em. 517 nm) (y-axis) for SiNWs (curve a), APTES-modified SiNWs (curve b), and HPTS-modified SiNWs (curve c). Note that only HPTS-modified SiNWs display a coherent fluorescent intensity ratio as a function of pH. (C) Schematic representation of the photoacid mechanism on the HPTS-modified SiNW FETs. In a more acidic environment, surface-confined HPTS molecules reside mainly in their phenol-like state. At this state, steady illumination at 405 nm will cause excitation of HPTS molecules (and the subsequent proton release) and shift to the phenolate-like negatively charged state, leading to increase in the Isd of a p-type FET. In more basic environment around pH 7.3, more HPTS molecules reside at their optically inactive phenolate state. Therefore, light excitation induces smaller changes of Isd due to the reduced extent of the photoinduced proton release. (D) Real time monitoring of current changes (y-axis, nA, x-axis, time (s)) as a function of pH, before, and after light irradiation. HPTS-modified SINW OG FET at (a) pH 6.0 (curve a) and (b) pH 8.0 (curve b), (c) APTES-modified SiNW FET at pH 6.0 (curve c). (E−G) Fluorescence, and phase-contrast images (insets), for APTES-modified SiNW FETs (E), and for HPTS-modified SiNW FETs (F) after chip fabrication. (G) Magnification of (F). Scale bars are (E,F) 10 μm, (G) 2.5 μm.

significant “opto-electrical gating” effect, under the given light wavelength, as the one commonly observed for the HPTSmodified optically active SiNW FETs. Accordingly, the light-to-electricity coupling of the HPTSrelated photoacid-effect to the well-known SiNW FET electrical devices will later on constitute the base for the development of

our self-calibrating OG-FET sensing devices. Because of its physiological pKa, HPTS may represent a suitable improved candidate to serve as a surface-confined pH indicator in physiological solutions. Changes in the pH value of a given solution will cause protonation−deprotonation of the phenolic group in the HPTS molecule, hence leading to changes in the E


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Figure 4. pH sensing versus current normalization. (A,C) Real-time current, Isd, measurement for different pHs at constant Vg = −0.1 for (A) a representative APTES-modified p-type SiNW FET and (C) a typical HPTS-modified SiNW FET. (B,D) Real-time pH sensing plotted as ΔI change (with the conductance value at pH = 6 as reference point), measured in the subthreshold regime (Vg) −0.1 V for (B) APTES-modified FET, and (D) HPTS-modified FETs. (E) Real time current, Isd, measurement for increasing and/or decreasing pH at constant Vg = −0.1 for HPTS-modified SiNW FETs.

electrostatic environment and surface potential of the onedimensional NWs, and subsequent changes in the measured device conductance. Figure 4A−D shows a comparison of the pH-response of SiNW-based FET devices, before and after their modification with the optically active HPTS derivative molecules. The amino-terminated SiNW FETs Figure 4A,B as well as HPTS-modified SiNW FETs Figure 4C,D effectively respond to changes in wide pH spectrum, pH = 6−9 at physiological conditions at high ionic strength (phosphate buffered saline (PBS 1×), 10 mM phosphate buffer, and 138 mM NaCl). However, the HPTS-modified SiNW FETs consistently exhibit a significantly higher sensitivity, ∼3-fold, to minor changes in pH, mainly around the physiological range, pH = 7−7.5, Figure 4C,D. In addition, the amino-terminated and the HPTS-modified SiNW FETs pH sensitivity was measured from different devices and different chips, Figure 4C−E. This may be the reason for the vast majority of HPTSmodified SiNW devices exhibit a considerably higher pH

sensitivity in this physiological range, than unmodified (surface silanol groups) or amino-modified (aminopropyl triethoxy silane APTES) SiNW FET devices. As reported in the past, SiNWs show exceptional sensitivity for the real time label-free detection of molecular species with single-molecule sensitivity.23,24,29,39 Although chemically unmodified and amino-modified SiNW FETs have been shown to be very sensitive to proton concentration in pH sensing applications,19,20 the device-to-device electrical variations, sensitivity to minor changes in the solution ionic strength and chemical composition, nonspecific adsorption of chemical and biological species, sensing limitations in complex physiological samples, temporal electrical drifts, electrical deterioration of device performance, and additional factors lead to detrimental artifacts in the real time measurement of the absolute pH values of the media in interest. While the pHdependent electrical calibration of each individual electrical device30 prior measurements, along with the use of stable F


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Figure 5. pH and acidity calibration curves versus current-to-transconductance normalization for individual FET devices. (A) pH and (B) acidity calibration curves of our PBS (1×) working solution at 37 °C by our HPTS-modified SiNW FETs. x-axis, pH/acidity; y-axis, normalized current-totransconductance values of HPTS-functionalized p-type SiNWs-FET (n = 11 devices).

further impede the application of traditional NW-based devices in real world applications of this type. With the purpose of overcoming these hampering limitations, we made use of our newly developed optically gated FETs and their advantages as self-calibrating sensing devices, Figure 6A,B. Intriguingly, absolute pH values can be directly measured by simply calculating the ratio between the measured “dark current” and the “light-induced” current measured after irradiation of the NW devices at 405 nm. As seen in Figure 6A, opto-electrical nanodevices with completely disparate electrical pH sensitivities, curves a and b, display almost identical pH calibration curves after calculating the above-mentioned current ratio, Figure 6B, curves a and b. Additionally, the calculated ratio is proven to be deviceindependent, and absolute pH values can be directly extrapolated from these calculated ratios. Note that the current changes experienced upon irradiation directly depend on the intensity of the irradiated light, and of course on its wavelength as well (with a maximum current change at the absorption maximum of HPTS molecules, ∼405 nm, see Experimental Methods section in Supporting Information for more details). These results, clearly demonstrate the capability of our OGFETs to readily self-calibrate and provide real absolute [H+] values without any prior requirement to electrically calibrate each device for its transconductance and pH sensitivity. In order to rule out potential artifacts originating from the nanowire FET-to-light interaction, we performed a similar series of experiments on bare-SiNW FETs, as well on aminomodified SiNWs FETs, Figure 6A curve c and Figure 6B curve c. These control experiments clearly show that no effect is experienced on the conductance of nanowire devices in the absence of surface-confined optically active HPTS molecules, and a dark-to-light current ratio of ∼1 is always observed, regardless the pH of the solution under test. Furthermore, we have tested the performance of our OG-FET devices in pH measurement experiments for periods of weeks with no observable degradation on their self-calibration capabilities and pH determination ability. Notably, even significant degradation of the electrical responsibility of the devices, decreased transconductance, did not lead to deterioration on sensing performances, still exhibiting an almost constant darkto-light current ratio value at a given pH. This phenomenon can be explained as follows: At each point in time, and at a given pH, there is a constant ratio between HPTS species in their phenol-like and phenolate-like state, [phenol]/[pheno-

reference electrodes, may partially alleviate few limitations, the current nanowire-based electrical sensing schemes cannot overcome most of the above-mentioned intrinsic restrictions leading to false measurements. In this aspect, it would be highly desirable to develop a novel generation of self-calibrating nanosensors capable of ratiometically measuring absolute pH values in real-time (and other chemical targets as well) at physiological conditions, without the requirement of insufficient precalibration of each sensing device and not susceptible to detrimental measurement artifacts due to the above-mentioned issues. Figure 5 shows transconductance-calibrated pH-dependency response curves for various representatives HPTS-modified SiNW FETs. Clearly, calibration of all nanodevices based on their transconductance30,41 does not solve the large variability in their current-topH change dependency, Figure 5A,B and Table 2. This intrinsic Table 2. The Linear Equations Obtained for the Final Calibration Curves for Each of the above Shown FET Devices device # 1 2 3 4 5 6 7 8 9 10 11

pH y y y y y y y y y y y

= = = = = = = = = = =

0.027x 0.044x 0.057x 0.047x 0.038x 0.020x 0.041x 0.036x 0.041x 0.041x 0.019x

HCI (μmol/mL) − − − − − − − − − − −

0.125 0.175 0.166 0.165 0.223 0.093 0.186 0.076 0.086 0.086 0.096

y y y y y y y y y y y

= = = = = = = = = = =

−0.004x −0.006x −0.008x −0.007x −0.008x −0.003x −0.006x −0.005x −0.006x −0.006x −0.003x

+ + + + + + + + + + +

0.087 0.166 0.279 0.203 0.074 0.061 0.132 0.207 0.234 0.230 0.055

handicapping limitation forces us to regularly calibrate each nanodevice for its pH sensitivity. Furthermore, the pH at the beginning of measurements, t0, must be known and measured ex situ (by the use of pH meter), if the pH value of the solution under test is needed to be measured at any given time. Please note that the requirement for calibrating each device separately for its pH sensitivity substantially hinder the deployment of these devices in real-world bioapplications. Also, the electrical drifts these devices typically experience, ionic strength changes during the course of pH masurement, and biofouling-related artifacts usually encountered when measuring biosamples G


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Figure 6. Optically gated FETs and their advantages as self-calibrating sensing devices (A) Isd versus time curves for the real time monitoring of pH and the effect of light irradiation at 405 and 455 nm (arrows denote the time of irradiation, blue for 455 and purple for 405) on (A) HPTS-modified SiNW FETs, curves a and b, and APTES-modified SiNW FETs, curve c. (B) Calculated [Isdlight]/[Isddark] at 405 nm continuous excitation (few seconds) for different pH values of HPTS-modified, electrically disparate, SiNW OG-FETs, curves a and b, and of APTES-modified FET at the same conditions, curve c. The calculated ratio is shown to be device-independent, and absolute pH values can be directly extrapolated from these calculated ratios.

Figure 7. Real time monitoring of the metabolic activity rate of Jurkat cells by our OG-FET devices. (A) Schematic representation of metabolic activity sensing. In contrast to normal differentiated cells, cancer cells primarily rely on aerobic glycolysis rather than on mitochondrial oxidative phosphorylation to generate ATP as the fuel for energy needed for cellular processes. The HPTS-modified SiNW FETs can reliably sense minor extracellular pH changes in physiological cell culture media around the HPTS pKa (∼7.3). (B) Relative normalized current changes recorded simultaneously from HPTS-functionalized p-type FET SiNWs. (C) Average of metabolic activity rate recorded from 11 devices after injection of control working solution (blue), Jurkat cells diluted in working solution (orange), and Jurkat cells in working solution after glucose injection (5 mM) (red). Significant changes were obtained after injection of glucose. (The Wilcoxon Two-Sample test, *p < 0.05, **p < 0.01, and ***p < 0.001; n.s. refers to nonsignificant differences, n = 11 devices). (D−F) Optically gated calibrated pH sensing recorded simultaneously from OG-FET devices after injection of control working solution, Jurkat cells diluted in working solution, and Jurkat cells in working solution after glucose injection (5 mM).

late]. Under dark conditions the pKa of HPTS is ∼7.3, and at any given pH value there is a constant [phenol]/[phenolate] ratio on the nanowire surface, defined merely by the pKa at the ground state.42,43 The pKa change to ∼0.4, caused by light irradiation of HPTS molecules, leads to a sharp change in the [phenol]*/[phenolate]* ratio on the nanowire’s surface, and this ratio is also merely defined by the pKa* of HPTS molecules

in the excited state. Upon irradiation, a given number of HPTS molecules residing in their protonated phenol-like state will transform into excited highly acidic species, thus expelling a proton and converting into negatively charged phenolate species. This optically gated electrostatic transformation mechanism is responsible for the modulation of surface potential of NW devices. Thus, at any point in time the H


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Figure 8. Orthogonally modified silicon nanotube-based OG-FET devices for intracellular chemical sensors. (A) Schematic illustration of the selective inner-wall modification of SiNT elements with HPTS molecules. (B,C) TEM images of germanium−silicon core−shell nanowires (B) and after germanium sacrificial cores are etched away (C). (D) Calibration curve of pH titration (x-axis) as a function of the fluorescence intensity ratio (Ex.405 nm, Em.517 nm/Ex.455 nm, Em. 517 nm) (y-axis) for APTES-modified SiNTs (curve b), and HPTS-modified SiNTs (curve a). Again, only HPTS-modified SiNTs display a coherent fluorescent intensity ratio as a function of pH. (E,F) Confocal fluorescence images of SiNTs chemically modified with APTES selectively confined to the inner void. (E) Focus mainly on the outer surface; (F) focus mainly on the inner void. (G,H) SiNTs chemically modified with HPTS molecules selectively confined to the inner void. (G) Focus mainly on the outer surface; (H) focus mainly on the inner void. Insets: Representative phase-contrast images of the same SiNTs. Scale bar (E−H) 2.5 μm.

measured ratio [Isdlight]/[Isddark] is shown to be constant at a given pH and allows for the absolute concentration measurement of the molecular species under interest, H+ in this case, without the need for electrical calibration of individual devices and without the influence of screening limiting factors as stated before. Clearly, the [Isdlight]/[Isddark] ratio depends exclusively on the surface-confined HPTS molecules opto-chemical activity, (only those in the phenol-like state). Ionic strength changes, nonspecific-adsorption of chemical species on the sensor’s surface, device electrical drift, and such factors do not influence on this extracted ratio. The successful demonstration of our self-calibrating nanosensors allows their utilization in several important biosensing applications. For instance, these devices are ideally suited for the real-time monitoring of cellular metabolic activity, through the measurement of pH in the extracellular space.44−46 As already stated, extracellular pH changes can be applied in the monitoring of the metabolic activity of cells in a broad range of metabolic-related processes and diseases. Using our devices, live cellular measurements can be performed under physiologic salt concentrations (∼150 mM) with high sensitivity, since monitoring the protonation and deprotonation of surfaceconfined HPTS molecules occur within the ∼0.7 nm Debye screening length.44,47 Furthermore, our system allows for the monitoring of pH in real-time in aerobic and anaerobic (oxygen-depleted) modes, namely open and close states. Figure 7A schematically depicts the basic principle for the monitoring of cellular metabolic activity through the measurement of extracellular pH from only a few cells, down to less than 50

cells, or in the vicinity of a single cell. During their metabolic activity living cells excrete acidic metabolites to the extracellular media, some volatile (CO2) and some nonvolatile (lactate and pyruvate). These excreted metabolites may under certain conditions accumulate in the extracellular space, leading to its acidification. In turn, the resulting acidification can be directly and accurately monitored when culturing cells on top of our nano-optoelectrical arrays. For this purpose, we followed in real-time the acidification of the extracellular media caused by the metabolic activity of nonadherent Jurkat cells, which are an immortalized line of T lymphocyte cells used to study acute T cell leukemia, T cell signaling, and also used to determine the mechanism of differential susceptibility of cancers to drugs and radiation.48 Figure 7B−F (see Supporting Information, Figure S4) demonstrates the capability of eleven OG-FET devices to monitor in real-time the metabolic activity of living cells through the resulting extracellular acidification process in complex cell culture conditions, Figure 7C, while simultaneously measuring real accurate pH values. No significant changes in the pH of the cells media is observed prior to cells addition, Figure 7D. Addition of cells to the media causes a slow decrease in the solution’s pH and devices measured current (less negatively charged phenolate-like species present on the nanowire surface), Figure 7E, as a consequence of basal cells’ metabolism and acidic metabolites excretion. Cancer cells4,49 and activated T cells50,51 rapidly hyperinduce glycolysis (“Warburg effect”),52 for example by overexpression of glucose transporter (GLUT).53 Therefore, further addition of the nutrient glucose, brings to a sharper and faster acidification rate I


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of the extracellular media (The Wilcoxon Two-Sample test, **p < 0.01, n = 11), Figure 7C,F (see Supporting Information, Figure S4), as a results of an increased metabolic cellular activity. Clearly, real time pH values extracted from 11 different devices do show almost identical trends and amplitude. Monitoring cells metabolism by extracellular probe have several advantages. First, there is a persistent acidic accumulation versus a relatively small average change in the transient intracellular responses due to homeostatic physiological regulation. Second, there is no physiological interference of the extracellular probe with intracellular processes, and third there is no background fluorescence in contrast to significant leakage of intracellular probes. Despite that, intracellular pH alters the function of many organelles and plays a crucial task in multiple cell metabolism processes, such as the proliferation, senescence, and apoptosis.1 Thus, measuring pH fluctuations with high temporal and spatial resolution inside a living cell is critical for broadening our understanding of cell biology. Molecular fluorophores, fluorescent proteins, quantum-dots, and polymers have been used for intracellular pH monitoring. Also, nanoparticle-based ratiometric pH sensors with simultaneously assembled different dyes (pH-sensitive together with pH-insensitive) on the same particle have been reported.54−56 This work emphasizes the advantages of our ratiometric approach for such extracellular and intracellular pH measurements mainly because of high signal-to-noise ratio. Toward this end, we employed our past-developed orthogonally modified silicon nanotube-based building blocks for the fabrication of intracellular-specific chemical sensors, as schematically illustrated in Figure 8A. First, germanium−silicon core−shell nanowires (TEM, Figure 8B) were selectively modified on their outer wall surfaces with a hydrophobic silane derivative, leading so to a pH-insensitive outer surface. Next, the germanium sacrificial cores were etched away (TEM, Figure 8C), followed by the modification of HPTS molecules confined to the interior walls of the nanotubular structure, resulting in a pH-sensitive inner void. This selective chemical modification scheme can be combined with standard device fabrication procedures on flexible PDMS substrates, leading to optoelectrical nanodevices for the monitoring of pH selectively occurring in the inside void of the nanotubular structure. This chemical modification strategy was further monitored by a series of XPS studies, verifying the successful orthogonal selective modification of Si nanotubes. Table 3 shows XPS spectroscopy results showing the successful orthogonal modification of the inner versus the outer wall of SiNT elements. Figure 8E-H shows representative fluorescence microscopy images of the unmodified, and inner-void

selectively modified Si nanotubular structures. Evidently, the fluorescence signal is strictly confined to the interior void of the nanotubes, Figure 8H. Suspension of HPTS-modified SiNTs, Figure 8D curve a, follows a similar pH-dependent optical behavior to that of a standard HPTS solution and of HPTSmodified SiNWs (see the Supporting Information Section, Figures S1 and S2). In contrast, suspensions of aminomodified-SiNTs do not show any coherent pH-dependent optical behavior, Figure 8D curve b. Physical obstruction of the nanotube’s open ends will lead to a device merely insensitive to changes in the pH media. Unobstructed nanotubular devices (with at least one open-end) allow for the inclusion of fluid inside the nanotubular inner void, leading to devices with high pH sensitivities. This will lead to chemical detection selectively confined to the inner void of the nanotubular structure. Thus, these nanodevices can be readily used for real-time intracellular monitoring of pH in single living cells. A further important advance in the field of cellular metabolic activity monitoring will represent the development of the simultaneous real-time monitoring of extracellular and intracellular pH, and other cellular metabolites by a single differential device. Toward this goal we made use of our past developed nanowire−nanotube, NW−NT, building blocks.57 Figure 9A exemplifies the mode of action of the dual-function NW−NT-based OG-FET devices for the simultaneous monitoring of extracellular and intracellular pH, performed by a single opto-electrical nanodevice. First, Si NW−NTs are prepared through the bottom-up synthesis of Si−Ge/Si core− shell heterostructures.58,59 Next, chemical modification of the whole nanostructure with HPTS leads to a dual-functionality building block, which allows the fabrication of devices for the simultaneous intracellular (done by the nanotube segment) and extracellular (done by the nanowire segment) monitoring of pH, and other chemical metabolites, as schematically illustrated in Figure 9A. Figure 9B shows SEM images of SiNW-NT. Inset represents a confocal fluorescence image of the nanotube segment selectively modified by HPTS. Figures 9C,D shows SEM images of SiNW-NT devices. Note that in the case of the nanotube segment, the source and drain electrodes, as well as the area in between the electrodes, are isolated by a dielectric layer from the outside solution. This will prevent current changes caused by chemical alterations in the outer wall of the nanotubular segment, only leaving the inner void free for the sensing process. In contrast, the nanowire sensing area, between source and drain electrodes, is left uncovered (and modified with HPTS), thus pH changes occurring in the extracellular media will bring to modulation of the measured S/D current, due to alteration in the surface electrostatics. Impalement of the nanotube segment into a living cell will thus allow for the simultaneous detection of intracellular and extracellular biochemical changes, all done by a single multifunctional nanodevice. Conclusions. To the best of our knowledge, we have demonstrated for the first time the coupling between a photoactive molecular species and potentiometric nanoelectronic devices. In these optically gated nanoeletrical devices, the photoactive molecular layer serves as the “gating-agent”, which alters the surface potential of the devices thus modulating its current flow. The new developed opto-electrical nanodevices, so-called OG-FETs, were shown to be very effective in the monitoring of pH and were demonstrated to perform “selfcalibration” by a single photoactive dye. The irradiation of light causes the excitation of HPTS photoacid molecular species on

Table 3. XPS Measurements of the Orthogonally-Modified SiNT Elements with HPTS Confined Selectively to the Inner Wallsa Si-nanotubes

C O S

Si-nanotubes-HPTS

23° take-off angle




34.64 4.25

17.48 2.22

31.13 9.43 1.13

17.39 6.63 0.68

a

ARXPS characterization of Si nanotubes (from GeSi core−shell NWS) with hydrophobic outer surface modification, with and without inner modification of HPTS on Au-substrate. J


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Figure 9. SiNW-NT dual-functionality sensor for the simultaneous intra- and extracellular sensing of pH. (A) Schematic description of the mode of action of the dual-function NW-NT-based OG-FET devices for the simultaneous monitoring of extracellular and intracellular pH, performed by a single optoelectrical nanodevice. (B) SEM image of representative SiNW-NT heterostructures (scale bar: 250 nm). Inset represents a confocal fluorescence image of the nanotube segment selectively modified by HPTS. (C,D) Representative SEM images of SiNW-NT bifunctional sensing devices at different magnifications. (Scale bar: (C) 2 μm and (D) 1 μm).

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the nanowire’s surface, turning into superacids that covert to negatively charged surface-confined species by expelling a proton. Measuring the ratio of the current under dark versus light conditions, [Isdlight]/[Isddark], allows for the measurement of absolute pH and device self-calibration. No need for calibration of each single device for its electrical sensitivity and pH sensitivity is required. Additionally, artifacts resulting from device electrical drift, ionic strength changes during measurements, nonspecific adsorption of molecular species on the nanowire surface and device degradation do not affect the measured [Isdlight]/[Isddark] ratio and thus do not hamper the monitoring of real pH under physiological conditions. We further applied these devices for the real-time simultaneous monitoring of intra- and extracellular pH monitoring in living cells by the use of diverse 1D building blocks such as nanowires, nanotubes, and nanowire−nanotubes multifunctional structures. This work can also be extended to the use of additional photoacids, photobases, and diverse switchable receptor molecules specific to different chemical analytes metabolites,60 interacting with light at multiple wavelengths. For instance, the self-calibrated sensing of ionic species can be envisioned by the use of surface-confined molecular layers consisting of photoswitchable ion-chelating molecules. Furthermore, the next generation of OG-FET devices will be used in the near future for the simultaneous optical activation of chemical and biological processes with nanoscale resolution, when used as nanometer-size proton (and hydroxyl) guns and the simultaneous electrical monitoring of the processes modulation by the same nanowire-based device. Conversely, the opto-electrical nanodevices may be used in future for the temporal study of photochemical process occurring on the device surface with a spatial resolution in the nanometer scale.

ASSOCIATED CONTENT

S Supporting Information *

Additional figures and information. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

H.P.-S. and A.P. contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Legacy Foundation and ISF (Israel Science Foundation) for financial support. We want to thank Dr Larisa Burstein for her help with XPS measurements and analysis.

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REFERENCES

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Optically-Gated Self-Calibrating Nanosensors: Monitoring pH and

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