Letter pubs.acs.org/NanoLett
An Ultrasensitive, Selective, Multiplexed Superbioelectronic Nose That Mimics the Human Sense of Smell Oh Seok Kwon,†,‡,◊ Hyun Seok Song,†,§,◊ Seon Joo Park,†,◊ Seung Hwan Lee,† Ji Hyun An,† Jin Wook Park,† Heehong Yang,† Hyeonseok Yoon,∥ Joonwon Bae,⊥ Tai Hyun Park,*,†,# and Jyongsik Jang*,† †
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-600, Republic of Korea § Division of Bioconvergence Analysis, Korea Basic Science Institute (KBSI), Yuseong, Daejeon 169-148, Republic of Korea ∥ School of Polymer Science and Engineering, and ◊Department of Polymer Engineering, Graduate SchoolChonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea ⊥ Department of Applied Chemistry, Dongduk Women’s University, Seongbuk-gu, Seoul, Republic of Korea # Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do 443-270, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Human sensory-mimicking systems, such as electronic brains, tongues, skin, and ears, have been promoted for use in improving social welfare. However, no significant achievements have been made in mimicking the human nose due to the complexity of olfactory sensory neurons. Combinational coding of human olfactory receptors (hORs) is essential for odorant discrimination in mixtures, and the development of hOR-combined multiplexed systems has progressed slowly. Here, we report the first demonstration of an artificial multiplexed superbioelectronic nose (MSB-nose) that mimics the human olfactory sensory system, leading to high-performance odorant discriminatory ability in mixtures. Specifically, portable MSBnoses were constructed using highly uniform graphene micropatterns (GMs) that were conjugated with two different hORs, which were employed as transducers in a liquid-ion gated field-effect transistor (FET). Field-induced signals from the MSB-nose were monitored and provided high sensitivity and selectivity toward target odorants (minimum detectable level: 0.1 fM). More importantly, the potential of the MSB-nose as a tool to encode hOR combinations was demonstrated using principal component analysis. KEYWORDS: Multiplexed bioelectronic nose, graphene micropatterns, field-effect transistor, olfactory receptor, human mimicking, odorant discrimination
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devices, are unable to encode human olfactory receptor (hOR) combinations representing distinct odor identities because these systems are unable to simultaneously discriminate more than two odorants. Furthermore, the reported transducers were irregularly deposited or immobilized onto the FET substrates, resulting in poor reproducibility. Recently, Food and Drug Administration (FDA)-approved odorants have been included in consumer products such as foods, beverages, and fragrances.17 Such odorants are characterized by their unique structures and are harmonized exactly with hORs; however, unexpected side-effects can occur in sensing odorant mixtures, off-odors, and antagonists.18,19 For example, unlike single odorants, the fragrance of a mixture can
umans can identify odors based on a combination of multiple olfactory signals in olfactory bulbs; these signals are collected through multiple olfactory sensory neurons (OSNs) that express different olfactory receptors.1,2 In particular, the human nose expresses a large family of olfactory receptors (approximately 390 different olfactory receptors) and can recognize and distinguish specific odorants by recognizing end-functional groups at a resolution equivalent to a single carbon atom.1,3,4 The exceptional receptor/odorant interactions that occur in the human olfactory system enables high selectivity and sensitivity to target odorants, even at low concentrations.5,6 In previous work, efforts at mimicking the human nose have inspired odorant-sensing systems that involve components such as field-effect transistors (FETs) and chemoresistors, resulting in single-channel bioelectronic-noses (B-nose).7−11 However, single-channel human-mimicking electronics, such as electronic-tongue,12−14 -skin,15 and -ear16 © XXXX American Chemical Society
Received: June 9, 2015 Revised: August 27, 2015
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Figure 1. Schematic diagram of functional anatomy of human olfactory system and components of MSB-noses simulating each functional stages of human nose. (a) Olfactory bulb, where the olfactory signals generated by OSNs are combined for the generation of combinatorial olfactory codes, matching with artificial olfactory codes generated by MSB-nose. (b) OSNs, where olfactory signals triggered by the specific binding of hORs and odorants, matching with GMs functionalized with hORs. (c) hORs for the specific recognition of odorants. (d) Illumination of specific interaction between hOR and odorant.
connected to glomeruli in the olfactory bulb (Figure 1c) such that OSNs expressing the same OR gene share one or several glomeruli.24 The binding of odorants to their specific hORs triggers olfactory signals, which are transferred to olfactory bulbs through multiple OSNs. Finally, the forebrain recognizes smell after combining multidimensional signals from the olfactory bulb.1,2,25,26 To mimic this feature of the human olfactory system, the MSB-nose was systematically constructed using the following components simulating each stage of the human olfactory system: (i) human hORs that interact with specific odorants as recognition elements; (ii) a multiplexed sensing geometry to generate olfactory signals that mimic those produced by human OSNs; and (iii) an artificial olfactory code that combines significant signals from multiple responses. Figure 1d shows the structure of a hOR that contains seventransmembrane helices and a binding pocket enabling specific ligand interaction. The hORs are class A GPCR proteins (the rhodopsin-like family), and high-resolution structures of some GPCRs, including rhodopsin and human adrenergic receptors, have been determined using X-ray crystallography.27−29 Using these well-characterized GPCR structures as templates, static structural models of hORs can be assumed. Several studies have examined the specific ligand binding of hORs using these structural models.30,31 Hypervariable regions comprising several amino acids in transmembrane helices form a pocket that specifically interacts with particular odorant. Thus, to develop an artificial B-nose that mimics the human nose and its odorant specificity, hORs should be integrated into a bioprobe that operates at single-atom resolution. In our previous reports, quantitative and qualitative odorant analyses were performed by B-noses comprising hORs and conductive nanomaterials in a liquid-ion gated FET system that exhibits high stability in solution.8−10 Several components are important when developing FET-type multiplexed B-noses: (i) uniform transducer arrays, (ii) hORs for odor recognition, and (iii) the odorants/hORs must interact stably in liquids.
be sensed as a completely different odor, and the odor intensity of the mixture can lie between the intensities of the unmixed components.20 Such odor changes in mixtures provide incorrect odor-combination information and interrupt odor coding; this represents a critical problem in mimicking the human nose.21 Although the difficulty in identifying mixed odorants has gradually been addressed, no attractive alternatives are available to meet the increasing demand for analytical methodologies that can simultaneously discriminate more than two odorants. Therefore, the development of an improved artificial human nose system remains challenging. Here, we report for the first time the creation of a “human nose” prototype using hOR-conjugated graphene micropattern (GM) geometries, referred to here as a multiplexed super bioelectronic-nose (MSB-nose). Due to the nature of controllable GM transducers, the MSB-nose can operate stably as a liquid-ion gated field-effect transistor (FET) system, and its superior capability in discriminating mixed odorants was demonstrated; the FET system exhibited high sensitivity and single-carbon atomic resolution and provided multiple responses toward mixed target odorants. The MSB-nose exhibited unprecedented reliability and reproducibility in control experiments on volatile organic compounds and odorant mixtures; thus, this MSB nose encoded hOR combinations that identify distinct odors. Our novel approach offers a multiplexed sensing geometry for complex biomimetic systems. The concept of a MSB-nose is based on simulations of a human olfactory system. Figure 1 illustrates the functional anatomy of the human olfactory system (Figure 1a), and the MSB-nose simulates each stage of this system.22,23 The olfactory neuroepithelium, which lies in the upper reaches of the nasal cavity, contains olfactory sensory neurons (OSN; Figure 1b) and a stem-cell population (termed basal cells). Each OSN expresses only one of approximately 390 functional OR genes, and the axons of these neurons are separately B
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Figure 2. Fabrication and characterization of MSB-nose. (a) Schematic illustration for the MBS-nose on silicon wafer (RIE: reactive-ion etching, GM: graphene micropattern, DAN: 1,5-diaminoaphthalene, GA: glutaraldehyde, and OR: olfactory receptor). (b) HR-TEM of single layer CVDgraphene. (c) Photograph of MSB-nose circuit. (d) Optical image of MSB-nose channel. Position A and B confirmed the presence of graphene via RIE process (The inset indicates high-magnification AFM images of PR removed GM). (e) Raman spectra of the different regions (A and B) indicated in panel d.
Edge observation was performed using high-resolution transmission electron microscopy (HR-TEM) to obtain the number of layers and the thickness of the graphene layer. Large-scale CVD-grown graphene comprised a single layer according to the HR-TEM observations (Figure 2b). The fabricated multiplexed electrodes are illustrated in the photographic image shown in Figure 2c. The optical image of the multiplexed substrate shows that the GM channel (50 × 50 μm) bridges the passivated electrodes. The multiplexed substrate was highly uniform, and the transducer design proved suitable for forming a multiplexed sensing geometry (Figure 2d), resulting in the desired surface areas. To confirm the presence of a stable GM after PR removal, high-magnification atomic force microscopy (AFM) was used to measure the average thickness of the GM channel; a vertical thickness of approximately 0.8 nm was determined at the cross-section shown in the inset of Figure 2d, consistent with the HR-TEM image. Raman spectra were obtained to distinguish the GM regions on the substrate after conducting the RIE process. Figure 2e compares Raman spectra of GMs at different locations on a silicon oxide substrate. The stability of the GM can be explained by the presence of peaks G (at 1587 cm−1) and 2D (at 2965 cm−1) in area B of Figure 2d. No significant peaks were present in the A region, suggesting that the graphene was perfectly etched by the RIE process. Although peaks G and 2D, which are characteristic of single-layer graphene, were present in the B region of the spectrum, the existence of GMs can be explained by the 2D/G intensities,
Consequently, size- and shape-controlled graphene micropatterns (GMs) were used as transducers in the B-nose system. Specifically, the multiplexed geometry used in the artificial human nose comprises two important components (GMs and hORs; see Figure 2a). A large-scale single graphene layer was first prepared on the silicon wafer using chemical vapor deposition (CVD) and dry-transfer methods (Supplementary Figure S1).32 The resistance of the graphene film was then determined using a four-point probe (approximately 0.6 kΩ per square). Uniform GMs were constructed on a four-inch silicon oxide wafer using conventional photolithography (PL) and reactive-ion etching (RIE) processes. After conducting standard thermal evaporation and lift-off, Au/Cr microelectrodes, which were used as the source (S) and drain (D), were gradually deposited on the GM channels to prepare sacrificial photoresist (PR) patterns; the electrodes were then passivated to block unexpected side-responses (Figure S2). To form a signal pathway between the odorants and the GM channels, the surfaces of the exposed GMs were formed with 1,5diaminonaphthalene (DAN) by π−π interactions and functionalized with glutaraldehyde (GA) via a Schiff-base reaction.9 Subsequently, two types of hORs (hOR2AG1 and hOR3A1), which specifically interact with the target odorants amyl butylate (AB) and helional (HE) were immobilized on the GA/DAN/GM substrate via similar Schiff-base reactions. Finally, the odorant sensory substrate that was based on the hOR-conjugated GMs was constructed on the silicon wafer. C
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Figure 3. Characterization of sub-SB-noses under MSB-nose. (a) Western blot analysis of hORs expression in E. coli. (b) Illustration of hORsattached GM electrodes by Schiff-base reaction. Typical FE-SEM image (c) after the introduction of hORs on GM electrodes. (d) Comparison of current−voltage (I−V) curves in processes of sub-SB-nose construction. (e) Schematic illustration of single sub-SB-noses operated by liquid gating. (f) Transfer curves of the FET sub-SB-noses.
hOR attachment. Importantly, no hORs were present on the microelectrodes due to the passivation and the blocking of side reactions, such as the adsorption or desorption of odorants and hORs on the gold electrodes, which would affect the contact resistance. To further understand the condensation reaction between the carboxylic groups of the hORs and the amine groups of DAN, current−voltage (I−V) changes were monitored during the hOR immobilization process. Figure 3d shows the I−V values for the GM configuration before and after hOR attachment. Although the effected change (dI/dV) was slightly decreased due to the contact resistance arising from biomolecular attachment, the I−V values for hOR-conjugated GMs were linear over a voltage range from −0.1 to +1.0 V, indicating stable ohmic behavior between the microelectrodes. Based on these results, hOR-conjugated GM transducers can maintain reliable electrical contact and provide an efficient conductive pathway due to specific odorant interactions. To exploit the potential of hOR-conjugated GMs as transducers in the liquid-ion gated FET-type MSB-nose, a liquid-ion gating system was constructed. Phosphate-buffered saline at pH 7.4 (PBS) was used as an electrolyte and surrounded the source (S) and drain (D) electrodes, and the gating electrode was immersed in the electrolyte (Figure 3e). This strategy enabled efficient and intimate contact between the target molecules and the biomarker-conjugated transducer, leading to signal amplification in electrical-sensing applications. hOR2AG1- and hOR3A1-conjugated super B-noses (sub-SB2and sub-SB3-noses) were suitable as single FET channel systems, as demonstrated by the Ids−Vg transfer curve at room temperature (Figure 3f). As observed in previous work, although our GM device exhibited ambipolar conductance, it exhibited stable operation and enhanced sensing performance in p-type (hole-transporting) regions. Subsequently, the remaining experimental tests reported here were conducted in the p-type region of the GMs.32 The real-time responses of each single sub-SB-nose in an MSB-nose were monitored and compared; this procedure should be performed before creating the multiplexed B-nose system, because sub-SB-noses can affect each other as
because graphene defects generally result in lower 2D/G integration values and higher D integration values. According to the 2D/G integration value (1.24), GMs were successfully designed on the substrate. These experimental observations show that the GM channels between source and drain can be utilized as transducers for FET-type biosensors. Two hORs, hOR2AG1 and hOR3A1, were produced using an E. coli expression system and were integrated using a MBE nose as a recognition element. High-levels of expression were confirmed by Western blot analysis (Figure 3a). A distinct band was observed at approximately 54 kDa, corresponding to the monomer size of GST-tagged hORs; this finding indicates that hORs were successfully expressed at high levels. Multimeric bands were also observed by Western blot analysis. Multimers are formed by nonspecific aggregation, even under the reducing conditions used for Western blot analysis, due to the formation of spurious disulfide bonds (this phenomenon is widely found for membrane proteins).33 Membrane proteins, including hORs, are difficult to overexpress at high levels in bacterial cells due to their strong hydrophobicity and toxicity, which is attributed to improper membrane insertion.34,35 However, in our previous studies, several GPCRs (including olfactory receptors and taste receptors) were overexpressed by optimizing the bacterial expression vectors and culture conditions used.7,9,10,13,36 The Schiff-base reaction was used to immobilize the hORs on a GM surface that had previously been modified with DAN via π−π stacking interactions because covalent bonding can provide greater stability than physical adsorption processes to the FET-sensing geometry in the liquid state (Figure 3b).37 The amount of hOR (hOR2AG1, 6.18 × 10−16 moles, approximately 3.72 × 108 OR molecules; and hOR3A1, 4.9 × 10−16 moles, approximately 2.94 × 108 OR molecules) to be loaded on a GM was determined using a bicinchoninic acid assay to obtain reproducible sensing properties.9,32 Fieldemission scanning electron microscopy (FE-SEM) images were obtained before (Figure S3) and after (Figure 3c) the introduction of hOR2AG1 (SEM images for hOR2A3 were not obtained). The GM surface was considerably roughened by D
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Figure 4. High-performance sub-SB-noses. Real-time responses of (a) sub-SB2-nose and (b) sub-SB3-nose toward various odorant concentrations (AB and HE: 0.1 fM to 10 pM). (c) Calibration curves of the FET sub-SB-noses. (d) Discrimination of sub-SB2 and SB3-noses toward various odorants which were organized by carbon-atomic differences and functional groups. (e) Real-time responses of sub-SB2-nose toward real-samples (apricot). (f) Electrostatic gating effect (shift of Dirac point) upon the increasing AB concentrations.
antagonists or agonists.8 The responses of the liquid-ion gated FET-type sub-SB2-nose toward various concentrations of amyl butylate (AB) are shown in Figure 4a. The current increased immediately in response to AB and reached an equilibrium state representing the interaction between the hORs and odorants. Based on the sensing performances of the sub-SB2nose, the minimum detectable level (MDL) was approximately 0.1 fM (signal-to-noise: 3.8), and the response was saturated at approximately 10 pM (signal-to-noise: 3.2). The response of the sub-SB3-nose to helional (HE) and the MDL for this response were similar to those for the sub-SB2-nose; however, the sensitivity of the sub-SB2-nose was 10 times higher than that of the sub-SB3-nose (Figure 4b). Moreover, the reversibility of odorant binding toward sub-SB-noses was also confirmed by reducing the odorant concentrations (Figure S4), which real-time responses with increasing current values were similar to the ones in Figure 4a and b. GM/DAN and pristine GMs were also evaluated as transducers in control experiments for use in preparing sensitive and selective sub-SB-noses for discriminating odorants. To quantify the sensing behaviors of sub-SB-noses, the equilibrium constant K was calculated from direct contact. The Langmuir isotherm model was used to estimate the K value, from which universal parameters for the analytical methodologies.38 The normalized sensitivity, N, can be calculated from the interaction between hORs and odorants as follows (see the Supporting Information): N=
where C indicates the concentration of the odorant in the solution. Thus, a K value can be obtained from N and C. Using this equation, normalized sensitivities (ΔI/I0) can be obtained from the real-time responses shown in Figure 4a and b. The K values were determined by curve fitting and were 2.93 × 1015 M−1 for the sub-SB2-nose and 9.6 × 1014 M−1 for the sub-SB3nose (Figure 4c).7 Although the K value for the sub-SB2-nose is slightly higher than that for the sub-SB3-nose (due to properties that naturally differ between hORs), similarly shaped signals were obtained for the odorant responses from both noses. The human olfactory system can perceive thousands of odorant molecules in an odorant mixture at low concentrations (0.01 ppt);39 therefore, high-resolution odorant recognition is essential for an artificial B-nose. To demonstrate single-atom resolution of sub-SB-noses, the selectivities of sub-SB2- and sub-SB3-noses were observed for similar odorant structures with (i) different numbers of carbon atoms (Figure 4d, upper right) and (ii) different terminal groups (Figure 4d, lower right). Superspecificity of the sub-SB2-nose toward AB was clearly exhibited at the lowest concentration of AB (0.1 fM; Figure 4d, upper left) in the presence of odorants with slightly different numbers of carbon atoms (propyl butyrate, PB; hexyl butyrate, HB and butyl butyrate, BB). Moreover, the sub-SB3nose exhibited a response toward HE (0.1 fM; Figure 4d, lower left) that was distinct from that toward other terminal groups (safrole, SA; phenyl propanol, PP and piperonal, PI). The realization of artificial intelligence in sub-SB-noses can be explained using the electrostatic gating effect from FET
C 1/K + C E
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Figure 5. High-performance MSB-nose. (a) Schematic illustration of MSB-nose in liquid-ion gated FET system. (b) Transfer curves of each subunits, sub-SB-noses, in the MSB-nose system. (c) and (d) Selectivity of MSB-nose toward various individual odorants and odorant mixtures (PB, HB, BB, PI, PP, and SA; 1 μM, AB and HE; 0.1 fM).
geometry but not by electronic doping.40 It is known that GPCRs, including ORs, are activated by specific ligand-GPCR binding events that initiate the structural rearrangement of GPCRs, resulting in a partial acquisition of a negative point charge (Figure S5).41,42 More interestingly, the real-samples were prepared from apricot, which has AB, by extraction.43,44 Comparing to the selectivity in Figure 4d, positively increasing current values in Figure 4e confirmed the existence of a target molecule (AB) in real-samples. Negative charges were effectively induced in the liquid-ion gate dielectric near the GM surface, resulting the accumulation of positive charge carriers in the GM channel. The enhanced negative gate voltage (Vg) induced by the hOR-odorant interaction increased the value of Ids for p-type sub-SB-noses. This effect is similar to an indirect gating effect and does not directly affect the GM channels. This type of molecular gating effect can be demonstrated with a shift of the Dirac point in the transfer curve of GM transistors (Figure 4f). The Dirac point voltage of a sub-SB2-nose, Vg,min (0.26 V, the point at which charge carriers change polarity) was slightly altered; the value of Vg,min for the electrostatic gating effect positively increased in response to increased AB concentrations. Therefore, the significant signals that are obtained using the sub-SB-noses in response to target odorants will cause positively increased Ids values due to the geometry of the FET. Innovative biomimetic materials must be effective, safe, and stable in the environment.45 The long-term stability of the subSB-noses was tested over a 10-week period with designed samples that were stored in a sealed vessel at 25 °C under dry air conditions (Figure S6). The real-time responses from the sub-SB-noses were measured every 2 weeks, and normalized sensitivity changes were calculated to examine their timedependence. The real-time responses from the sub-SB-noses
decreased slightly, and the sensitivity differed by approximately 5% after 10 weeks due to degradation of the active hORs. This result demonstrated the excellent lifetime and stability of the sub-SB-noses. A major advance achieved by MSB-noses is their ability to simultaneously discriminate target odorants in mixtures; this is achieved using a multichannel array device that might be useful in a multiplexed sensing geometry (Figure S7).46−49 The MSBnose with a liquid-ion gate dielectric exhibited similar transfer curves to those of the single sub-SM-noses in Figure 3f, indicating that the MSB-nose can also be adopted as a transducer in a multiplexed liquid-ion gated FET system (Figure 5a and b). Based on the FET system, the real-time responses from the MSB-noses were highly sensitive and selective toward various odorants containing different numbers of carbon atoms (PB, HB, BB, and AB) and different functional groups (PI, PP, SA, and HE). The specific selectivity of the MSB-nose was synchronized to that of the single channel SBnose (Figure 4d). The sub-SB2-nose was responsive only to AB, whereas the sub-SB3-nose selectively interacted with HE (Figure 5c). Interestingly, each hOR in the MSB-nose accurately discriminated target odorants in the mixture at femtomolar concentration. Nontarget odorants, which were present at even higher (micromolar) concentrations, caused no significant responses. The MSB-nose operated as an actual human nose system toward target odorants, including AB and HE, producing simultaneous and analogous responses (Figure 5d). To further support the outstanding sensing performances of the artificial MSB-nose, the sensing performance of hOR2AG1conjugated-conducting polymer nanotubes (CPNTs) was compared against that of the sub-SB-noses. The MSB-nose was constructed using the following four nanomaterials as F
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Figure 6. Discrimination of MSB-nose consisted of sub-SB-noses and CP NTs as sensing nanomaterials, and artificial olfactory coding through the analysis of MSB-nose. Selectivity of MSB-nose with four-different transducers toward (a) and (b) various odorants and (c) vapor organic compounds. (d) Comparison histogram of response values characterized from a, b, and c. (e), Principle component analysis plots of the data set of response intensities inputted from four sensing materials to 12 odorant analytes (all of the sample concentrations were fixed at around 1 μM).
the MSB-nose produced no signal change while attempting to discriminate between various VOCs due to the attachment of hORs to the graphene surface. To analyze these results, the Isd values in Figure 6a, b, and c were evaluated simultaneously using two types of methodologies. First, a bar diagram demonstrates the excellent odorant discrimination obtained using the MSB-nose, even at extremely low concentrations of AB and HE (0.1 fM). With only AB or HE, the sub-SB2- and SB3-noses produced a much greater signal than that produced by CPNTs/hOR2AG1, indicating that the MSB-nose can distinguish specific odorants in mixtures (Figure 6d). Finally, the collected sensing data (with sample concentrations fixed at 1 μM) from transducers constructed from four types of materials was subjected to principal component analysis (PCA). The first three principal component scores are plotted in Figure 6e, and the accuracy of the simulation results was satisfactory (>99%). The analytes exhibited unique signatures, as shown by their segregation into separate regions of the plot, thus enabling the identification of individual analytes. In particular, components of AB and HE were clearly distributed into different regions when using MSBnoses, validating the discriminative power of the MSB-nose. These results demonstrate that a simple and portable MSBnose can encode olfactory receptor combinations for distinct odor identification. Conclusions. The aim of this research was to produce a human-like artificial MSB-nose that encodes human olfactory receptors for distinct odor identification. To create an MSBnose that was selective at single-carbon resolution, controlled GMs were designed as transducers, and two types of hORs were attached to the GMs as bioprobes. A field-induced MSBnose simultaneously allowed the sensitive and selective
transistors (see the Supporting Information): (a) GM/ hOR3A1 (sub-SB3-nose), (b) GM/hOR2AG1 (sub-SB2nose), (c) carboxylated poly(3,4-ethylenedioxythiophene) nanotubes (CPEDOT NTs)/hOR2AG1, and (d) carboxylated polypyrrole nanotubes (CPPy NTs)/hOR2AG1.10,50 Figure S8 includes SEM images of four different transducers deposited on the multiplexed electrodes, and Figure S9 shows the remarkably rough surfaces of CPEDOT NTs and CPPy NTs to which hORs had been added and the comparably smooth surface of pristine NTs. The stimulus from the artificial MSB-nose was monitored by measuring the gating-induced current changes in response to target odorants; the results obtained were consistent with those obtained in response to nontarget odorants. No significant signals were obtained from CPNT transducers with hOR2AG1 and sub-SB3-noses when target odorants (0.1 fM AB) were incorporated into the MSB-nose geometry; by contrast, an immediate change (
