Letter pubs.acs.org/NanoLett
Manipulating and Monitoring On-Surface Biological Reactions by Light-Triggered Local pH Alterations Hagit Peretz-Soroka,† Alexander Pevzner,† Guy Davidi,† Vladimir Naddaka,† Moria Kwiat,† Dan Huppert,† and Fernando Patolsky*,†,‡,§ †
School of Chemistry, the Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel The Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel § Department of Materials Science and Engineering, the Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel ‡
S Supporting Information *
ABSTRACT: Significant research efforts have been dedicated to the integration of biological species with electronic elements to yield smart bioelectronic devices. The integration of DNA, proteins, and whole living cells and tissues with electronic devices has been developed into numerous intriguing applications. In particular, the quantitative detection of biological species and monitoring of biological processes are both critical to numerous areas of medical and life sciences. Nevertheless, most current approaches merely focus on the “monitoring” of chemical processes taking place on the sensing surfaces, and little efforts have been invested in the conception of sensitive devices that can simultaneously “control” and “monitor” chemical and biological reactions by the application of on-surface reversible stimuli. Here, we demonstrate the light-controlled fine modulation of surface pH by the use of photoactive molecularly modified nanomaterials. Through the use of nanowire-based FET devices, we showed the capability of modulating the on-surface pH, by intensity-controlled light stimulus. This allowed us simultaneously and locally to control and monitor pH-sensitive biological reactions on the nanodevices surfaces, such as the local activation and inhibition of proteolytic enzymatic processes, as well as dissociation of antigen−antibody binding interactions. The demonstrated capability of locally modulating the on-surface effective pH, by a light stimuli, may be further applied in the local control of on-surface DNA hybridization/dehybridization processes, activation or inhibition of living cells processes, local switching of cellular function, local photoactivation of neuronal networks with single cell resolution and so forth. KEYWORDS: bioaffinity interactions, antibody, light switch, nanowire, field effect transistors, pH measurement, biosensors
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exceptional sensitivity for the real time label-free electrical detection of molecular species.29−31 In particular, silicon nanowirebased FET devices have been largely explored and shown to be very sensitive in pH sensing applications.25−28 Nevertheless, most current approaches are merely focused on “monitoring” and controlling of chemical processes taking place on the sensing surfaces, by reversible stimuli. For instance, no studies were reported on the development of controlled strategies for achieving surface-triggered local pH alterations and their potential applications. In this framework, light-triggered proton transfer occurs reversibly in various photoacid molecular species, which could therefore provide effective ways to controllably induce on-surface local pH changes. Photoacids are aromatic organic molecules that display properties of weak acids in their ground electronic state, but exhibit acidity greater by many orders of magnitude in their first excited electronic state. Although the great potential of photoacids has yet to be explored,
ver the past decades, significant attention has been devoted to the integration of biological species with nanoelectronic elements to yield biochemoelectronic devices. The integration of DNA, proteins, or whole cells with electronic devices has great potential for future applications.1−9 As such, the quantitative detection of biological species and monitoring of biological processes can serve as a valuable tool in numerous areas of medical and life sciences. In this context, pH-related information is of central importance in multiple areas, from chemical analysis through biomedical basic studies and medicine, to industrial applications.10−12 Various methods have been developed for measuring and monitoring pH, such as pH-sensitive fluorescent or radiolabeled probes,13−15 pH-selective microelectrodes (metal-based, glass-based, or liquid membrane-based),16−20 31P NMR spectroscopy,21 electrochemical probes,22 nanoparticles-based optical probes,23,24 ion-sensitive field-effect transistor (FETs) devices,25−28 and so on. Among them, sensing devices based on one-dimensional (1D) semiconducting nanomaterials, such as nanowires and nanotubes, have shown a great promise.29−47 These devices, owing to their remarkably high surface-to-volume ratios, display © 2015 American Chemical Society
Received: April 23, 2015 Revised: June 5, 2015 Published: June 18, 2015 4758
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Figure 1. Schematic diagram showing the chemical modification of silicon nanowires (SiNWs) by photoactive HPTS derivative molecules, followed by biomolecules such as enzymes. (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) Acyl-protecting groups react with sodium acetate to expose the phenol functional group. (3) APTES free-amino groups are coupled with glutheraldehyde. (4) Enzymes (or antibodies) are immobilized to the glutaraldehydeterminated surface, followed by passivation of free-aldehyde groups with ethanolamine.
This paradigm is demonstrated by two biological models; first, modulation and monitoring of pH-dependent enzymatic activity, and second, controlling and monitoring pH-sensitive antibody− antigen binding events. Results and Discussion. Figure 1 schematically describes the creation of our light-triggered nanowire-based multifunctional devices. First, a chemical derivative (2) of the photoacid molecule HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) was chemically anchored to the surface of amino-terminated silicon nanowires, modified with aminopropyl triethoxysilane (APTES) to create the photoreactive SiNW elements (see the experimental methods section in the Supporting Information for detailed information). HPTS is a water-soluble pH indicator, with a pKa of ∼7.3. Notably, the first excited state of HPTS is significantly more acidic than its ground state, pKa ∼0.4, and thus it is commonly used as a photoactivable source of protons in various studies.56−58 The successful modification of SiNWs with HPTS derivative molecules was verified by the use of fluorescence microscopy measurements, Figure 2A and B, as well as by chemical characterization by XPS spectroscopy.54,55 These results show that HPTS photochemical activity is not adversely affected during its chemical manipulation and surface attachment and remains highly fluorescent. Also, the photochemical characteristics of the HPTS derivative greatly resemble those of the parent HPTS molecules. Furthermore, using quartz crystal microbalance (QCM, see the experimental methods section in the Supporting Information for detailed information), we determined an APTES coverage of ∼1.2 × 1014 molecules/cm2, whereas a controlled surface coverage of 0.2−1.5 × 1013 molecules/cm2 can be achieved for HPTS, depending on the period of time allowed for its coupling to the surface-confined APTES molecules, although a surface coverage
photoacids have been widely studied since the 1970s in several applications.48−53 In a recent report from our group, we proposed the application of optically switchable photoacid-modified SiNW devices for the ratiometric sensing of pH under physiological conditions.54,55 We showed that upon photoexcitation, these devices could detect a photoresponse due to the pH-jump by proton transfer to the solvent via surface-linked photoacid molecules. In these optically gated nanoelectrical devices, the photoactive molecular layer served as a pH-dependent “gating agent”, altering the surface potential of the devices, and thus modulating their current flow. Besides pH sensing, the light-triggered pH jump caused by photoactivation of the surface-confined photoacid molecules may be simultaneously applied for on-surface modulation, activation or deactivation, of pH dependent chemical and biological processes. Here, we report on the on-surface light-triggered modulation, activation and deactivation, of various chemical and biological processes, by using multifunctional photoacid-modified SiNW FET devices as nanometer-size proton gun sources. The light-triggered on-surface pH modulation and the monitoring of biological pH-sensitive processes is done by the same nanowire-based device, which is also used as a pH sensor. We demonstrate that the pH in close vicinity of the nanowire surface can be readily modulated by the surface density of the surface-confined photoacid molecules, as well as by the intensity of the irradiated photoexcitation light source. Photoexcitation of the surface-confined photoacid species causes rapid pH changes, in a pH range, whose its extent is controlled by modulation of the light intensity used for the photoactivation. 4759
DOI: 10.1021/acs.nanolett.5b01578 Nano Lett. 2015, 15, 4758−4768
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Figure 2. (A) (Top) Fluorescent SiNWs after their chemical modification with 8-acetoxy-pyrene-1,3,6-trisulfonyl chloride (HPTS derivative), and (Bottom) SiNWs chemically modified with APTES. No fluorescence is observed under confocal visualization conditions. (B) Fluorescence images for two representative HPTS-modified SiNW FETs after chip fabrication. Scale bar 10 μm. (C) 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. (D) Ids versus Vg (Vg = water gate) for a p-type HPTS-modified SiNW FET at a constant Vds of −0.1 V. (E) Real time monitoring of HPTS-modified FET current changes (y axis, percent of current change; x axis, time (sec)) as a function of pH, before and after light irradiation. (F) Calculated [Isdlight]/[Isddark], at 400 nm continuous excitation (for a period of several seconds), for different pH values, of five representative HPTS-modified, electrically disparate, SiNW FETs. The calculated ratio is shown to be device-independent, and absolute pH values can be directly extrapolated from these calculated ratios. (G) Reversible chemical and electrical behaviors of a representative HPTS-modified FET device upon irradiation at the excitation wavelength of 400 nm. (H) Controlling the HPTS-modified FET device light responsivity by modulation of the light intensity source at 400 nm.
of ∼2 × 1013 HPTS molecules/cm2 was employed for all subsequent experiments. The photoacid-modified SiNWs can now serve as building blocks for the fabrication of FET devices,
Figure 1, based on a recently published procedure using standard photolithography steps.55,59 Notably, the HPTS molecules exhibit high stability and retain intact fluorescence and ratiometric 4760
DOI: 10.1021/acs.nanolett.5b01578 Nano Lett. 2015, 15, 4758−4768
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Nano Letters pH-dependent activity after SiNW-FETs fabrication,54,55 as verified by spectroscopic experiments (for more details about the optical behavior of HPTS versus HPTS-modified SiNWs samples please refer to the Supporting Information section). The chemical modification of SiNWs prior to their transfer to the device substrate allows the exclusive confinement of the photoactive and biological molecules to the surface of SiNWs.55 Clearly, the use of HPTS-modified p-type-SiNW building blocks lead to the creation of devices with optimal electrical characteristics and gating behavior, Figure 2B, C, and D, as well as effective pH-dependent photogating activity, Figure 2E and F, as previously reported.55 In Figure 2F and G, the significant pH-dependent reversible changes in the HPTS-modified FET devices conductance, due to UV irradiation (400 nm), are indicative of the surface-associated photoacid molecules. Moreover, our experiments have shown that modulation of the light intensity indeed influences the light-to-dark current measurements, Figure 2H. As extracted from surface coverage QCM measurements, only 3−15% of the APTES amino groups are anchored to HPTS molecules. Thus, the resulting SiNW surface displays a large number of unbound free amino groups, available for further chemical coupling of biomolecules. Accordingly, we used the HPTS-modified SiNW FET devices as a platform for local monitoring and controlling of different pH-sensitive enzymatic processes, though the anchoring of the respective pH-sensitive enzyme molecules, applying well-known coupling procedures (see the experimental methods section in the Supporting Information for detailed information) , Figure 1, steps 3 and 4. Enzymes are large biological molecules responsible for the chemical interconversions that sustain life. In this context, the ability to control enzymatic activity is of central importance in multiple areas from the medical to the food and military industries. Enzymes display activity only over a limited pH range, and most have a particular pH at which their catalytic activity is optimal. Nevertheless, prior to engaging in comprehensive sensing experiments, we performed a series of experiments, and examined the enzymatic process of trypsin (or pepsin) and trypsin (or pepsin)-modified SiNWs suspensions with their specific substrate (BAEE for trypsin and Z-L-glutamyl-L-tyrosine for pepsin) by simple fluorescence scanning measurements. Trypsin has an optimal operating pH of about 7.5−8.5, whereas pepsin displays its optimal activity at pH 2−3. In particular, we measured kinetically the pH changes of the enzymatic process by using our simple ratiometric HPTS assay.55 Please note that both hydrolytic enzymes cause acidification of the electrolyte medium, a decrease in pH, as a result of the release of acidic products.60 This acidification can be monitored in solution by the use of pH-sensitive ratiometric HPTS units anchored to the nanowire surfaces, eqs 1 and 2. As mentioned before, HPTS exhibits a pH-dependent adsorption shift and allows the performance of fluorescence ratiometric pH measurements by using the two excitations at 405 and 450 nm. As seen, HPTS−trypsin modified SiNW suspensions, Figure 3A (green line), follow a pH-dependent optical behavior similar to that of a standard Trypsin solution in the presence of the enzyme substrate, Figure 3B. In contrast, and as expected, control suspensions of only HPTS-modified SiNWs (or in the presence of the enzymatic substrate BAEE) do not show the coherent pH-dependent optical behavior of the surfaceattached enzymatic system, Figure 3B (blue and red lines, respectively)
These results clearly show that surface-confined HPTS− trypsin molecules retain both their intact fluorescence ratiometric pH-dependent behavior (protonation−deprotonation reaction at HPTS phenolic site), as well as their enzymatic functionality (media acidification caused by trypsin hydrolysis of its substrate). Clearly, these findings are very important prerequisites for the further development of multifunctional dualway communication devices. Similar measurements were performed using the PepsinHPTS modified SiNW suspensions, and exhibited comparable enzymatic temporal acidification results. In these conventional measurements HPTS molecules have been only used for the pH monitoring (or as a pH-calibrated acidification monitor), but not yet as a solution pH controller. Next, we examined the dual use of HPTS, for both monitoring and controlling of the enzymatic process at the surface around 4761
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sensitive to environmental changes, other than pH. Thus, only minor changes upon the addition of different enzymatic substrates, in particular the addition of BAEE (or Z-L-glutamyl-Ltyrosine) at concentrations up to 2 mM (Supporting Information Figure 1S) could be observed. Second, surface functionalization with the enzyme does not have a detrimental effect on the electrical properties of the devices. The detection of charges in high ionic strength solutions, as in most biosamples, is severely hampered by ionic screening effects.32,61,62 In ionic solutions, a charged surface attracts counterions forming an electrical double layer (EDL) that effectively screens off the surface charges within the range of the Debye length (∼3 nm in 10 mM ionic solution). Although the distance of the HPTS, and its linker APTES, to the SiNW is only about 6 Å, the distance between the FET sensing surface to the enzyme is ∼2−3 nm. As a consequence, in these BioFETs, the relative percent of conductance change measured as a result of the enzymatic activity, sensitivity to pH, may still be strongly dependent on the ionic strength of the sensing buffer. Figure 3C shows that enzymatic hydrolytic activity, by means of temporal acidification, can be detected in real-time by the trypsin−HPTS-modified SiNW devices, in the presence of its substrate BAEE, at pH 7. The observed conductance decrease upon enzymatic activity originates from the formation of acidic enzymatic products, and a concomitant pH decrease, in the close vicinity of the p-type nanowire-based devices. As we proceeded, it should be noted that in 10 mM phosphate buffer the relative percent conductance change of the enzyme activity was estimated at 5−10% (Figure 3C), whereas in 1 mM phosphate buffer the estimated percentage change was 30−50% (Figure 3C). Thus, the biosensing measurements that followed were all conducted under this low ionic strength condition, 1 mM, where higher pH sensitivity is achieved. Similar sensing results are observed for Pepsin-HPTS modified SiNW FET devices in the presence of the enzyme substrate Z-L-glutamyl-L-tyrosine. Additionally, we tested the enzymatic activity of the on-wire pepsin molecules, their temporal medium acidification, as a function of the buffer pH, from pH 7 to pH 3, Figure 4A. These results clearly show, as expected, that the enzyme pepsin remains inactive in medium at pH values higher than 3.5 but exhibits enhanced hydrolytic activity at pH values lower than 3.5; this conductance change results from the medium acidification caused by the hydrolytic cleavage of the enzyme substrate at these lower pH values. In contrast, nanowires FET devices that were only modified with the photoacid HPTS molecules without the enzyme, Figure 4B, do not show any response to the addition of the enzymatic substrate, regardless of the pH value of the medium tested. These results on the pH dependency of the on-wire enzymatic activity will later on serve as a basis for comparison to subsequent measurements of light-triggered enzymatic activity switching. Accordingly and as expected, UV-excitation of the surfaceconfined HPTS photoacid molecules on SiNW FET devices should induce a sharp local acidification in the vicinity of the nanowire-attached enzyme molecules, due to a photoactivated pH-jump, thus revealing a characteristic activation, or inhibition, of the corresponding enzymatic activity under pH reduction, Figure 5A. By using substrates that yield acidic or basic reaction products, as described before, the enzyme activity can be both, temporally, light-controlled and sensed by the calibrated conductance of the multifunctional HPTS-enzyme attached SiNWs.
Figure 3. (A) Real time monitoring of the enzymatic process by means of pH changes (green line) using HPTS−trypsin-modified SiNWs and standard substrate solution (final concentration of 2 mM BAEE). As control experiments, we measured the pH changes only on HPTSmodified SiNWs (blue line) and on HPTS-modified SiNWs mixed with the standard substrate solution (BAEE concentration), (red line). The PBS calibration curves of pH and acidification as a function of the fluorescence intensity ratio values were obtained by samples of pH titration by known amounts of 1 M HCl, (λex, 405 nm; λem, 517 nm/λex, 455 nm; λem, 517 nm). (B) Real time monitoring of the enzymatic process by means of pH changes (green line) using standard HPTS solution (1 μM), standard trypsin solution (final concentration of 25 units) and standard substrate solution (final concentration of 2 mM N,α-benzoyl-L-arginine ethyl ester (BAEE)). For control experiments we measured the pH changes, only on the standard trypsin solution (blue line) and only on its specific substrate (BAEE) (red line). (C) Relative change in conductance versus enzymatic activity for an HPTS−trypsin functionalized p-type SiNWs FETs at increasing buffer concentration. The enzymatic activity was measured in 1 mM sensing phosphate buffer (red and blue lines) and in 10 mM sensing phosphate buffer (red and blue dash dots). B = Blank solution of phosphate buffer 1 mM or 10 mM, pH = 7. S = the enzymatic substrate (BAEE) at a final concentration of 2 mM diluted in 1 mM or 10 mM phosphate buffer.
the nanoscale nanowire elements. First, the calibrated conductance of the HPTS-modified SiNWs was found not considerably 4762
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surface potential and finally leading to a modulation in the measured device current. As clearly seen in Figure 5B and C, points hυon and S2, irradiation of the HPTS−pepsin modified nanoFET devices at 400 nm leads to a clear conductance decrease after the addition of the enzyme substrate. This process causes the measured temporal conductance decreased observed at point S2, under light irradiation. These results clearly confirm the capability of measuring enzymatic activity on the nanoFET devices while simultaneously being capable of switching “on” and “off” this activity by applying light stimulus. Light illumination at wavelengths other than these required for excitation of HPTS molecules do not cause any change in the enzymatic activity at pH 6.5, and no subsequent decrease in conductance, even in the presence of the enzymatic substrate. This further corroborates the key task of surface-confined photoacid HPTS molecules in the light-triggered surface-controlled pH modulation process. Notably, the hydrolytic enzyme activity can be switched from a completely inactive state, at pH 6.5, to a highly active hydrolytic state after illumination of the nanoFET devices at 400 nm for multiple cycles, without any observable degradation of the devices performance. These results directly imply that continuous irradiation of the multifunctional HPTS−pepsin-modified nanoFET devices at 400 nm causes a sharp “pH jump” in the close surroundings of the nanowire surfaces due to deprotonation of photoexcited photoacid molecules from a baseline pH of 6.5 to an “effectivepH” value probably lower than 3.5, suitable for the activation of the enzyme hydrolytic activity. To further support this notion, trypsin-modified SiNW FET devices show that the enzymatic activity of the surface-confined trypsin molecules can be both monitored and light-switched, from an active state at a baseline pH of 7 to an inactive state after irradiation of the devices at 400 nm, Figure 5D. A simple calculation, taking into consideration the surface coverage of HPTS photoacid units and the intensity of illuminated light, further validates that pH jumps of the necessary extent, down to an effective pH of approximately 2.5, can be achieved in the volume at close vicinity to the nanowire surface (assuming that photoreleased protons are limited to diffuse within a thin water layer with a thickness of 10 nm) (for a detailed calculation, please see Supporting Information methods sections). Furthermore, these approximate calculations show the capability of directly controlling the surface “effective pH” by a simple modulation of the intensity of the irradiated light source (or alternatively by surface density modulation of photoacid species), Figure 6A. Additionally, confocal fluorescence experiments performed using HPTS-modified SiNWs, together with the diffusional pH-sensitive pHrodo Red fluorescence molecule (0.5 mM in 1 nM phosphate buffer pH 6.5), proved that a fast and steady pH jump is experienced in the close vicinity of the nanowire device surfaces, as described before, Figure 6B. pHrodo Red displays a pH-sensitive fluorescence emission that sharply increases in intensity at increasing acidity63,64 (excitation wavelength 560 nm and emission 590 nm, Figure 6C). pHrodo is slightly fluorescent at neutral pH; however, increasing the acidity of the environment elicits a bright, red fluorescent signal. Also, its excitation and emission characteristics allow for its separate fluorescent monitoring, unaffected by excitation at the wavelength of HPTS molecules, 380−400 nm. Irradiation of HPTS molecules brings a sharp decrease of pH near the surface of nanowire elements, which in turn causes an increase in the fluorescent signal reported by the pHrodo molecules, Figure 6D. These measurements demonstrate that a pH decrease is indeed
Figure 4. Real time curves showing that the enzymatic activity of onwire pepsin molecules is preserved and pH dependent. (A) Normalized conductance (current divided by transconductance gm) versus time of HPTS−pepsin-modified SiNWs and (B) of only HPTS-modified p-type SiNWs. These curves exhibit FET sensors following the addition of pepsin substrate (S = Z-L-glutamyl-L-tyrosine) diluted in phosphate buffer (1 mM) at different pH solutions (pH 7, 5.3 and 3.5). The conductance of the HPTS-attached SiNWs is insensitive to the addition of standard substrate solutions at different pH. S: point at different pH where the enzyme substrate was added.
Therefore, for these purposes, we used the enzyme pepsin (which cleaves amide bonds with optimal activity at pH 2−3) as a model for the on-surface light-activated switching-on enzymatic hydrolysis mechanism, (Figure 5A), and trypsin (with optimal activity at pH 7−9) for the light-induced switching-off inhibition mechanism (Figure 5D). Figure 5B depicts the results of two representative full enzymatic cycles of on-FET pepsin activity controlled by external light-stimuli, of eight representative devices, at a baseline medium of pH 6.5. At pH 6.5, and as previously measured in Figure 4A, no enzyme hydrolytic activity is expected to be detected by the nanoFET devices, as pepsin displays enzymatic activity at pH values lower than 3.5. Accordingly, no temporal changes of the device conductance are observed in the absence, or presence, of the enzyme substrate Z-L-glutamyl-L-tyrosine, points B and S1 on the graph, at the nonoptimal pH of 6.5. On the contrary, under UV irradiation at a wavelength of 400 nm, HPTS molecules are brought to their excited state where they display a dramatic change in their pKa, from 7.4 to ∼0.4, turning into superacid molecules which rapidly expel their phenolic protons. The HPTS-modified SiNW FETs consequently face a dramatic “gating” effect after UV irradiation, as a result of the fast deprotonation of protonated HPTS species, thus activating the surfaceattached enzyme molecules by decreasing the pH around the NW. This light-triggered continuous activation process yields acidic enzymatic products that change the electrostatic environment of the SiNW FET devices, causing a sharp change in their 4763
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Figure 5. (A) Schematic representation of the dual mechanism of the p-type HPTS-enzyme-modified SiNW FETs. By using substrates that yield acidic/ basic products the enzyme activity can be sensed by the calibrated conductance of the HPTS-enzyme-attached SiNWs. In parallel, steady illumination at 400 nm causes excitation of the photoacid HPTS molecules, and therefore, the subsequent proton release will cause the pH-dependent activation or inhibition of the enzyme activity. (B and C) Pepsin Switching “ON” mechanism. Real time monitoring of the electrical response of representative HPTS-enzyme-SiNW FET devices (y axis, I/gm(v); x axis, time (s), gm is transconductance). (n = 8 devices). (B) Two cycles of pepsin activation and three cycles of light irradiation. (C) Magnification of one full representative cycle of pepsin enzymatic activity activation caused by light-irradiation of onwire HPTS molecules, light-on condition inside the dotted-line blue square, and light-off condition inside dotted-line orange square. B = Blank solution of phosphate buffer (1 mM, pH 6). S = the substrate (Z-L-glutamyl-L-tyrosine) at a concentration of 2 mM diluted in phosphate buffer 1 mM. hυ and hυoff are points in time when light irradiation is initiated or stopped, respectively. (D) Trypsin switching “OFF” mechanism. Real time monitoring of current changes (y axis, I/gm (V); x axis, time (s)), before and after the addition of the enzyme substrate under dark conditions and after light irradiation while the enzyme substrate flows.
interaction.65 In practice, elution buffers occasionally cause some loss of antibody or antigen function, limiting the number of times an affinity support can be reused. The most widely used elution buffer for affinity purification of proteins is 0.1 M glycine HCl, pH 2.5−3.0. This buffer is known to effectively dissociate most protein−protein and antibody−antigen binding interactions, preserving protein structure and binding affinity in most cases. Thus, based on our approach, local pH jumps on the surface of nanowire devices, caused by the local photoexcitation of surface-confined HPTS molecules, are expected to decrease the antibody−antigen binding interaction strength. As a model system, we chose the troponin antigen, a cardiac marker clinically used for the detection of myocardial infraction. First, we examined two anti-troponin antibody clones by using the traditional ELISA assay, in order to measure their binding characteristics against cardiac troponin I (cTnI) antigen. Both antibody clones display similar binding characteristics against the cTnI antigen (Supporting Information Figure 2S). Engaging into sensing experiments, Figure 7A displays results on the pH dependency of anti-troponin antibody binding affinity, against
occurring near the sensors surface, and that the corresponding pH decrease depends on the intensity of irradiating light source. In Figure 6C, we observe that the pHrodo fluorescence intensity increases by a factor of ∼2.7 upon acidification from pH 7.5 to pH 3, respectively. Notably, the fluorescence intensity from the light-of f state (no HPTS excitation on the nanowires surface at 390 nm) at initial pH of 7.5 increases by a factor of 2.5 upon irradiation of HPTS surface species at 390 nm. Thus, we can confidently assume that a surface pH jump down to pH ∼3.3−3.5 is experienced in close vicinity to the nanowire surface. In addition, we have applied our approach to control the binding affinity of surface-confined antibody molecules toward their specific antigen on photoacid-modified surfaces by the simple use of light stimuli. Antibody−antigen binding interactions are optimal in aqueous buffers at physiological pH and ionic strength, such as in phosphate-buffered saline (PBS) pH 7. Consequently, in conventional affinity methods, elution can be often accomplished by lowering the pH (or increasing the pH) or altering the ionic state to disrupt the antigen−antibody binding 4764
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Figure 6. (A) Graph displaying the “calculated” on-surface pH values expected to be experienced as a function of the intensity of the irradiated light source at a wavelength of 400 nm (please see Supporting Information for further details). (B) Schematic diagram exhibiting confocal fluorescence experiments, using HPTS-modified SiNWs together with the diffusional pH-sensitive pHrodo Red fluorescence (left scheme) molecule. As shown, a steady pH jump in the close vicinity of the nanowire device surfaces is experienced upon excitation of HPTS at 380 nm, causing a sharp increase in emission intensity, at increasing acidity, of the pHrodo dye (excitation wavelength 560 nm and emission 590 nm, right scheme). Thus, pHrodo can be used as a local reporter for the pH jump event caused by photoexcitation of surface-confined HPTS molecules. (C) pHrodo Red displays a pH-sensitive fluorescence emission that sharply increases in intensity at greater acidity. pHrodo is lowly fluorescent at neutral pH; however, increasing the acidity of the environment elicits a bright, red fluorescent signal, blue curve (excitation 560 nm and emission 590 nm). When excited at 380−400 nm (the excitation wavelength of HPTS), almost no fluorescence emission is observed for pHrodo molecules, red curve. Thus, HPTS molecules can be photoexcited, and the pH-jump effect caused by HPTS can be simultaneously monitored through the emission of the diffusional pHrodo molecules. (D) Graph displaying confocal fluorescence measurements of the dye pHrodo (excitation 560 nm and emission 590 nm) with simultaneous excitation of the surface confined HPTS molecules (excitation 390 nm) at different light source intensities of HPTS excitation (at 390 nm). (Red curve) Confocal fluorescence intensity of the dye pHrodo measured at areas covered by HPTS molecules (on-wire measurements), and (Blue curve) confocal fluorescence intensity of the dye pHrodo measured at areas not covered by HPTS molecules (off-wire measurements). These results show that irradiation of surface confined HPTS molecules brings to a local decrease of pH near the surface of nanowire elements, which in turn causes a lightintensity-dependent increase in the fluorescent signal reported by the pHrodo molecules.
binding interaction strength. Lowering pH causes a sharp decrease on the binding affinity of the surface-confined antibody molecules toward the antigen species. Furthermore, at pH values lower
cardiac troponin I (cTnI) antigen, at different pH values, using HPTS−antibody-modified SiNW devices. These results clearly illustrate the influence of pH on the antibody−antigen pair 4765
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Letter
Nano Letters
(40 nM) to the antibody/HPTS-modified NW FETs, under dark conditions, demonstrates a higher binding affinity (larger signal change) than the second binding cycle performed under low power (25% light source intensity, at an estimated photogenerated pH of 3.5−4) light irradiation. Notably, the isoelectric point (pI), of cTnI is 9.87. Thus, in a physiological pH of 7.3 the antigen is positively charged, and therefore, the conductivity of the SiNWs decreases upon binding of the positively charged antigen to the surface-confined antibody units. Irradiation at higher light intensities (100% light source intensity, at an estimated photogenerated pH < 3) causes a complete binding inhibition of the antigen to the antibody receptors, Figure 7C. Importantly, ceasing irradiation in the presence of the antigen, restores the effective binding of the cTnI antigen, as observed for the first under-dark conditions cycle. Also, light-triggered binding inhibition cycles could be repeated multiple times, without any considerable degradation of the antibody binding affinity as measured under dark conditions. Taken together, these results pinpoint the capability of modulating the binding affinity of surface-confined pH dependent biomolecular receptors, such as protein, antibodies, and DNA,66,67 by a light-triggered local switching of pH conditions caused by surface-confined photoacid species. Besides the demonstrated dual-communication mechanism using biomolecule−photoacid modified nanoFET devices, we can apply this approach for the creation of multifunctional lightresponsive nanoparticle-based systems, of potential future applications in drug delivery and theranostics. For instance, 200 nm diameter aminated silica nanobeads were comodified with the photoacid HPTS and the pepsin enzyme, as earlier described for SiNW elements. A suspension containing the photoacid−enzyme modified nanobeads, in phosphate buffer pH 7, was examined for its enzymatic catalytic activity in the presence of the enzymatic substrate Z-L-glutamyl-L-tyrosine, under dark and illuminated conditions. Figure 8, curve a, shows that no pH changes of the nanobeads suspension are observed in the presence of the enzyme substrate under dark conditions. As before (Figure 3A), the pH of the suspension was kinetically measured using the ratiometric pH behavior of HPTS molecules attached to the nanobeads, as well as by direct measurement of the pH by a conventional pH meter device.55 In contrast, illumination of the multifunctional nanobeads suspension leads to a steady temporal decrease in the suspension’s pH, Figure 8, curve b. This measured decrease in the medium pH is a direct consequence of the photoactivation of the surface-confined photoacid molecules, and the subsequent sharp pH jump in the vicinity of nanobeads surface, from an initial pH of 6 to an estimated pH < 3. This photoactivated proton release switches a “turn-on” effect of the surface-bound pepsin enzyme molecules, now capable of performing their optimal hydrolytic activity, consequently releasing acidic products to the bulk medium and causing a steady decrease in its pH. This approach could potentially be used in the future for selective light-triggered activation or deactivation of enzymatic processes at the cellular level in biological, drug delivery, and imaging applications. The same nanoparticle-based system could be easily used for the photoactivated elution of antigen species bound to surfaceconfined antibody receptors, as commonly performed in affinity chromatography applications. In this case, the photoactivation of surface-confined photoacid species brings a sharp decrease of the local pH in the intimate surrounding of the nanoparticle surfaces, from the bulk medium of pH 6−7 to a local effective pH of
