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Nanoplasmonic monitoring of odorants binding to olfactory proteins from honeybee as biosensor for chemical detection
Sensors and Actuators B 221 (2015) 341–349
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Nanoplasmonic monitoring of odorants binding to olfactory proteins from honeybee as biosensor for chemical detection Diming Zhang a , Yanli Lu a , Qian Zhang a , Yao Yao a , Shuang Li a , Hongliang Li b , Shulin Zhuang c , Jing Jiang d , Gang Logan Liu d , Qingjun Liu a,∗ a Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China b College of Life Sciences, China Jiliang University, Hangzhou 310018, PR China c College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, PR China d Micro and Nanotechnology Lab, University of Illinois at Urbana-Champaign, IL 61801, USA
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Article history: Received 13 April 2015 Received in revised form 10 June 2015 Accepted 13 June 2015 Available online 26 June 2015 Keywords: Odorant binding protein (OBP) Nanocup arrays Localized surface plasmon resonance (LSPR) Small odorant molecules Nitro-explosives
a b s t r a c t Odorant binding proteins (OBPs) are key soluble sensitive proteins for odor detections in animal olfactory systems. Here, we present a nanosensor based localized surface plasmon resonance (LSPR) to monitor binding of small odorant molecules to OBPs from honeybees. The binding of odorants molecules to OBPs, can be quantified by nanocup arrays from 10 nM to 1 mM, with a high refractive index sensing of LSPR wavelength shift in transmission spectra. Linear dose-dependent behavior can be observed in relationship of LSPR wavelength versus analyte concentrations. More than floral odorants, this type of biofunctionalized nanocups also showed potential applications in explosive detections for nitro-compounds such as 2,4,6-trinitrotoluene, 2,4-dinitrotoluene and 3-mononitrotoluene. This study demonstrated a LSPR device to monitor interactions between small molecules and proteins, which provided a great biosensor platform for healthcare diagnosis, environment monitoring and food analysis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Honeybees have been demonstrated to distinguish thousands of floral odors at low concentrations by their remarkable olfactory systems [1,2]. Theoretically, honeybees can detect floral scents containing hundreds of different types in their living environment, which helps them navigate rapidly and accurately to food sources kilometers away. Thus, bio-components from olfactory systems of honeybees, such as antenna and sensing proteins, could be widely used in odor detections [3–5]. More interestingly, the olfactory perceptions of honeybees were reported even out of ranges of floral odors existing in their living environment [6–8]. Honeybees showed a great discrimination for several compounds that they never suffered in natural conditions, while trained to detect explosives and diagnose diseases. Thus, the remarkable olfaction should have more important and practical applications in chemical detections, more than just detecting for floral scents. Over last couple of decades, significant efforts were made to develop biosensors that combined biosensing components to
∗ Corresponding author. Tel.: +86 571 87951676; fax: +86 571 87951676. E-mail address: [email protected] (Q. Liu). http://dx.doi.org/10.1016/j.snb.2015.06.091 0925-4005/© 2015 Elsevier B.V. All rights reserved.
physicochemical transducers, in the field of environmental monitoring, food safety, and medical diagnosis [5,9,10]. Now, the lack of selective and sensitive biosensing components became a key challenge, making the biosensors inadequate in many applications. For proteins-based biosensors, enzymes, antigens–antibodies, and receptors were common sensitive elements to bind target molecules and then elicit sensor responses [11,12]. However, these complex proteins in biological organisms were often difficult to purify, almost impossible to synthesize, and thus limited their usage for biosensors. Thus, it was a sustaining work to obtain biosensitive component in easy and low-cost way. However, odorant binding proteins (OBPs) were one kind of chemical sensing proteins in olfactory system [13–15]. They were thought to deliver odor molecules to olfactory receptors, and were the first biochemical step in odor reception capable of some level of odor discrimination. OBPs often showed an affinity to a wide range odorants and had a broad specificity for different analytes simultaneously. Notably, different from membrane proteins, OBPs as one kind of soluble proteins, could be expressed at low cost, purified easily, and kept great bioactivities in vitro [16,17]. With these advantages, OBPs from animals, such as bovine and swine, have been used to detect alcohols and pheromone [18,19]. Besides these common chemicals, OBPs often had multiple binding sites simultaneously with
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conformational changes for diverse molecules such as pesticides and explosives [20,21]. Thus, OBPs should have more biosensor applications by monitoring interactions between target analyte and OBPs. Detection for specific binding of small molecules to proteins was of substantial importance because of its great application in drug screening, biochemical analysis and physiological study [22–24]. In recent years, several nanostructure sensors (e.g., nanoparticles, nanoholes, and nanowires) have been reported as excellent platforms for the biodetection for small molecules with high sensitivity, excellent reproducibility, real-time responses, and label-free detection [25–28]. They were based on collective oscillations of conduction electrons of localized surface plasmon resonance (LSPR), at nanostructures with high confined electrical field extended on their surfaces. Using this type of sensors, biointeractions could be monitored quantifiably and ultrasensitively, even at single molecular level, through observing wavelength shifts in LSPR before and after binding of the target ligands. It was especially helpful to quantify small molecules which only elicited little changes in bio-interactions. Moreover, optical LSPR observation had advantages that allowed multiple-component analysis in array without labels on targets and physical connections to detecting elements. Thus, the LSPR sensing with nanostructures provided an interesting and promising approach to quantify small molecule–protein interactions in biosensing. Here, we developed a novel biosensor based on nanocup arrays (nanoCA) to monitor the bindings of small molecules to proteins through LSPR spectroscopy. The nanoCA sensor can recognize the conformation changes of the OBPs in presence of typical floral odorants such as -ionone in a dose-dependent response, while its biosensing mechanism was simulated with Mie scattering model. More interestingly, bindings of 2,4,6-trinitrotoluene (TNT) to the OBPs can be observed in molecular docking and nanoCA monitoring, respectively. The nanoCA with OBPs had significant wavelength shift in detection for TNT, as well as 2,4-dinitrotoluene (DNT) and 3-mononitrotoluene (1NT), which suggested potential application of the nanoCA for nitro-explosive detections. 2. Materials and methods 2.1. Proteins and agents In our previous study, OBPs of Acer-ASP2 from honeybee have been demonstrated to bind floral odors and semiochemicals (e.g. -ionone, geraniol, benzaldehyde, and isoamyl acetate) by electrochemical impedance measurement [5,29]. Thus, the OBPs were also immobilized on nanoCA in this study to bind typical odorant of -ionone to demonstrate LSPR monitoring for small molecule–protein interaction. The OBPs solution used in experiments was at concentration of 500 g/ml in phosphate buffered saline (PBS; pH = 7.4), while -ionone was diluted into different concentrations ranging from 10 nM to 1 mM with methanol. As the negative control, bull serum albumin (BSA) was prepared at 500 g/ml and immobilized on the device by same method as that of OBPs. Moreover, in nitro-explosive detections, TNT, DNT, and 1NT were diluted with methanol into increasing concentrations of 10 nM, 1 M, and 100 M. All other agents were purchased from Sigma and of analytical grades. 2.2. Fabrication of nanoCA NanoCA devices were fabricated by nanoimprint with a template containing nanocone arrays and then deposited with Au nanoparticles. The detailed processes have been described in our previous study [30]. In brief, a 250 m flexible (Poly)ethylene
terephthalate (PET) sheet was used as supporting substrate, and UV curable polymer was evenly distributed on the template. A UV lightcuring flood lamp system (Dymax, EC-Series) was used at average power density of 105 mW/cm2 for 60 s to solidify the UV polymer. Au nanoparticles were deposited on sidewalls of nanocups with a six pocket e-beam evaporation system (Temescal), after using 5 nm thick titanium as adhesive layer. The depth and opening diameter of nanocup were 500 nm and 200 nm, respectively, while diameters of the particles were about 20 nm. NanoCA could have good reproducibility if the sizes of nanocup arrays and nanoparticles were well controlled in the same fabrication conditions. 2.3. Expression and purification of OBPs The recombinant OBPs of Acer-ASP2 were cloned from the full-length cDNA of adult worker bees, Apis cerana cerana. The recombinant expressed plasmid pET-Acer-ASP2 was transformed into Escherichia coli BL21 (DE3) competent cells after 450 bp fragment was excised with BamH I and Hind III from the pGEMAcer-ASP2 plasmid. The cells were grown in Luria–Bertani broth (including 30 g/ml kanamycin) at 37 ◦ C with 1.5 mM isopropyl-d-thiogalactopyranoside to induce expression of the protein. After 5 h at 28 ◦ C, the bacterial cells were harvested and lysed by sonication and centrifuged into crude cell extracts (pellet and supernatant). The pellets of crude cell extracts, containing recombinant proteins, were severely precipitated in 1.5 M urea in ddH2 O and finally freeze-dried. Finally, the protein was resuspended (500 g/ml) in PBS and stored under 4 ◦ C for biosensor experiments. More details could be found in our previous study about expression of the OBPs from honeybee [31]. 2.4. Self-assembled immobilization with PEG NanoCA device was firstly washed with mixture of H2 SO4 and H2 O2 (7:3), and DI water respectively, to remove the organic residues. Subsequently, the NanoCA device was immersed and reacted with 2 ml poly(ethylene glycol) 2-mercaptoethyl ether acetic acid (HOOC-PEG-SH, 2 kDa) at 1 mg/ml overnight (about 18 h) at room temperature to form Au S semi-covalent bond on the surface of the nanoCA device. Then, 2 ml mixing solution of 8 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma) and 12 mg/ml N-hydroxy-succinimide (NHS) in 0.1 M 2-(N-Morpholino)ethanesulfonic acid (MES) buffer (pH = 5) was immersed for 15 min to activate carboxylate groups of HOOC-PEGSH after extra HOOC-PEG-SH was washed off with DI water. Finally, this mixture solution was adjusted into pH = 8.0 with saturated NaHCO3 , added with 1 ml OBPs, and incubated at room temperature for 2 h. The nanoCA was washed with DI water by gently shaking for 60 s, followed by air drying with nitrogen and stored at 4 ◦ C for following experiments. 2.5. Simulation of nanoCA with OPBs for odorant binding With OBP immobilization on nanoCA, the device was converted into a biosensor for odorant from a transducer sensitive to refractive index change. The biofunctionalized nanoCA (bio-nanoCA) could detect small odorant molecule binding to the protein by monitoring wavelength shift of LSPR peak in transmission spectra. The surface plasmon resonance peak in the transmission spectra of bionanoCA could be modeled by Mie light scattering of single coated gold sphere. The dielectric constant of Au in the spectral range of 300–1100 nm was from Johnson and Christy’s review [32]. The dielectric constant of coated bio-component ε1 changed from 5 to 7, while dielectric constant of solution environment ε0 was fixed on 2.0736 in methanol. In the simulation, the inner and outer radiuses of coated sphere were set up at 20 nm and 30 nm, respectively. The
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calculation was performed with software programed by the group of García de Abajo [33]. 2.6. Electrochemical and optical measurement Electrochemical impedance sensing was performed with standard three electrode system. NanoCA was used as working electrode, while platinum electrode and Ag/AgCl electrode were used as counter electrode and reference electrode, respectively. 5 mM K4 [Fe(CN)6 ]/K3 [Fe(CN)6 ] (1:1) was employed as redox couple for electrochemical detecting. Electrical signals were measured by electrochemical workstation (CHI660E, Chenhua Instruments Co., China) in AC impedance mode. The tested frequency was set from 1 Hz to 1 kHz with a 5 mV alternating voltage. Before the optical measurement, 30 l analyte solutions were added in the well of the plate. The solutions could be diffused and distributed evenly on the nanoCA surface due to tension. The optical detection was based on a multi-mode detection platform (SpectraMax Paradigm, Molecular Devices Co., United Sates). Normal transmission model was applied to measure the spectra of bio-nanoCA when scanning range was set from 300 nm to 900 nm and step was fixed on 1 nm. Considering harmful effect of repeated elution for immobilization and bio-activity of the protein, the bionanoCA device was used only one time in optical measurement to keep best performance for the odorant detection. 3. Results 3.1. NanoCA monitoring with LSPR spectroscopy Periodic nanostructures had outstanding optical features such as absorption and transmission peaks in visible spectroscopy [26,34]. These features were elicited by plasmon resonance and could be utilized in optical sensors. As shown in Fig. 1a, the nanoCA chips were integrated into 96 well plates and could be performed in high throughput measurements. On the surface of nanoCA chip, nanocups were subwavelength opening on transparent plastic substrate, fabricated periodically in arrays and decorated with Au nanoparticles by electron beam evaporation (Fig. 1b). Fig. 1c showed photo of nanoCA device obtained with scanning electron microscope. Transmission detection was applied to measure the LSPR spectroscopy of nanoCA. NaCl at different concentrations were employed as increasing refractive index solutions, to determine the refractive-index sensing properties of nanoCA (Fig. 1d). With synergetic LSPR effects of periodical array structure and nanoparticles deposited on the nanocups, the nanoCA showed a high sensitivity for refractive index change with ∼104 nm per refractive index unit (RIU), which was much greater than typical nanoparticle sensors and nanohole sensors based on plasmon resonance [35–37]. Thus, a high sensitive LSPR device based on nanoCA can be obtained and used to monitor binding of small molecules to OBPs as a highthroughput biosensor. 3.2. Fabrication of bio-nanoCA for odorant binding detection In this study, OBPs of AcerASP2 were produced from E. coli and used to modify nanoCA. Based on Au S semi-covalent linkage, a self-assembled film of HS PEG COOH was used to fix OBPs on the surface of nanoCA for small odorant molecule binding (Fig. 2a). The immobilization of OBPs on surface of nanoCA was important for fabrication of the nanoplasmonic biosensor monitoring the odorant binding. Thus, two methods, including optical and electrochemical measurements, were used to characterize property changes of the biosensor during the protein immobilization, in order to verify successful immobilization of OBPs. Fig. 2b showed optical transmission spectra measurement, when PEG and OBPs were immobilized on
Fig. 1. The nanosensor device and its optical measurement system. (a) Schematic diagram of the optical detection using a 96-well plate instrument in transmission mode. (b) Structure of the periodic nanocups on the nanoCA chip, with Au nanoparticles deposited along the sidewalls. (c) SEM image of the cup arrays. (d) Transmission spectra of nanoCA in presence of NaCl at different concentrations. The inset showed wavelength shifts with concentration increasing. WS: wavelength shift, RI: refractive index.
nanoCA step by step. Obvious wavelength shifts, more than 20 nm, could be observed in LSPR peak around 500 nm, which suggested well immobilization of OBPs. Moreover, in our previous work, nanoCA device was demonstrated as a good electrode for electrochemical measurement [38]. Thus, as one of the most powerful and sensitive techniques, electrochemical impedance spectroscopy was also chosen to verify the protein immobilization. The most important parameter of impedance spectroscopy is a semicircle portion corresponding to the electron transfer limiting process, the diameter of which represented interface impedance change on device surface. As shown in Fig. 2c, significant increase in semicircle diameter can be observed in Nyquist plot with interface impedance changed by the protein immobilization. These both indicated that the OBPs were immobilized on the nanoCA surface efficiently. After well immobilization of the OBPs on nanoCA, the bionanoCA could be used to detect odorant binding to the proteins. As shown in Fig. 3a, OBPs on nanoCA could bind small molecule ligands and then had property changes (e.g., conformation, volume, and electron distribution), which could modulate LSPR in electrical field near nanoCA surface. The LSPR spectroscopy of nanoCA was from complex plasmonic effects of surface plasmon polaritons-Bloch
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Fig. 2. Immobilization of OBPs on the sidewalls of nanoCA device. (a) Schematic diagram of the nanoCA functionalized by the self-assembled proteins. (b) Transmission spectra of nanoCA immobilized with the proteins. Significant wavelength shift max can be observed in shift from 523 nm to 551 nm. (c) Nyquist plots for immobilization of nanoCA with PEG and protein.
wave, Wood’s anomaly, and Mie scattering on hybrid structures of nanocups and nanoparticles [30]. The peak around 600 nm was due to Mie scattering from nanoparticles. Thus, a model of coated sphere was used to describe the Mie scattering of bio-nanoCA (Fig. 3b). In theoretical simulation, Mie light scattering curve of the model showed high similarity to the resonance peak around 600 nm in the transmission spectra of nanoCA modified by proteins in the experiment (Fig. 3c). It was also found that the wavelength had linear shifts with dielectric constant changes, from 5 to 7, of coated biosensing elements (Fig. 3d). Although RIU sensitivity in
simulation was lower than that of experiments, the linear relationship shown in simulation gave a possible guarantee to dose-dependent responses of bio-nanoCA in monitoring of binding between small odorant molecules and OBPs. 3.3. Bio-nanoCA monitoring for binding of odorants to OBPs Several chemical molecules could specifically bind into hydrophobic pockets of OBPs immobilized on the nanoCA surface, which could modulate protein conformation and change relative
Fig. 3. Biosensing mechanism of the bio-nanoCA. (a) Schematic diagram of the nanoparticles on the surface of nanoCA functionalized by biosensing elements. (b) Model of coated sphere representing the bio-component modified nanoparticles. ε0 , ε1 , and ε2 are relative dielectric constants of solution environment, bio-component, and Au nanoparticle respectively, while (a) and (b) are inner and outer radiuses of the particles. (c) Simulated transmission spectra from Mie scattering of coated particles and experimental LSPR spectra. (d) Linear relationship between wavelength shift and dielectric constant changes of the bio-component immobilized on the nanoCA.
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Fig. 4. Examination of nanoCA monitoring odorant molecule–OBPs binding. (a) Chemical structures of -ionone, butanedione and acetic acid, and smell they had. (b, c) Transmission spectra of OBPs- and BSA-modified nanoCA, respectively. The OBPs-modified device shows specific response to -ionone, while no significant responses were observed in other conditions. (d, e) Statistic of p and max for responses of OBPs- and BSA-modified nanoCA, respectively (mean ± SD, n = 15; *P < 0.05).
dielectric constants of the bio-coating [23,39]. In odorant measurement, one kind of aromatic odorants, -ionone, was used as modeling molecule to test responses of the bio-nanoCA with OBPs. Butanedione and acetic acid, having different pungent and buttery smell, were used as the control to assess nanoCA monitoring of odorant molecules–OBPs binding (Fig. 4a). As illustrated in Fig. 4b, transmission spectra of bio-nanoCA device, especially intensity and location of transmission peak, was notably changed by specific binding of -ionone and OBPs. In contrast, there were no significant variations observed in transmission spectra in presence of acetic acid and butanedione at same concentration without specific binding. To further examine whether change of transmission spectra was caused by specific interactions between odorant molecules and the proteins, we also carried out same experiments on BSA-modified nanoCA. The BSA-modified device was used as nonspecific-binding group to show transmission spectra changes of devices elicited by own refractive index of solution rather than specific interactions between OBPs and target molecules. Indeed, BSA-modified nanoCA showed no remarkable responses in presence of all compounds of butanedione, acetic acid and -ionone (Fig. 4c). Moreover, transmission intensity change p and LSPR wavelength shift max , were both calculated into statistics with the OBPs- and BSA-modified devices (Fig. 4d and e). In accordance with the result of transmission spectra, the statistics showed that OBPs-modified device could selectively respond to -ionone and BSA-modified nanoCA had no response to all compounds. These results all demonstrated that nanoCA could monitor specific binding of odorant molecule of -ionone to OBPs with transmission intensity change and wavelength shift in LSPR peak. Then, the dose-dependent behavior of bio-nanoCA was analyzed by transmission intensity changes and wavelength shifts from responses of the devices to -ionone at increasing concentrations. -Ionone at concentrations from 10 nM to 1 mM were used to bind OBPs on the device to modulate LSPR in transmission spectra (Fig. 5a). As shown in Fig. 5b and c, wavelength shift
and transmission intensity decreasing obtained from OBPs modified nanoCA, both showed similar dose-dependent responses to -ionone, while no significant responses can be found from the nanoCA with BSA. However, compared to the intensity decreasing, the wavelength shift had broader linear responses from 10 nM to 1 mM. Considering wavelength shifts observed in theoretical simulation, thus, the wavelength shift was analyzed as effective responses of bio-nanoCA. With the dose-dependent curve, the detection limit of bio-nanoCA to -ionone was determined at 26.7 pM based on 3ı/slope method with wavelength shifts. However, as shown in Fig. 5d, -ionone detection without nanoCA showed overlap of 10 M and the control of methanol in absorption spectrum, which meant higher detection limit than 10 M in nanoCA-absent measurement. Therefore, the nanosensor really had ultrasensitive capacity to monitor the binding of small odorant molecules to the proteins. 3.4. Bio-nanoCA for nitro-explosive detection Several studies recently reported about training honeybees and using their sensing proteins to find nitro-explosives [40–42]. Especially, different OBPs have been used to modify biosensor for explosive detections [21,43,44]. Thus, we tried to explore the binding of nitro-explosive molecules to the OBPs of AcerASP2 by the bio-nanoCA. Fig. 6a showed the molecular docking of specific binding of TNT to OBPs. TNT molecules could enter protein’s cavities, interact with amino acid residues and form the complex with OBPs. This process might elicit same conformations change of OBPs as that of the -ionone binding and modulate the LSPR on the nanoCA in the optical measurement. Fig. 6b showed transmission spectrum of the bio-nanoCA with OBPs in presence of TNT at different concentrations. TNT at high concentrations of 100 M and 1 M elicited significant wavelength shifts in comparison to the control, while TNT at 10 nM had no effect on the device. In order to check whether the shifts came from specific binding of TNT, the nanoCA
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Fig. 5. Bio-nanoCA with OBPs for -ionone at increasing concentrations. (a) LSPR spectra of OBPs-modified nanoCA in presence of -ionone from 10 nM to 1 mM. (b) Dose-dependent profiles of bio-nanoCA with OBPs in intensity decreasing of transmission peak. (c) Dose-dependent profiles of bio-nanoCA with OBPs in wavelength shift of transmission peak. The wavelength shifts have good linear responses to the concentrations of -ionone, when methanol is used as blank control. (d) Measurement for -ionone binding in the absorption spectrum without nanoCA. The curves overlap of 10 M -ionone and the control means higher detection limit than 10 M (mean ± SD, n = 15).
Fig. 6. Bio-nanoCA with OBPs for explosive detection. (a) Molecular docking of TNT in the binding pocket of the protein. (b) Transmission spectrum of OBPs-modified nanoCA for TNT at different concentrations. (c) Comparing responses of the bio-nanoCA with and without OBP to TNT at 100 M. (d) Wavelength shift for responses of the bio-nanoCA with OBPs and BSA to explosives of TNT, DNT, and 1NT (mean ± SD, n = 15; *P < 0.05; NS, not significant, P > 0.05).
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without OBPs was also exposed to TNT. As shown in Fig. 6c, the bio-nanoCA with OBPs showed obvious wavelength shift in presence of TNT, while responses of the nanoCA without OBPs had no reliable difference between TNT and blank control of methanol. It suggested that binding of TNT and OBPs could really be monitored by the device and used for explosive detection. Besides TNT, two kinds of nitro-explosive analogs, DNT and 1NT, were carried out in experiments to test bio-nanoCA. Fig. 6d indicated high responses of the bio-nanoCA to three kinds of nitro-explosives of TNT, DNT and 1NT at different concentrations. The responses to three nitroexplosives were almost equal to those from -ionone, although their responses were similar at 100 M and 1 M without dosedependence behaviors.
4. Discussion In our study, nanoCA was successfully used to monitor and quantify binding of small molecules to proteins. The bio-nanoCA was proved to detect interaction between odorant molecules of ionone and OBPs in linear dose-dependence. The detection limit of the device for -ionone was 26.7 pM, which was much lower than our previous study using electrochemical impedance spectroscopy for odor detections [5,29]. The higher sensitivity might come from the combination of OBPs and nanostructured LSPR device. On one hand, similar to SPR technique, LSPR was an excellent method to monitor the bio-molecular binding event. As reported, with several nanostructures, the sensor can even detect single molecular binding on the sensor surface [45–47]. On the other hand, the special binding of small molecules to OBPs also was the reason of higher sensitivity of the device than the previous electrochemical method. Different from common biomolecular binding on bio-component surface, the binding of small molecule to OBPs happened on the pocket of the protein, which elicited lower electrochemical impedance change than that of the common binding. However, the binding in the pocket and subsequent conformation change can be significantly monitored by the nanoCA device with excellent refractive sensing capacity. Thus, LSPR method using nanoCA with OBPs showed higher sensitivity than conventional electrochemical methods. Thus, it really suggested an efficient and ultrasensitive approach to detect small odorant molecules. The bio-nanoCA was also demonstrated to recognize binding of OBPs and nitro-explosives such as TNT, DNT and 1NT, although the selectivity was in broad range and the responses were not dosedependent. In the measurement, the response of the bio-nanoCA to TNT was larger than that of DNT and 1NT. It might come from the difference of interaction between OBP and explosive molecules. As reported, the binding of explosive molecules, such as TNT, DNT, and 1NT, was due to interaction of the adjacent methyl and nitro groups of the explosive molecule and amino acid residues [21,48]. Thus, as shown in Fig. S1, TNT, with more adjacent groups than DNT and 1NT, would had higher affinity to OBPs, give rise to larger conformation change, and produce more significant response signals of the sensor. In the rough, the bio-nanoCA could at least respond TNT at 1 M, which showed better performance than several reported biosensors for TNT (Table. S1). Beside the sensitivity, broad selectivity was a great challenge for the bio-nanoCA to distinguish TNT from other analytes such as -ionone. From molecular dicking, we could know that affinity of the OBPs to nitro-explosives might be attributed to some amino acids in the binding pocket of the protein. For instance, several amino acid sequences like His-Arg and Leu-Met-Pro were reported to have sensitivities to TNT molecules [21,49,50]. Thus, OBPs could be redesigned into more sensitive and selective proteins for certain analyte such as TNT by reengineering binding sites on natural OBPs. Then, utilizing ultrasensitive
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monitoring of nanoCA for binding of small molecules to proteins, a practical biosensor for nitro-explosives could be developed in foreseeable future. Moreover, our study showed a promising method to obtain excellent biosensing components by using easy-to-obtain OBPs from biological olfactory system. In fact, animal olfaction can provide distinguished responses to a variety of chemicals. For example, different kinds of insects, just like honeybees, can sensitively and selectively detect thousands of odors at very low concentrations. The perception ranges were from common gases like ammonia and carbon dioxide to some compounds, even if these were not occurring in the animal’s natural environments, such as volatiles from cancer cells [42,51–53]. Thus, more than hundreds of OBPs, all having affinities to various chemicals, can be screened and redesigned into specific proteins to provide a great source of excellent bio-sensitive components. With our nanobiosensor platform monitoring interaction of small molecules and proteins, more excellent sniffer abilities of animals can be screened, integrated, and used for practical detections. Now, several groups have tried to utilize animals’ olfaction to diagnose cancers, which means that biological organisms, such as dogs and fruit flies, may have bio-components sensitive to biomarkers of the diseases [53–55]. Therefore, more than security inspection for nitro-explosives, the sensor also has promising potential in cancer diagnosis. 5. Conclusion A LSPR sensor was developed for chemical detections by utilizing nanoCA to monitor specific binding between small molecules and proteins. We demonstrated the quantification between LSPR wavelength shifts and the binding by using theoretical simulation and model experiment with odorant binding to OBPs. More than detecting floral odorants, the nanosensor also showed potential application for several nitro-explosive detections. This study provided a powerful approach to quantify interactions between small molecules and proteins, which allowed further bio-detections in environment monitoring, food safety, and medical diagnosis. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81371643; No. 31372254) and the Zhejiang Provincial Natural Science Foundation of China for Distinguished Young Scholars (Grant No. LR13H180002). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.06.091 References [1] M. Laska, C.G. Galizia, M. Giurfa, R. Menzel, Olfactory discrimination ability and odor structure–activity relationships in honeybees, Chem. Senses 24 (1999) 429–438. [2] C.G. Galizia, S. Sachse, A. Rappert, R. Menzel, The glomerular code for odor representation is species specific in the honeybee Apis mellifera, Nat. Neurosci. 2 (1999) 473–478. [3] K.W. Wanner, A.S. Nichols, K.K. Walden, A. Brockmann, C.W. Luetje, H.M. Robertson, A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 14383–14388. [4] M. Schott, C. Wehrenfennig, T. Gasch, A. Vilcinskas, Insect antenna-based biosensors for in situ detection of volatiles, in: Yellow Biotechnology II, Springer, 2013, pp. 101–122. [5] Y. Lu, H. Li, S. Zhuang, D. Zhang, Q. Zhang, J. Zhou, et al., Olfactory biosensor using odorant-binding proteins from honeybee: ligands of floral odors and pheromones detection by electrochemical impedance, Sens. Actuators B: Chem. 193 (2014) 420–427.
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Biographies Diming Zhang received his bachelor degree in Zhejiang University, in 2011. Now he is a Ph.D. student of biomedical engineering of Zhejiang University. His work includes design of nanoplasmonic biosensor and electronic measurement. Yanli Lu received her bachelor degree in Xi’an Jiaotong University, PR China in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes OBPs-based biosensors and electronic measurement. Qian Zhang received her bachelor degree in Zhejiang University in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Yao Yao received her bachelor degree in Zhongnan University in 2013. Now she is an M.Sc. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Shuang Li received her bachelor degree in Hunan Normal University in 2014. Now she is an M.Sc. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Hongliang Li received his Ph.D. degree in entomology from Zhejiang University, PR China in 2007. He is currently an associate professor in Academy of Life Science, China Jiliang University. His research interests concentrate on the olfactory sensation and chemosensing system of honeybee.
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Shulin Zhuang received his Ph.D. degree in chemical biology from Zhejiang University, PR China in 2007. He is currently an associate professor in Academy of Environment and Resources, Zhejiang University. His research interests concentrate on chemical biology and biophysics.
Livermore national lab. In 2008, he joined University of Illinois at Urbana-Champaign as an assistant professor in Department of Electrical and Computer Engineering and Department of Bioengineering. His now research focuses on designing integrative bionano and microfluidic technologies.
Jing Jiang received his B.S. degree from the University of Illinois in 2011 with highest honors. He is now a Ph.D. candidate under supervision of Prof. Logan Liu. His researches include the design of mobile sensor system and nanomaterials for optical and biological application.
Qingjun Liu received his Ph.D. degree in biomedical engineering from Zhejiang University, PR China in 2006. He is currently a professor in Biosensor National Special Laboratory, Zhejiang University. He is also a visiting scholar in the Micro and Nanotechnology Laboratory (MNTL) at the University of Illinois at Urbana-Champaign (UIUC). He published the book of Cell-Based Biosensors: Principles and Applications, by Artech House Publishers USA in October 2009. His research interests concentrate on the biosensors (e.g. living cell sensor, DNA sensor and protein sensor) and BioMEMS system.
Gang Logan Liu obtained his Ph.D. degree in bioengineering from University of California Berkeley and UC-San Francisco. He finished his postdoctoral training in the Helen Diller comprehensive cancer center at San Francisco as well as Lawrence
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Nanoplasmonic monitoring of odorants binding to olfactory proteins from honeybee as biosensor for chemical detection Diming Zhang a , Yanli Lu a , Qian Zhang a , Yao Yao a , Shuang Li a , Hongliang Li b , Shulin Zhuang c , Jing Jiang d , Gang Logan Liu d , Qingjun Liu a,∗ a Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China b College of Life Sciences, China Jiliang University, Hangzhou 310018, PR China c College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, PR China d Micro and Nanotechnology Lab, University of Illinois at Urbana-Champaign, IL 61801, USA
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Article history: Received 13 April 2015 Received in revised form 10 June 2015 Accepted 13 June 2015 Available online 26 June 2015 Keywords: Odorant binding protein (OBP) Nanocup arrays Localized surface plasmon resonance (LSPR) Small odorant molecules Nitro-explosives
a b s t r a c t Odorant binding proteins (OBPs) are key soluble sensitive proteins for odor detections in animal olfactory systems. Here, we present a nanosensor based localized surface plasmon resonance (LSPR) to monitor binding of small odorant molecules to OBPs from honeybees. The binding of odorants molecules to OBPs, can be quantified by nanocup arrays from 10 nM to 1 mM, with a high refractive index sensing of LSPR wavelength shift in transmission spectra. Linear dose-dependent behavior can be observed in relationship of LSPR wavelength versus analyte concentrations. More than floral odorants, this type of biofunctionalized nanocups also showed potential applications in explosive detections for nitro-compounds such as 2,4,6-trinitrotoluene, 2,4-dinitrotoluene and 3-mononitrotoluene. This study demonstrated a LSPR device to monitor interactions between small molecules and proteins, which provided a great biosensor platform for healthcare diagnosis, environment monitoring and food analysis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Honeybees have been demonstrated to distinguish thousands of floral odors at low concentrations by their remarkable olfactory systems [1,2]. Theoretically, honeybees can detect floral scents containing hundreds of different types in their living environment, which helps them navigate rapidly and accurately to food sources kilometers away. Thus, bio-components from olfactory systems of honeybees, such as antenna and sensing proteins, could be widely used in odor detections [3–5]. More interestingly, the olfactory perceptions of honeybees were reported even out of ranges of floral odors existing in their living environment [6–8]. Honeybees showed a great discrimination for several compounds that they never suffered in natural conditions, while trained to detect explosives and diagnose diseases. Thus, the remarkable olfaction should have more important and practical applications in chemical detections, more than just detecting for floral scents. Over last couple of decades, significant efforts were made to develop biosensors that combined biosensing components to
∗ Corresponding author. Tel.: +86 571 87951676; fax: +86 571 87951676. E-mail address: [email protected] (Q. Liu). http://dx.doi.org/10.1016/j.snb.2015.06.091 0925-4005/© 2015 Elsevier B.V. All rights reserved.
physicochemical transducers, in the field of environmental monitoring, food safety, and medical diagnosis [5,9,10]. Now, the lack of selective and sensitive biosensing components became a key challenge, making the biosensors inadequate in many applications. For proteins-based biosensors, enzymes, antigens–antibodies, and receptors were common sensitive elements to bind target molecules and then elicit sensor responses [11,12]. However, these complex proteins in biological organisms were often difficult to purify, almost impossible to synthesize, and thus limited their usage for biosensors. Thus, it was a sustaining work to obtain biosensitive component in easy and low-cost way. However, odorant binding proteins (OBPs) were one kind of chemical sensing proteins in olfactory system [13–15]. They were thought to deliver odor molecules to olfactory receptors, and were the first biochemical step in odor reception capable of some level of odor discrimination. OBPs often showed an affinity to a wide range odorants and had a broad specificity for different analytes simultaneously. Notably, different from membrane proteins, OBPs as one kind of soluble proteins, could be expressed at low cost, purified easily, and kept great bioactivities in vitro [16,17]. With these advantages, OBPs from animals, such as bovine and swine, have been used to detect alcohols and pheromone [18,19]. Besides these common chemicals, OBPs often had multiple binding sites simultaneously with
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conformational changes for diverse molecules such as pesticides and explosives [20,21]. Thus, OBPs should have more biosensor applications by monitoring interactions between target analyte and OBPs. Detection for specific binding of small molecules to proteins was of substantial importance because of its great application in drug screening, biochemical analysis and physiological study [22–24]. In recent years, several nanostructure sensors (e.g., nanoparticles, nanoholes, and nanowires) have been reported as excellent platforms for the biodetection for small molecules with high sensitivity, excellent reproducibility, real-time responses, and label-free detection [25–28]. They were based on collective oscillations of conduction electrons of localized surface plasmon resonance (LSPR), at nanostructures with high confined electrical field extended on their surfaces. Using this type of sensors, biointeractions could be monitored quantifiably and ultrasensitively, even at single molecular level, through observing wavelength shifts in LSPR before and after binding of the target ligands. It was especially helpful to quantify small molecules which only elicited little changes in bio-interactions. Moreover, optical LSPR observation had advantages that allowed multiple-component analysis in array without labels on targets and physical connections to detecting elements. Thus, the LSPR sensing with nanostructures provided an interesting and promising approach to quantify small molecule–protein interactions in biosensing. Here, we developed a novel biosensor based on nanocup arrays (nanoCA) to monitor the bindings of small molecules to proteins through LSPR spectroscopy. The nanoCA sensor can recognize the conformation changes of the OBPs in presence of typical floral odorants such as -ionone in a dose-dependent response, while its biosensing mechanism was simulated with Mie scattering model. More interestingly, bindings of 2,4,6-trinitrotoluene (TNT) to the OBPs can be observed in molecular docking and nanoCA monitoring, respectively. The nanoCA with OBPs had significant wavelength shift in detection for TNT, as well as 2,4-dinitrotoluene (DNT) and 3-mononitrotoluene (1NT), which suggested potential application of the nanoCA for nitro-explosive detections. 2. Materials and methods 2.1. Proteins and agents In our previous study, OBPs of Acer-ASP2 from honeybee have been demonstrated to bind floral odors and semiochemicals (e.g. -ionone, geraniol, benzaldehyde, and isoamyl acetate) by electrochemical impedance measurement [5,29]. Thus, the OBPs were also immobilized on nanoCA in this study to bind typical odorant of -ionone to demonstrate LSPR monitoring for small molecule–protein interaction. The OBPs solution used in experiments was at concentration of 500 g/ml in phosphate buffered saline (PBS; pH = 7.4), while -ionone was diluted into different concentrations ranging from 10 nM to 1 mM with methanol. As the negative control, bull serum albumin (BSA) was prepared at 500 g/ml and immobilized on the device by same method as that of OBPs. Moreover, in nitro-explosive detections, TNT, DNT, and 1NT were diluted with methanol into increasing concentrations of 10 nM, 1 M, and 100 M. All other agents were purchased from Sigma and of analytical grades. 2.2. Fabrication of nanoCA NanoCA devices were fabricated by nanoimprint with a template containing nanocone arrays and then deposited with Au nanoparticles. The detailed processes have been described in our previous study [30]. In brief, a 250 m flexible (Poly)ethylene
terephthalate (PET) sheet was used as supporting substrate, and UV curable polymer was evenly distributed on the template. A UV lightcuring flood lamp system (Dymax, EC-Series) was used at average power density of 105 mW/cm2 for 60 s to solidify the UV polymer. Au nanoparticles were deposited on sidewalls of nanocups with a six pocket e-beam evaporation system (Temescal), after using 5 nm thick titanium as adhesive layer. The depth and opening diameter of nanocup were 500 nm and 200 nm, respectively, while diameters of the particles were about 20 nm. NanoCA could have good reproducibility if the sizes of nanocup arrays and nanoparticles were well controlled in the same fabrication conditions. 2.3. Expression and purification of OBPs The recombinant OBPs of Acer-ASP2 were cloned from the full-length cDNA of adult worker bees, Apis cerana cerana. The recombinant expressed plasmid pET-Acer-ASP2 was transformed into Escherichia coli BL21 (DE3) competent cells after 450 bp fragment was excised with BamH I and Hind III from the pGEMAcer-ASP2 plasmid. The cells were grown in Luria–Bertani broth (including 30 g/ml kanamycin) at 37 ◦ C with 1.5 mM isopropyl-d-thiogalactopyranoside to induce expression of the protein. After 5 h at 28 ◦ C, the bacterial cells were harvested and lysed by sonication and centrifuged into crude cell extracts (pellet and supernatant). The pellets of crude cell extracts, containing recombinant proteins, were severely precipitated in 1.5 M urea in ddH2 O and finally freeze-dried. Finally, the protein was resuspended (500 g/ml) in PBS and stored under 4 ◦ C for biosensor experiments. More details could be found in our previous study about expression of the OBPs from honeybee [31]. 2.4. Self-assembled immobilization with PEG NanoCA device was firstly washed with mixture of H2 SO4 and H2 O2 (7:3), and DI water respectively, to remove the organic residues. Subsequently, the NanoCA device was immersed and reacted with 2 ml poly(ethylene glycol) 2-mercaptoethyl ether acetic acid (HOOC-PEG-SH, 2 kDa) at 1 mg/ml overnight (about 18 h) at room temperature to form Au S semi-covalent bond on the surface of the nanoCA device. Then, 2 ml mixing solution of 8 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma) and 12 mg/ml N-hydroxy-succinimide (NHS) in 0.1 M 2-(N-Morpholino)ethanesulfonic acid (MES) buffer (pH = 5) was immersed for 15 min to activate carboxylate groups of HOOC-PEGSH after extra HOOC-PEG-SH was washed off with DI water. Finally, this mixture solution was adjusted into pH = 8.0 with saturated NaHCO3 , added with 1 ml OBPs, and incubated at room temperature for 2 h. The nanoCA was washed with DI water by gently shaking for 60 s, followed by air drying with nitrogen and stored at 4 ◦ C for following experiments. 2.5. Simulation of nanoCA with OPBs for odorant binding With OBP immobilization on nanoCA, the device was converted into a biosensor for odorant from a transducer sensitive to refractive index change. The biofunctionalized nanoCA (bio-nanoCA) could detect small odorant molecule binding to the protein by monitoring wavelength shift of LSPR peak in transmission spectra. The surface plasmon resonance peak in the transmission spectra of bionanoCA could be modeled by Mie light scattering of single coated gold sphere. The dielectric constant of Au in the spectral range of 300–1100 nm was from Johnson and Christy’s review [32]. The dielectric constant of coated bio-component ε1 changed from 5 to 7, while dielectric constant of solution environment ε0 was fixed on 2.0736 in methanol. In the simulation, the inner and outer radiuses of coated sphere were set up at 20 nm and 30 nm, respectively. The
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calculation was performed with software programed by the group of García de Abajo [33]. 2.6. Electrochemical and optical measurement Electrochemical impedance sensing was performed with standard three electrode system. NanoCA was used as working electrode, while platinum electrode and Ag/AgCl electrode were used as counter electrode and reference electrode, respectively. 5 mM K4 [Fe(CN)6 ]/K3 [Fe(CN)6 ] (1:1) was employed as redox couple for electrochemical detecting. Electrical signals were measured by electrochemical workstation (CHI660E, Chenhua Instruments Co., China) in AC impedance mode. The tested frequency was set from 1 Hz to 1 kHz with a 5 mV alternating voltage. Before the optical measurement, 30 l analyte solutions were added in the well of the plate. The solutions could be diffused and distributed evenly on the nanoCA surface due to tension. The optical detection was based on a multi-mode detection platform (SpectraMax Paradigm, Molecular Devices Co., United Sates). Normal transmission model was applied to measure the spectra of bio-nanoCA when scanning range was set from 300 nm to 900 nm and step was fixed on 1 nm. Considering harmful effect of repeated elution for immobilization and bio-activity of the protein, the bionanoCA device was used only one time in optical measurement to keep best performance for the odorant detection. 3. Results 3.1. NanoCA monitoring with LSPR spectroscopy Periodic nanostructures had outstanding optical features such as absorption and transmission peaks in visible spectroscopy [26,34]. These features were elicited by plasmon resonance and could be utilized in optical sensors. As shown in Fig. 1a, the nanoCA chips were integrated into 96 well plates and could be performed in high throughput measurements. On the surface of nanoCA chip, nanocups were subwavelength opening on transparent plastic substrate, fabricated periodically in arrays and decorated with Au nanoparticles by electron beam evaporation (Fig. 1b). Fig. 1c showed photo of nanoCA device obtained with scanning electron microscope. Transmission detection was applied to measure the LSPR spectroscopy of nanoCA. NaCl at different concentrations were employed as increasing refractive index solutions, to determine the refractive-index sensing properties of nanoCA (Fig. 1d). With synergetic LSPR effects of periodical array structure and nanoparticles deposited on the nanocups, the nanoCA showed a high sensitivity for refractive index change with ∼104 nm per refractive index unit (RIU), which was much greater than typical nanoparticle sensors and nanohole sensors based on plasmon resonance [35–37]. Thus, a high sensitive LSPR device based on nanoCA can be obtained and used to monitor binding of small molecules to OBPs as a highthroughput biosensor. 3.2. Fabrication of bio-nanoCA for odorant binding detection In this study, OBPs of AcerASP2 were produced from E. coli and used to modify nanoCA. Based on Au S semi-covalent linkage, a self-assembled film of HS PEG COOH was used to fix OBPs on the surface of nanoCA for small odorant molecule binding (Fig. 2a). The immobilization of OBPs on surface of nanoCA was important for fabrication of the nanoplasmonic biosensor monitoring the odorant binding. Thus, two methods, including optical and electrochemical measurements, were used to characterize property changes of the biosensor during the protein immobilization, in order to verify successful immobilization of OBPs. Fig. 2b showed optical transmission spectra measurement, when PEG and OBPs were immobilized on
Fig. 1. The nanosensor device and its optical measurement system. (a) Schematic diagram of the optical detection using a 96-well plate instrument in transmission mode. (b) Structure of the periodic nanocups on the nanoCA chip, with Au nanoparticles deposited along the sidewalls. (c) SEM image of the cup arrays. (d) Transmission spectra of nanoCA in presence of NaCl at different concentrations. The inset showed wavelength shifts with concentration increasing. WS: wavelength shift, RI: refractive index.
nanoCA step by step. Obvious wavelength shifts, more than 20 nm, could be observed in LSPR peak around 500 nm, which suggested well immobilization of OBPs. Moreover, in our previous work, nanoCA device was demonstrated as a good electrode for electrochemical measurement [38]. Thus, as one of the most powerful and sensitive techniques, electrochemical impedance spectroscopy was also chosen to verify the protein immobilization. The most important parameter of impedance spectroscopy is a semicircle portion corresponding to the electron transfer limiting process, the diameter of which represented interface impedance change on device surface. As shown in Fig. 2c, significant increase in semicircle diameter can be observed in Nyquist plot with interface impedance changed by the protein immobilization. These both indicated that the OBPs were immobilized on the nanoCA surface efficiently. After well immobilization of the OBPs on nanoCA, the bionanoCA could be used to detect odorant binding to the proteins. As shown in Fig. 3a, OBPs on nanoCA could bind small molecule ligands and then had property changes (e.g., conformation, volume, and electron distribution), which could modulate LSPR in electrical field near nanoCA surface. The LSPR spectroscopy of nanoCA was from complex plasmonic effects of surface plasmon polaritons-Bloch
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Fig. 2. Immobilization of OBPs on the sidewalls of nanoCA device. (a) Schematic diagram of the nanoCA functionalized by the self-assembled proteins. (b) Transmission spectra of nanoCA immobilized with the proteins. Significant wavelength shift max can be observed in shift from 523 nm to 551 nm. (c) Nyquist plots for immobilization of nanoCA with PEG and protein.
wave, Wood’s anomaly, and Mie scattering on hybrid structures of nanocups and nanoparticles [30]. The peak around 600 nm was due to Mie scattering from nanoparticles. Thus, a model of coated sphere was used to describe the Mie scattering of bio-nanoCA (Fig. 3b). In theoretical simulation, Mie light scattering curve of the model showed high similarity to the resonance peak around 600 nm in the transmission spectra of nanoCA modified by proteins in the experiment (Fig. 3c). It was also found that the wavelength had linear shifts with dielectric constant changes, from 5 to 7, of coated biosensing elements (Fig. 3d). Although RIU sensitivity in
simulation was lower than that of experiments, the linear relationship shown in simulation gave a possible guarantee to dose-dependent responses of bio-nanoCA in monitoring of binding between small odorant molecules and OBPs. 3.3. Bio-nanoCA monitoring for binding of odorants to OBPs Several chemical molecules could specifically bind into hydrophobic pockets of OBPs immobilized on the nanoCA surface, which could modulate protein conformation and change relative
Fig. 3. Biosensing mechanism of the bio-nanoCA. (a) Schematic diagram of the nanoparticles on the surface of nanoCA functionalized by biosensing elements. (b) Model of coated sphere representing the bio-component modified nanoparticles. ε0 , ε1 , and ε2 are relative dielectric constants of solution environment, bio-component, and Au nanoparticle respectively, while (a) and (b) are inner and outer radiuses of the particles. (c) Simulated transmission spectra from Mie scattering of coated particles and experimental LSPR spectra. (d) Linear relationship between wavelength shift and dielectric constant changes of the bio-component immobilized on the nanoCA.
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Fig. 4. Examination of nanoCA monitoring odorant molecule–OBPs binding. (a) Chemical structures of -ionone, butanedione and acetic acid, and smell they had. (b, c) Transmission spectra of OBPs- and BSA-modified nanoCA, respectively. The OBPs-modified device shows specific response to -ionone, while no significant responses were observed in other conditions. (d, e) Statistic of p and max for responses of OBPs- and BSA-modified nanoCA, respectively (mean ± SD, n = 15; *P < 0.05).
dielectric constants of the bio-coating [23,39]. In odorant measurement, one kind of aromatic odorants, -ionone, was used as modeling molecule to test responses of the bio-nanoCA with OBPs. Butanedione and acetic acid, having different pungent and buttery smell, were used as the control to assess nanoCA monitoring of odorant molecules–OBPs binding (Fig. 4a). As illustrated in Fig. 4b, transmission spectra of bio-nanoCA device, especially intensity and location of transmission peak, was notably changed by specific binding of -ionone and OBPs. In contrast, there were no significant variations observed in transmission spectra in presence of acetic acid and butanedione at same concentration without specific binding. To further examine whether change of transmission spectra was caused by specific interactions between odorant molecules and the proteins, we also carried out same experiments on BSA-modified nanoCA. The BSA-modified device was used as nonspecific-binding group to show transmission spectra changes of devices elicited by own refractive index of solution rather than specific interactions between OBPs and target molecules. Indeed, BSA-modified nanoCA showed no remarkable responses in presence of all compounds of butanedione, acetic acid and -ionone (Fig. 4c). Moreover, transmission intensity change p and LSPR wavelength shift max , were both calculated into statistics with the OBPs- and BSA-modified devices (Fig. 4d and e). In accordance with the result of transmission spectra, the statistics showed that OBPs-modified device could selectively respond to -ionone and BSA-modified nanoCA had no response to all compounds. These results all demonstrated that nanoCA could monitor specific binding of odorant molecule of -ionone to OBPs with transmission intensity change and wavelength shift in LSPR peak. Then, the dose-dependent behavior of bio-nanoCA was analyzed by transmission intensity changes and wavelength shifts from responses of the devices to -ionone at increasing concentrations. -Ionone at concentrations from 10 nM to 1 mM were used to bind OBPs on the device to modulate LSPR in transmission spectra (Fig. 5a). As shown in Fig. 5b and c, wavelength shift
and transmission intensity decreasing obtained from OBPs modified nanoCA, both showed similar dose-dependent responses to -ionone, while no significant responses can be found from the nanoCA with BSA. However, compared to the intensity decreasing, the wavelength shift had broader linear responses from 10 nM to 1 mM. Considering wavelength shifts observed in theoretical simulation, thus, the wavelength shift was analyzed as effective responses of bio-nanoCA. With the dose-dependent curve, the detection limit of bio-nanoCA to -ionone was determined at 26.7 pM based on 3ı/slope method with wavelength shifts. However, as shown in Fig. 5d, -ionone detection without nanoCA showed overlap of 10 M and the control of methanol in absorption spectrum, which meant higher detection limit than 10 M in nanoCA-absent measurement. Therefore, the nanosensor really had ultrasensitive capacity to monitor the binding of small odorant molecules to the proteins. 3.4. Bio-nanoCA for nitro-explosive detection Several studies recently reported about training honeybees and using their sensing proteins to find nitro-explosives [40–42]. Especially, different OBPs have been used to modify biosensor for explosive detections [21,43,44]. Thus, we tried to explore the binding of nitro-explosive molecules to the OBPs of AcerASP2 by the bio-nanoCA. Fig. 6a showed the molecular docking of specific binding of TNT to OBPs. TNT molecules could enter protein’s cavities, interact with amino acid residues and form the complex with OBPs. This process might elicit same conformations change of OBPs as that of the -ionone binding and modulate the LSPR on the nanoCA in the optical measurement. Fig. 6b showed transmission spectrum of the bio-nanoCA with OBPs in presence of TNT at different concentrations. TNT at high concentrations of 100 M and 1 M elicited significant wavelength shifts in comparison to the control, while TNT at 10 nM had no effect on the device. In order to check whether the shifts came from specific binding of TNT, the nanoCA
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Fig. 5. Bio-nanoCA with OBPs for -ionone at increasing concentrations. (a) LSPR spectra of OBPs-modified nanoCA in presence of -ionone from 10 nM to 1 mM. (b) Dose-dependent profiles of bio-nanoCA with OBPs in intensity decreasing of transmission peak. (c) Dose-dependent profiles of bio-nanoCA with OBPs in wavelength shift of transmission peak. The wavelength shifts have good linear responses to the concentrations of -ionone, when methanol is used as blank control. (d) Measurement for -ionone binding in the absorption spectrum without nanoCA. The curves overlap of 10 M -ionone and the control means higher detection limit than 10 M (mean ± SD, n = 15).
Fig. 6. Bio-nanoCA with OBPs for explosive detection. (a) Molecular docking of TNT in the binding pocket of the protein. (b) Transmission spectrum of OBPs-modified nanoCA for TNT at different concentrations. (c) Comparing responses of the bio-nanoCA with and without OBP to TNT at 100 M. (d) Wavelength shift for responses of the bio-nanoCA with OBPs and BSA to explosives of TNT, DNT, and 1NT (mean ± SD, n = 15; *P < 0.05; NS, not significant, P > 0.05).
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without OBPs was also exposed to TNT. As shown in Fig. 6c, the bio-nanoCA with OBPs showed obvious wavelength shift in presence of TNT, while responses of the nanoCA without OBPs had no reliable difference between TNT and blank control of methanol. It suggested that binding of TNT and OBPs could really be monitored by the device and used for explosive detection. Besides TNT, two kinds of nitro-explosive analogs, DNT and 1NT, were carried out in experiments to test bio-nanoCA. Fig. 6d indicated high responses of the bio-nanoCA to three kinds of nitro-explosives of TNT, DNT and 1NT at different concentrations. The responses to three nitroexplosives were almost equal to those from -ionone, although their responses were similar at 100 M and 1 M without dosedependence behaviors.
4. Discussion In our study, nanoCA was successfully used to monitor and quantify binding of small molecules to proteins. The bio-nanoCA was proved to detect interaction between odorant molecules of ionone and OBPs in linear dose-dependence. The detection limit of the device for -ionone was 26.7 pM, which was much lower than our previous study using electrochemical impedance spectroscopy for odor detections [5,29]. The higher sensitivity might come from the combination of OBPs and nanostructured LSPR device. On one hand, similar to SPR technique, LSPR was an excellent method to monitor the bio-molecular binding event. As reported, with several nanostructures, the sensor can even detect single molecular binding on the sensor surface [45–47]. On the other hand, the special binding of small molecules to OBPs also was the reason of higher sensitivity of the device than the previous electrochemical method. Different from common biomolecular binding on bio-component surface, the binding of small molecule to OBPs happened on the pocket of the protein, which elicited lower electrochemical impedance change than that of the common binding. However, the binding in the pocket and subsequent conformation change can be significantly monitored by the nanoCA device with excellent refractive sensing capacity. Thus, LSPR method using nanoCA with OBPs showed higher sensitivity than conventional electrochemical methods. Thus, it really suggested an efficient and ultrasensitive approach to detect small odorant molecules. The bio-nanoCA was also demonstrated to recognize binding of OBPs and nitro-explosives such as TNT, DNT and 1NT, although the selectivity was in broad range and the responses were not dosedependent. In the measurement, the response of the bio-nanoCA to TNT was larger than that of DNT and 1NT. It might come from the difference of interaction between OBP and explosive molecules. As reported, the binding of explosive molecules, such as TNT, DNT, and 1NT, was due to interaction of the adjacent methyl and nitro groups of the explosive molecule and amino acid residues [21,48]. Thus, as shown in Fig. S1, TNT, with more adjacent groups than DNT and 1NT, would had higher affinity to OBPs, give rise to larger conformation change, and produce more significant response signals of the sensor. In the rough, the bio-nanoCA could at least respond TNT at 1 M, which showed better performance than several reported biosensors for TNT (Table. S1). Beside the sensitivity, broad selectivity was a great challenge for the bio-nanoCA to distinguish TNT from other analytes such as -ionone. From molecular dicking, we could know that affinity of the OBPs to nitro-explosives might be attributed to some amino acids in the binding pocket of the protein. For instance, several amino acid sequences like His-Arg and Leu-Met-Pro were reported to have sensitivities to TNT molecules [21,49,50]. Thus, OBPs could be redesigned into more sensitive and selective proteins for certain analyte such as TNT by reengineering binding sites on natural OBPs. Then, utilizing ultrasensitive
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monitoring of nanoCA for binding of small molecules to proteins, a practical biosensor for nitro-explosives could be developed in foreseeable future. Moreover, our study showed a promising method to obtain excellent biosensing components by using easy-to-obtain OBPs from biological olfactory system. In fact, animal olfaction can provide distinguished responses to a variety of chemicals. For example, different kinds of insects, just like honeybees, can sensitively and selectively detect thousands of odors at very low concentrations. The perception ranges were from common gases like ammonia and carbon dioxide to some compounds, even if these were not occurring in the animal’s natural environments, such as volatiles from cancer cells [42,51–53]. Thus, more than hundreds of OBPs, all having affinities to various chemicals, can be screened and redesigned into specific proteins to provide a great source of excellent bio-sensitive components. With our nanobiosensor platform monitoring interaction of small molecules and proteins, more excellent sniffer abilities of animals can be screened, integrated, and used for practical detections. Now, several groups have tried to utilize animals’ olfaction to diagnose cancers, which means that biological organisms, such as dogs and fruit flies, may have bio-components sensitive to biomarkers of the diseases [53–55]. Therefore, more than security inspection for nitro-explosives, the sensor also has promising potential in cancer diagnosis. 5. Conclusion A LSPR sensor was developed for chemical detections by utilizing nanoCA to monitor specific binding between small molecules and proteins. We demonstrated the quantification between LSPR wavelength shifts and the binding by using theoretical simulation and model experiment with odorant binding to OBPs. More than detecting floral odorants, the nanosensor also showed potential application for several nitro-explosive detections. This study provided a powerful approach to quantify interactions between small molecules and proteins, which allowed further bio-detections in environment monitoring, food safety, and medical diagnosis. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81371643; No. 31372254) and the Zhejiang Provincial Natural Science Foundation of China for Distinguished Young Scholars (Grant No. LR13H180002). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.06.091 References [1] M. Laska, C.G. Galizia, M. Giurfa, R. Menzel, Olfactory discrimination ability and odor structure–activity relationships in honeybees, Chem. Senses 24 (1999) 429–438. [2] C.G. Galizia, S. Sachse, A. Rappert, R. Menzel, The glomerular code for odor representation is species specific in the honeybee Apis mellifera, Nat. Neurosci. 2 (1999) 473–478. [3] K.W. Wanner, A.S. Nichols, K.K. Walden, A. Brockmann, C.W. Luetje, H.M. Robertson, A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 14383–14388. [4] M. Schott, C. Wehrenfennig, T. Gasch, A. Vilcinskas, Insect antenna-based biosensors for in situ detection of volatiles, in: Yellow Biotechnology II, Springer, 2013, pp. 101–122. [5] Y. Lu, H. Li, S. Zhuang, D. Zhang, Q. Zhang, J. Zhou, et al., Olfactory biosensor using odorant-binding proteins from honeybee: ligands of floral odors and pheromones detection by electrochemical impedance, Sens. Actuators B: Chem. 193 (2014) 420–427.
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Biographies Diming Zhang received his bachelor degree in Zhejiang University, in 2011. Now he is a Ph.D. student of biomedical engineering of Zhejiang University. His work includes design of nanoplasmonic biosensor and electronic measurement. Yanli Lu received her bachelor degree in Xi’an Jiaotong University, PR China in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes OBPs-based biosensors and electronic measurement. Qian Zhang received her bachelor degree in Zhejiang University in 2012. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Yao Yao received her bachelor degree in Zhongnan University in 2013. Now she is an M.Sc. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Shuang Li received her bachelor degree in Hunan Normal University in 2014. Now she is an M.Sc. student of biomedical engineering of Zhejiang University. Her work includes biosensors and electronic measurement. Hongliang Li received his Ph.D. degree in entomology from Zhejiang University, PR China in 2007. He is currently an associate professor in Academy of Life Science, China Jiliang University. His research interests concentrate on the olfactory sensation and chemosensing system of honeybee.
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Shulin Zhuang received his Ph.D. degree in chemical biology from Zhejiang University, PR China in 2007. He is currently an associate professor in Academy of Environment and Resources, Zhejiang University. His research interests concentrate on chemical biology and biophysics.
Livermore national lab. In 2008, he joined University of Illinois at Urbana-Champaign as an assistant professor in Department of Electrical and Computer Engineering and Department of Bioengineering. His now research focuses on designing integrative bionano and microfluidic technologies.
Jing Jiang received his B.S. degree from the University of Illinois in 2011 with highest honors. He is now a Ph.D. candidate under supervision of Prof. Logan Liu. His researches include the design of mobile sensor system and nanomaterials for optical and biological application.
Qingjun Liu received his Ph.D. degree in biomedical engineering from Zhejiang University, PR China in 2006. He is currently a professor in Biosensor National Special Laboratory, Zhejiang University. He is also a visiting scholar in the Micro and Nanotechnology Laboratory (MNTL) at the University of Illinois at Urbana-Champaign (UIUC). He published the book of Cell-Based Biosensors: Principles and Applications, by Artech House Publishers USA in October 2009. His research interests concentrate on the biosensors (e.g. living cell sensor, DNA sensor and protein sensor) and BioMEMS system.
Gang Logan Liu obtained his Ph.D. degree in bioengineering from University of California Berkeley and UC-San Francisco. He finished his postdoctoral training in the Helen Diller comprehensive cancer center at San Francisco as well as Lawrence