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Piezoelectric olfactory receptor biosensor prepared by aptamer-assisted immobilization
Sensors and Actuators B 187 (2013) 481–487
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Piezoelectric olfactory receptor biosensor prepared by aptamer-assisted immobilization Liping Du a , Chunsheng Wu a , He Peng b , Ling Zou a , Luhang Zhao b , Liquan Huang c , Ping Wang a,∗ a Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Ministry of Education, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China b Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, China c Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, USA
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Article history: Available online 13 February 2013 Keywords: Olfactory receptor Piezoelectric biosensor Aptamer Immobilization Odor detection
a b s t r a c t Inspired by the very high sensitivity and specificity of biological olfactory system, engineers pay much attention to biomimetic olfactory biosensors due to their excellent performance and great commercial prospects. In this study we presented an olfactory receptor (OR)-based piezoelectric biosensor with high sensitivity and specificity for the odorant detection, in which aptamers were employed for the specific and effective olfactory receptor immobilization to improve the overall performance of biosensors. An olfactory receptor of C. elegances, ODR-10, was expressed heterologously in HEK-293 cells with a hexahistidine (His6 ) tag on the N terminus. His6 -tagged ODR-10 was extracted from the plasma membrane of transfected HEK-293 cells and used as the sensing element of biosensors. Anti-His6 aptamers were immobilized covalently onto the gold surface of piezoelectric quartz crystal microbalance (QCM) electrode to capture the His6 -tagged ODR-10 specifically, as well as achieve the purification of ODR-10 simultaneously. The immobilization procedures were characterized by electrochemical methods and scanning probe microscopy (SPM). Additionally, physical adsorption method was used as a control method to illustrate the influences of immobilization methods on the performances of OR-based biosensors. The results demonstrated that the piezoelectric biosensor prepared by aptamer-assisted immobilization method showed high specificity and improved sensitivity. The detection limit was as low as 1.5 ppm (v/v). Additionally, the aptamer-assisted receptor immobilization method has great potential to become a universal protein immobilization technique in biosensing, and be applied into the development of OR-based biosensor array for the simultaneous detection of several odorants. This aptamer-assisted olfactory receptor biosensor has great application prospects in many fields, such as food safety, environmental monitoring, and disease diagnosis. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The nature’s olfactory system has an excellent capacity in recognizing and discriminating various odorants. Since the concept of “bio-electronic nose” was proposed in 1998 by Göpel [1], mimicking nature’s olfactory system has become a new trend of biosensors for odorant detection. In the biological olfactory system, olfactory receptors (ORs) act as the sensitive elements of olfactory sensory neurons (OSNs), which play a great role in odorant specific recognition and discrimination [2]. ORs belong to the G protein-coupled receptor (GPCR) family. The binding of odorants and ORs will trigger the intracellular signal transduction cascades and induce the depolarization of OSNs. The unique functions of
∗ Corresponding author. Tel.: +86 571 87952832; fax: +86 571 87952832. E-mail address: [email protected] (P. Wang). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.02.009
ORs interacting with the specific odorant molecules make them ideal candidates as sensitive elements of biomimetic gas sensors toward odorant detection [3]. Glatz and Bailey-Hill have made a comprehensive comment on mimicking nature’s nose, and discussed various olfactory biosensors as well as their applications [4]. Various transduction techniques have been employed in the research of OR-based biosensors, including field effect transistor (FET) [5,6], quartz crystal microbalance (QCM) [7–9], surface acoustic wave (SAW) device [10,11], light addressable potentiometric sensor (LAPS) [12,13], surface plasmon resonance (SPR) [14], bioluminescence resonance energy transfer (BRET) [15], and intracellular calcium responses [16]. Almost all the odorant-induced information can be utilized for olfactory biosensing. However, one major issue in this field is how to achieve functional immobilization of defined ORs with high efficiency on the solid surface of secondary transducers. The antibody-directed specific immobilization of ORs is more specific and efficient than that of physical adsorption method,
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but the procedure is relative complex and the antibody is much expensive [17]. Recent years, aptamers have been widely viewed as promising complementary molecules to antibodies, which are single-stranded oligonucleic acid molecules [18]. Compared with antibodies, aptamers have a lot of advantages, such as more resistant, being more easily modified with functional groups or tags, and less expensive. They have been used to replace antibodies in many applications, such as enzyme-linked immunosorbent assay (ELISA) [19], Western blot [20], biosensing applications [21], and protein micro-arrays [22]. Concerning the unique advantages of aptamers, this study explore the possibility of using aptamers to achieve the high specific and effective functional immobilization of ORs onto the surface of secondary transducers. We present an OR-based piezoelectric biosensor prepared by the aptamer-assisted immobilization method. Quartz crystal microbalance (QCM) is a commonly used mass sensitive piezoelectric transducer [9], whose oscillation frequency changes with the mass loading on the crystal. The interactions between ORs and odorants can be detected by monitoring the oscillation frequency changes of QCM. A well-characterized olfactory receptor of C. elegances, ODR-10, is employed as a model of ORs. Diacetyl is the only volatile ligand to activate ODR-10 [23], and can induce the intracellular calcium elevation of HEK-293 cells expressing ODR-10 in a dose-dependent way [16]. In the present study, ODR-10 was expressed on the plasma membrane of HEK-293 cells with a His6 -tag on its N-terminus. The surface of gold electrodes of QCM was modified by specific anti-His6 aptamers via Au S bonds, which could only capture His6 -tagged ODR-10 specifically through anti-His6 aptamers. Purification of ODR-10 was also achieved simultaneously due to the specific interactions between aptamers and His6 -tags. The immobilization procedures of ODR10 were characterized by electrochemical methods and scanning probe microscopy (SPM). The specificity and sensitivity of this ORbased piezoelectric biosensor were tested. In addition, physical adsorption method was used as a control method to illustrate the influences of immobilization methods on the performances of ORbased biosensors.
2. Materials and methods 2.1. Preparation of functional olfactory receptors Here an olfactory receptor of C. elegances, ODR-10, was employed as a model of ORs, which were produced by cellular heterologous expression techniques. The full-length cDNA of odr-10 was amplified by polymerase chain reaction (PCR) from pEGFPN1/rho-tag/odr-10 [24]. A pair of specific primers were designed: the forward primer 5 -AGAGGTACCGATGCACCACCACCACCACCA CATGAACGGGACCGAGGGCCCAA-3 , containing the KpnI restriction enzyme cutting site (the underlined part) and His6 sequence (the dotted part); and the reverse primer 5 -TGTGGATCCTCATTACTACGTCGGAACTTGAGACAAATTGG-3 , containing the BamHI enzyme cutting site (the underlined part). PCR was performed in a 50 L reaction volume using 1.0 L of the plasmid template pEGFP-N1/rho-tag/odr-10, 1 L of the forward primer, 1 L of the reverse primer, 4 L of 10 mM dNTP mixture, 0.5 L of STARTM polymerase, 10 L of 5× PCR buffer (containing Mg2+ ), and 32.5 L of deionized water. The reaction cycle conditions were 30 cycles of 94 ◦ C denaturation for 30 s, 60 ◦ C annealing for 1 min, and 72 ◦ C extension for 1 min. The obtained His6 -tag/rho-tag/odr-10 sequence was subcloned into the KpnI and BamHI sites of the expression vector pcDNA 3.1(+). The rho-tag import sequence was used to facilitate the localization of ODR-10 on the cell plasma membrane. His6 -tag can indicate the expression level of ODR-10 and assist the functional specific immobilization of ORs on the sensor surface.
The expression vector pcDNA 3.1(+)/His6 -tag/rho-tag/odr-10 was confirmed by restriction enzyme mapping and DNA sequencing. Then the plasmid pcDNA 3.1(+)/His6 -tag/rho-tag/odr-10 was transfected into the heterologous HEK-293 cells to express ODR-10 on the plasma membrane. HEK-293 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) (Gibco, UK) supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 g/mL), at 37 ◦ C in the 5% CO2 incubator. HEK-293 cells were seeded and cultured in 6-well plates. When being 80% confluent, cells were transfected with 4 g of the expression plasmid and 10 L of LipofectamineTM 2000 (Invitrogen) for each well according to the LipofectamineTM 2000 instructions. After 48 h, transfected cells were collected for subsequent assays. Reverse transcription polymerase chain reaction (RT-PCR) was conducted to indicate the expression of ODR-10 at mRNA level. Total RNA was collected from transfected cells by TRIZOL and analyzed with PrimeScript® 1st Strand cDNA Synthesis Kit (Takara). The cDNAs were used as templates for PCR amplification. Forward primer: 5 -AGAGGTACCGATGCACCACCACCACCACCACATGAACGG GACCGAGGGCCCAA-3 , and reverse primer: 5 -TGTGGATCCTCATTACTACGTCGGAACTTGAGACAAATTGG-3 . The reaction products were resolved by electrophoresis. The expected length of the target product was 1120 bp. Western blot was conducted to verify the expression of His6 tagged ODR-10 at the protein level. Transfected cells were collected and lysed. The protein supernatants were loaded on a SDS-PAGE gel and transferred onto a PVDF membrane. Then the membrane was incubated with the primary antibody anti-His6 mouse IgG, followed by incubation with the secondary antibody DyLight 680 conjugated goat anti-mouse IgG. The bands were visualized with the Odyssey infrared imaging system. The expected size of target protein was about 45 kDa. The membrane protein extraction kit (Sangon Biotech, China) was used to extract the functional His6 -tagged ODR-10 from the plasma membrane of transfected HEK-293 cells. About 5 × 108 cells were collected from 6-well plates and washed with ice cold cell wash buffer for three times. After resuspended in 1 mL extraction buffer (containing 1 L protease inhibitor and 1 L DTT), cells were sonicated with a sonicator for 3–4 times and centrifuged at 14,000 rpm for 10 min at 4 ◦ C. The supernatant was transferred to a new vial and centrifuged at 13,000 rpm for 5 min at room temperature. The pellet was the total cellular membrane protein, containing proteins from both plasma membrane and cellular organelle membrane. By further centrifuging, 10 L of the plasma membrane proteins were harvested. This solution was the mixture of total plasma membrane proteins, including His6 -tagged ODR-10, and stored at −70 ◦ C for the development of biosensors. 2.2. Aptamer-assisted olfactory receptor immobilization and characterization The aptamer-assisted immobilization method was proposed to immobilize the His6 -tagged ODR-10 efficiently and specifically. Its schematic was shown in the inset of Fig. 1. Before immobilization, the gold electrode surface of QCM was cleaned thoroughly to remove inorganic and organic contaminants, and then incubated with a mixture solution for 21 h to form a self assembly monolayer (SAM) through Au S bonds, which contained 5 M of thiol-modified anti-His6 aptamer molecules (5 -HS-(CH2 )6 GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-3 ) [25] and 50 M of blocker molecules 11-mercaptoundecanoic acid (11-MUA) (Sigma, USA). 11-MUA was used to block free sites of the electrode surface and provide suitable steric spaces for the aptamers. Thereafter the gold electrode was rinsed with ethanol thoroughly and dried under the nitrogen flow. 10 L of the plasma membrane extraction protein solution, containing the His6 -tagged
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Fig. 1. Piezoelectric olfactory receptor biosensor and measurement system. The inset figure illustrated the aptamer-assisted ODR-10 immobilization mechanism.
ODR-10, was dissolved in 90 L of phosphate buffer solution (PBS) containing 0.5% Trion X-100 and incubated onto the aptamermodified electrode for 3 h in 37 ◦ C bath. Then the functionalized gold electrode was rinsed by deionized water thoroughly to remove the unspecific binding proteins. In this study, immobilized anti-His6 aptamers on the electrode were able to capture the His6 -tagged ODR-10 specifically. Other irrelative proteins without His6 -tags cannot be captured onto the electrode. Therefore this method achieved the purification, pre-concentration and specific immobilization of ODR-10 simultaneously. To compare the different immobilization methods, another piezoelectric biosensor was also developed using the physical adsorption method to immobilize His6 -tagged ODR-10 [8]. 10 L of the plasma membrane extraction protein solution was evenly spread onto the electrode of the quartz crystal for 3 h at 37 ◦ C. The aptamer-assisted immobilization process was monitored by electrochemical cyclic voltammetry (CV) and electrochemical impedance spectrum (EIS). The gold electrode modified with His6 -tagged ODR-10 was the working electrode. A Pt plate and a saturated calomel electrode (SCE) were used as the counter electrode and reference electrode, respectively. All the electrochemical measurements were performed in PBS (pH 7.0) with 5 mM K4 [Fe(CN)6 ] and 5 mM K3 [Fe(CN)6 ]. The potential range of CV was from −0.3 to +0.7 V versus SCE at a rate of 50 mV/s. EIS was performed in the frequency range from 1 Hz to 100 kHz at an open loop potential with a frequency modulation of 5 mV. All electrochemical measurements were performed in a Faraday cage. The impedance data was fitted with a commercial available software Zview/Zplot. The equivalent circuit was shown in the inset of Fig. 2b, which included four elements: (1) Rs, the ohmic resistance of the electrolyte solution; (2) Rct , the charge transfer resistance; (3) CPE, the constant phase elements. Two fitting parameters, CPE-T and CPE-P, represented the double layer capacitance and its phase angle, respectively. The impedance of CPE was expressed by ZCPE = 1/[T(i × ω)P ], i: the imaginary unit, ω: the circular frequency, T: CPE-T, P: CPE-P; and (4) Zw , the generalized finite Warburg impedance in the open circuit model. W–R, W–T, and W–P were three fitting parameters of Zw . Its impedance was given by Zw = R × ctnh([i × T × ω]P )/(i × T × ω)P .
Scanning probe microscopy (SPM) (VEECO, USA) was used to characterize the surface morphology of the ODR-10 modified electrode. An antimony (n) doped silicon tip with spring constant of 30 N/m was used. The thickness and length of the cantilever are 4 m and 130 m, respectively. The free resonance frequency is 318 kHz. The image is taken in tapping mode, which can minimize the potentially destructive shear and adhesive forces on the sample. All the experiments were performed under ambient conditions with relative humidity of 60%. The image was first-order flattened. 2.3. Piezoelectric olfactory receptor biosensor and measurement system QCM200 system (Stanford Research Systems, Inc., USA), which has 5 MHz AT-cut quartz crystals with polished gold electrodes, was used as a piezoelectric transducer. The QCM200 is a standalone instrument with a built-in detection circuit and frequency counter. The resonant frequency changes as a linear function of the mass of material deposited on the crystal surface. The schematic of measurement system was shown in Fig. 1. After immobilization process, the ODR-10 modified QCM sensor was rinsed with deionized water to remove excess proteins and dried with nitrogen. Then it was placed in a detection chamber with inlet and outlet ports. The frequency data was detected by QCM200 detection system and transmitted into a computer via the RS-232 interface. All the odorants was freshly prepared in Tedlar bags (SKC Inc., USA) by a liquid organic gas blender (MF-3B, Huanchen Instruments, China). A syringe pump associated with valves controlled the injection of odorants into the detection chamber. The fresh air was used as the reference gas to simulate real-world conditions and to refresh the detection chamber after each measurement. The flow rate of odorants kept at 1 mL/s. Gas injection time was 1 min and the minimal interval for the next injection was 5 min. All the measurements were carried out at the same environmental conditions at 25 ◦ C. Six odorants were applied to test the specificity of this ODR10 based QCM biosensor, which include diacetyl, isoamyl acetate, anisole, lavender, butanone, and 2,3-pentanedione. Isoamyl acetate, anisole, and lavender belong to three different odorant groups according to their effectiveness and their molecular
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experimental data was presented as means ± standard deviation (SD). The significant difference analysis was carried out by performing Student’s t-test and p < 0.05 was considered as significant difference.
3. Results and discussion 3.1. Preparation results of olfactory receptors Functional ORs are the most crucial sensing materials of ORbased biosensor. In this study we prepared His6 -tagged ODR-10 by extracting proteins from pcDNA 3.1(+)/His6 -tag/rho-tag/odr-10 transfected HEK-293 cells. During the expression process, RT-PCR and Western blot were performed to validate the expression of His6 -tagged ODR-10 at the mRNA and protein levels. Fig. S1 was the result of RT-PCR, in which an obvious right band can be observed above 1000 bp this result verified the expression of ODR-10 in HEK293 cells at the mRNA level. Fig. S2 showed the result of Western blot and one obvious band was found between 43 and 55 kDa. It was consistent with the theoretical value of ODR-10 (45 kDa). The negative control group (HEK-293 cells without any transfection) showed no band. All these results inferred that ORs with His6 -tags were expressed successfully and there was no other His6 -tagged proteins in the extracted protein solution.
3.2. Characterization of aptamer-assisted olfactory receptor immobilization process
Fig. 2. Electrochemical characteristions of the ODR-10 immobilization procedure. (a) CV measurements. (b) Nyquist plots of impedance spectra in the frequency range from 1 Hz to 100 kHz. The inset of (b) shows the equivalent circuit model. All the electrochemical measurements were conducted in PBS solution (pH 7.0) with 5 mM K4 [Fe(CN)6 ] and 5 mM K3 [Fe(CN)6 ].
structure in the electro-physical experiments [26,27], while the other two odorants differ from diacetyl only by the addition of a methyl group (2,3-pentanedione) or the absence of a keto group (butanone). Therefore they were selected for the specificity test. Fresh air was used as the negative control group. All the odorants were applied at the concentration of 100 ppm (v/v) for the specificity test. The sensitivity assay was performed by detecting different concentration of diacetyl ranging from 10 to 100 ppm. All the odorants were purchased from Sigma–Aldrich (USA). 2.4. Data processing and statistical analysis All the measurements were conducted after the frequency baseline stabilized at f0 Hz. When injecting one odorant, the frequency will change and stabilize at f1 Hz again. Then frequency shift (f = f0 − f1 ) was the response of biosensor to this odorant. In the specificity test, data normalization was conducted to compare visually the different responses of various odorants, namely the maximum frequency shift was normalized as 1 and other results were calculated by dividing the maximum frequency shift. The
The aptamer-assisted His6 -tagged ODR-10 immobilization was monitored by electrochemical techniques (CV and EIS). Fig. 2a showed CV results of each step, from which a good reversible cyclic voltammogram was observed in the bare electrode. After the modification of thiolized anti-His6 aptamer and blocker molecules, a mixed SAM was formed on the surface of gold electrode, accompanying by a considerable decrease in the amperometric responses of the electrode and an increase in the peak to peak separation between the cathodic and anodic waves of the redox probe. This indicates the formation of SAM resulted in an insulating surface and the electron transfer kinetics of [Fe(CN)6 ]4−/3− are perturbed. When His6 -tagged ODR-10 was captured by anti-His6 aptamers, a slight current response was observed. The current increase indicated that His6 -tagged ODR-10 was captured by anti-His6 aptamers and the stable immobilization was formed on the surface of electrode. It was probably due to the conformation changes of aptamers and structural re-arrangement on the electrode after binding with His6 -tagged ODR-10. Another possible reason was that the amino group of His6 -tagged ODR-10 interacted with the carboxylic terminal group of blocker molecules, which was negatively charged in the condition of pH 7.0 and might repulse the negative redox ions [Fe(CN)6 ]4−/3− . Nyquist plots of impedance spectra of the immobilization procedure were shown in Fig. 2b, in which significant differences between each step of immobilization were observed. Comparing with the bare gold electrode, the deposition of the mixed aptamer and block molecules produced a significant increase of the charge transfer resistance (Rct ), indicating the good insulating properties of the mixed monolayer. After the immobilization of His6 -tagged ODR-10, a decrease in polarization resistance was observed and consistent with the CV result. This indicated a recovery in the efficiency of the mass transfer phenomenon with formation of a new layer. The fitting data of the electrochemical procedure was shown in Table S1 and consistent with results of CV. All these data verified the successful immobilization of His6 -tagged ODR-10 on QCM electrode. All the results suggested this aptamer-based method has
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Fig. 3. SPM images (3 m × 3 m) of QCM surface before (a), and after (b) the immobilization of ODR-10.
high efficiency for the specific immobilization of His6 -tagged ODR10. SPM was employed to characterize the surface morphology of QCM electrode before and after the immobilization of ODR-10, as shown in Fig. 3. The area is 3 m × 3 m. Fig. 3a illustrates the rather uniform aptamer-modified sensor surface without His6 tagged ODR-10. The average height is around 2.4 nm, which is close to the size of aptamer molecules. Fig. 3b is the morphology of the sensor surface with His6 -tagged ODR-10. Evenly distributed grainy features can be observed and the sizes range from 30 nm to 100 nm. The average height is around 8 nm. These sizes are consistent with the theoretical value of membrane proteins. The thickness of the His6 -tagged ODR-10 layer is approximately equivalent to that of the cell membrane, indicating that the captured His6 -tagged ODR-10 may still fold in the cellular membrane fractions. This could provide hydrophobic environment for the immobilized ODR-10 to maintain the natural structures and functions. All these results demonstrate that His6 -tagged ODR-10 has been evenly immobilized onto the sensor surface.
ODR-10 were used as a negative control group. Isoamyl acetate, anisole, lavender, butanone, and 2,3-pentanedione were selected to test the specificity of the biosensor. From the normalized specificity result in Fig. 5, it can be found that the response of ODR-10 immobilized QCM biosensor to diacetyl was significantly higher than those of other odorants tested, while the group without ODR10 responded to each odorant without significant difference. It
3.3. Performance of piezoelectric olfactory receptor biosensor Using the aptamer-assisted immobilization method, ODR-10 based QCM biosensor has been well developed for the odorant detection. The typical response–time curve of an odorant detection cycle was presented in Fig. 4a, consisting of the odorant-puffing stage, the stabilization stage, the refreshing stage I, and the stabilization stage II. Once diacetyl was introduced into the detection chamber at t0 , the frequency changed quickly and then stabilized gradually at another frequency. The moment of 90% stabilization frequency is t1 , and the response time of this biosensor was defined as t1 − t0 . Fresh air was introduced at t2 to refresh the detection chamber, and the frequency returned to the stabilization phase at t3 . The recover time was defined as t3 − t2 . The results demonstrated that the response time and recover time of this piezoelectric ODR-10 based biosensor are about 21 ± 3.2 s and 25 ± 2.9 s (n = 4), respectively. The fluctuation of frequency in the stabilization phase is about ±1 Hz. Fig. 4b showed four diacetyl detection cycles, and the curve in the dotted rectangle was one complete detection cycle shown in Fig. 4a. In the diacetyl detection cycle, the average frequency shift between t0 and t3 is about ±3 Hz (n = 4), indicating the good reversibility of this biosensor. The specificity of immobilized ODR-10 was tested by recording responses to several odorants. Diacetyl is the only natural ligand of ODR-10, which has been identified by cellular assays [16,23]. Therefore diacetyl was the detection target of this biosensor. Fresh air was used as a background gas to detect the influence of the air flow. The extracted membrane proteins of HEK-293 without
Fig. 4. (a) One typical odorant response cycle curve of the ODR-10 based QCM biosensor; (b) QCM responses to 100 ppm of diacetyl. (a) is taken from one cycle of (b).
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Fig. 5. The selectivity test of ODR-10 based QCM biosensor to various odorants. All the data are represented by means ± SD (standard deviation), n = 4. ** significant difference, p < 0.05, Student’s t-test.
was indicated that aptamer-assisted immobilization method kept the odorant detection function of ODR-10 and this ODR-10 based QCM biosensor had good specificity to its natural ligand, diacetyl. Based on this idea, various ORs can be tagged by different tags and captured by the corresponding aptamers on the biosensor surface. Therefore various olfactory receptor molecular sensor arrays can be achieved for the high sensitive and high-throughput detection of specific odorants. The sensitivity of this ODR-10 immobilized QCM biosensor was determined by testing various concentrations of diacetyl. Fig. 6 showed the dose-dependent responses of two ODR-10 based biosensors, whose production procedures were same completely except the immobilization method of ODR-10. One was aptamer-assisted immobilization method (the upper blue line); while the other was physical adsorption method without specific covalent immobilization (the lower black dotted one). The linear response range is from 10 to 100 ppm. It can be found that the aptamer-assisted immobilization technique has improved the sensitivity of this biosensor compared to the physical adsorption group. The detection limit was estimated and calculated from 3*
the standard deviation of the blank response frequencies (fresh air). The estimated detection limit result is 1.5 ppm. Many ODR-10 based biosensors have been developed by utilizing various techniques, such as QCM [8], SAW [10,11], BRET [15] in cell-free format, nematode chemotaxis assay [23], Ca2+ imaging [16] and SPR technique [14] in whole cell. The summarized results were shown in Table S2. It can be found that the reported most sensitive one is the BRET system by inserting a bioluminescent donor and a fluorescent acceptor protein into the sequence of ODR-10 with the detection limit of 3.55 fM, which is equivalent to 0.31 ppq [15]. Other ODR-10 based mass sensitive biosensor can detect gaseous diacetyl in ppq levels with wide linear response range (approximately 8 log units) [8,10,11]. The previous work has also demonstrated that the efficient immobilization of ODR-10 will improve the sensitivity of biosensor [11]. The sensitivity of biosensor is determined by many factors, such as secondary transducers, the amount and immobilization methods of sensitive materials. This aptamer-assisted ODR-10 biosensor is relative sensitive among the same kind of biosensors [9], and its sensitivity can also been improved by increasing the oscillation frequency of QCM. For the hazard gases, the detection in low concentration range is more meaningful. Therefore we pay more attention to the low linear detection range of the biosensor (10–100 ppm) in this work, where it has a good linear relationship, not ‘S’ response curves in the wide concentration range [15,16,23]. Besides, the short response and recovery time make this biosensor very convenient and useful in food safety and direct environmental detections. The performance of reproducibility has been evaluated by analyzing the sensitivities of several biosensors fabricated by the exact same procedures. The statistical results of five biosensors were shown in Table S3. The average sensitivity and limit of detection are 0.553 ± 0.0236 Hz/ppm and 1.524 ± 0.0321 ppm. The small standard deviation means that these ODR-10 biosensors have fine reproducibility performance. Additionally, ODR-10 biosensors were stored in a dry chamber at 4 ◦ C and tested the responses to 100 ppm of diacetyl for two weeks. The results demonstrate that aptamer-assisted ODR-10 biosensors have stable responses within one week, better than the physical adsorption group (2–3 days). While after one week, the responses become much more variable. The reasons may be due to the structure changes of ODR-10 and the loss of odorant detection functions. That is still the major challenge of olfactory receptor based biosensors for the practical applications and commercialization. The potential solutions maybe focus on the mimicking natural environments of olfactory receptors on the sensor surface. All these results demonstrated that the aptamer-assisted olfactory receptor immobilization was an effective method and can improve the performance of biosensor effectively. Additionally, aptamer-assisted immobilization method, as a general protein immobilization technique, can also be applied into the development of olfactory receptor sensor array for different ORs immobilization.
4. Conclusion
Fig. 6. Concentration-dependent responses lines of two ODR-10 based QCM biosensors. All the data are represented by means ± SD (standard deviation), n = 4.
In this study, we investigated a piezoelectric olfactory receptor biosensor for the odorant detection, which was prepared with the aptamer-assisted immobilization method. The results demonstrated that His6 -tagged ODR-10 was successfully immobilized onto the sensor surface by anti-His6 aptamers with high efficiency and specificity. The sensitivity and selectivity have been greatly improved compared with the physical adsorption group. This ODR-10 based QCM biosensor has lower detection limit and good repeatability for the real-time odorant detection. Using
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this aptamer-assisted immobilization method, different olfactory receptors could be immobilized onto the sensor surface to develop biosensor arrays. Considering the fast response time and the good reversibility, this aptamer-assisted ODR-10 based QCM biosensor has great application potential in many fields, such as food safety, environment monitoring, and poison gas detection.
[18]
[19]
Acknowledgments
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This work was supported by grants from the National Natural Science Foundation of China (grant nos. 31000448, 81027003, 31228008), the China Postdoctoral Science Foundation (grant no. 201104734), the National Public Welfare Project (no. 201305010), and the 973 Program of Ministry of Science and Technology of China (no. 2009CB320303).
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[22] [23] [24]
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.2013.02.009.
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Biographies Liping Du received her B.S. degree from China Medical University in 2008. She is currently a Ph.D. candidate at the Department of Biomedical Engineering in Zhejiang University. Her research interests focus on olfactory receptor-based biosensor and olfactory electrophysiology. Chunsheng Wu received the Ph.D. degree of Biomedical Engineering from Zhejiang University, Hangzhou, China in 2009. He is currently an Associate Researcher of Biosensor National Special Laboratory at Zhejiang University. He was a joint Ph.D. student of Micro Systems Laboratories at University of California, Los Angeles (UCLA) in 2008. He was a Post-doctoral Fellow of Instrument Science and Technology at Zhejiang University from 2010 to 2012. His research is focused on the development and application of cell and molecule-based biosensors, instruments, and systems. He Peng received her M.S. degree in Biochemistry and Molecular Biology from Zhejiang University, Hangzhou, China, in 2012. Her research interests focus on the heterologous expression of membrane receptors and functional assays. Ling Zou is a Ph.D. candidate of biomedical engineering in Zhejiang University. Her research is focused on the heterologous expression of receptor proteins and the development of receptor-based biosensors. Luhang Zhao received the B.S., M.S., and Ph.D. degrees in Biochemistry and Molecular Biology from Zhejiang University, Hangzhou, China, in 1985, 1998, and 2011, respectively. He is currently an Associate Professor with the Department of Biochemistry and Genetics, Zhejiang University. His current research interests include Genetic Engineering and Glycobiology. Liquan Huang received his Ph.D. degree in Molecular Biology from Yale University. His research is directed at the molecular mechanisms that underlie taste and olfaction sensations. The focus is on identifying molecules involved in the recognition and transmission of chemical stimulus. He is currently an associate member of Monell Chemical Senses Center, USA. Ping Wang received the B.S. degree, M.S. degree and Ph.D. degree from Electrical Engineering of Harbin Institute of Technology, Harbin, China in 1984, 1987 and 1992, respectively. From 1992 to 1994 he is Post-Doctoral Fellow in Biosensor National Special Lab, Dept. of Biomedical Engineering, Zhejiang University, Hangzhou, China. At present, He is Professor of Biomedical Engineering of Zhejiang University, while he is Director of Biosensor National Special Laboratory and Director of Key Lab for Biomedical Engineering of National Education Ministry of China, Zhejiang University. He is a member of The International Society for Olfaction and Chemical Sensing and a member of steering committee of Asia Chemical Sensors Society. He is also a Director of Biomedical Measurement Society of China, Vice-Director of Ion and Biological Sensing Society and Vice-Director of Biomedical Sensors Technique Society of China. Besides, he is a visiting scholar at Edison Sensors Laboratory of Case Western Reserve University, USA and Biosensor and Bioinstrumentation Laboratory in University of Arkansas, USA, in 2002 and 2005, respectively.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Piezoelectric olfactory receptor biosensor prepared by aptamer-assisted immobilization Liping Du a , Chunsheng Wu a , He Peng b , Ling Zou a , Luhang Zhao b , Liquan Huang c , Ping Wang a,∗ a Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Ministry of Education, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China b Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, China c Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, USA
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Article history: Available online 13 February 2013 Keywords: Olfactory receptor Piezoelectric biosensor Aptamer Immobilization Odor detection
a b s t r a c t Inspired by the very high sensitivity and specificity of biological olfactory system, engineers pay much attention to biomimetic olfactory biosensors due to their excellent performance and great commercial prospects. In this study we presented an olfactory receptor (OR)-based piezoelectric biosensor with high sensitivity and specificity for the odorant detection, in which aptamers were employed for the specific and effective olfactory receptor immobilization to improve the overall performance of biosensors. An olfactory receptor of C. elegances, ODR-10, was expressed heterologously in HEK-293 cells with a hexahistidine (His6 ) tag on the N terminus. His6 -tagged ODR-10 was extracted from the plasma membrane of transfected HEK-293 cells and used as the sensing element of biosensors. Anti-His6 aptamers were immobilized covalently onto the gold surface of piezoelectric quartz crystal microbalance (QCM) electrode to capture the His6 -tagged ODR-10 specifically, as well as achieve the purification of ODR-10 simultaneously. The immobilization procedures were characterized by electrochemical methods and scanning probe microscopy (SPM). Additionally, physical adsorption method was used as a control method to illustrate the influences of immobilization methods on the performances of OR-based biosensors. The results demonstrated that the piezoelectric biosensor prepared by aptamer-assisted immobilization method showed high specificity and improved sensitivity. The detection limit was as low as 1.5 ppm (v/v). Additionally, the aptamer-assisted receptor immobilization method has great potential to become a universal protein immobilization technique in biosensing, and be applied into the development of OR-based biosensor array for the simultaneous detection of several odorants. This aptamer-assisted olfactory receptor biosensor has great application prospects in many fields, such as food safety, environmental monitoring, and disease diagnosis. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The nature’s olfactory system has an excellent capacity in recognizing and discriminating various odorants. Since the concept of “bio-electronic nose” was proposed in 1998 by Göpel [1], mimicking nature’s olfactory system has become a new trend of biosensors for odorant detection. In the biological olfactory system, olfactory receptors (ORs) act as the sensitive elements of olfactory sensory neurons (OSNs), which play a great role in odorant specific recognition and discrimination [2]. ORs belong to the G protein-coupled receptor (GPCR) family. The binding of odorants and ORs will trigger the intracellular signal transduction cascades and induce the depolarization of OSNs. The unique functions of
∗ Corresponding author. Tel.: +86 571 87952832; fax: +86 571 87952832. E-mail address: [email protected] (P. Wang). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.02.009
ORs interacting with the specific odorant molecules make them ideal candidates as sensitive elements of biomimetic gas sensors toward odorant detection [3]. Glatz and Bailey-Hill have made a comprehensive comment on mimicking nature’s nose, and discussed various olfactory biosensors as well as their applications [4]. Various transduction techniques have been employed in the research of OR-based biosensors, including field effect transistor (FET) [5,6], quartz crystal microbalance (QCM) [7–9], surface acoustic wave (SAW) device [10,11], light addressable potentiometric sensor (LAPS) [12,13], surface plasmon resonance (SPR) [14], bioluminescence resonance energy transfer (BRET) [15], and intracellular calcium responses [16]. Almost all the odorant-induced information can be utilized for olfactory biosensing. However, one major issue in this field is how to achieve functional immobilization of defined ORs with high efficiency on the solid surface of secondary transducers. The antibody-directed specific immobilization of ORs is more specific and efficient than that of physical adsorption method,
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but the procedure is relative complex and the antibody is much expensive [17]. Recent years, aptamers have been widely viewed as promising complementary molecules to antibodies, which are single-stranded oligonucleic acid molecules [18]. Compared with antibodies, aptamers have a lot of advantages, such as more resistant, being more easily modified with functional groups or tags, and less expensive. They have been used to replace antibodies in many applications, such as enzyme-linked immunosorbent assay (ELISA) [19], Western blot [20], biosensing applications [21], and protein micro-arrays [22]. Concerning the unique advantages of aptamers, this study explore the possibility of using aptamers to achieve the high specific and effective functional immobilization of ORs onto the surface of secondary transducers. We present an OR-based piezoelectric biosensor prepared by the aptamer-assisted immobilization method. Quartz crystal microbalance (QCM) is a commonly used mass sensitive piezoelectric transducer [9], whose oscillation frequency changes with the mass loading on the crystal. The interactions between ORs and odorants can be detected by monitoring the oscillation frequency changes of QCM. A well-characterized olfactory receptor of C. elegances, ODR-10, is employed as a model of ORs. Diacetyl is the only volatile ligand to activate ODR-10 [23], and can induce the intracellular calcium elevation of HEK-293 cells expressing ODR-10 in a dose-dependent way [16]. In the present study, ODR-10 was expressed on the plasma membrane of HEK-293 cells with a His6 -tag on its N-terminus. The surface of gold electrodes of QCM was modified by specific anti-His6 aptamers via Au S bonds, which could only capture His6 -tagged ODR-10 specifically through anti-His6 aptamers. Purification of ODR-10 was also achieved simultaneously due to the specific interactions between aptamers and His6 -tags. The immobilization procedures of ODR10 were characterized by electrochemical methods and scanning probe microscopy (SPM). The specificity and sensitivity of this ORbased piezoelectric biosensor were tested. In addition, physical adsorption method was used as a control method to illustrate the influences of immobilization methods on the performances of ORbased biosensors.
2. Materials and methods 2.1. Preparation of functional olfactory receptors Here an olfactory receptor of C. elegances, ODR-10, was employed as a model of ORs, which were produced by cellular heterologous expression techniques. The full-length cDNA of odr-10 was amplified by polymerase chain reaction (PCR) from pEGFPN1/rho-tag/odr-10 [24]. A pair of specific primers were designed: the forward primer 5 -AGAGGTACCGATGCACCACCACCACCACCA CATGAACGGGACCGAGGGCCCAA-3 , containing the KpnI restriction enzyme cutting site (the underlined part) and His6 sequence (the dotted part); and the reverse primer 5 -TGTGGATCCTCATTACTACGTCGGAACTTGAGACAAATTGG-3 , containing the BamHI enzyme cutting site (the underlined part). PCR was performed in a 50 L reaction volume using 1.0 L of the plasmid template pEGFP-N1/rho-tag/odr-10, 1 L of the forward primer, 1 L of the reverse primer, 4 L of 10 mM dNTP mixture, 0.5 L of STARTM polymerase, 10 L of 5× PCR buffer (containing Mg2+ ), and 32.5 L of deionized water. The reaction cycle conditions were 30 cycles of 94 ◦ C denaturation for 30 s, 60 ◦ C annealing for 1 min, and 72 ◦ C extension for 1 min. The obtained His6 -tag/rho-tag/odr-10 sequence was subcloned into the KpnI and BamHI sites of the expression vector pcDNA 3.1(+). The rho-tag import sequence was used to facilitate the localization of ODR-10 on the cell plasma membrane. His6 -tag can indicate the expression level of ODR-10 and assist the functional specific immobilization of ORs on the sensor surface.
The expression vector pcDNA 3.1(+)/His6 -tag/rho-tag/odr-10 was confirmed by restriction enzyme mapping and DNA sequencing. Then the plasmid pcDNA 3.1(+)/His6 -tag/rho-tag/odr-10 was transfected into the heterologous HEK-293 cells to express ODR-10 on the plasma membrane. HEK-293 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) (Gibco, UK) supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 g/mL), at 37 ◦ C in the 5% CO2 incubator. HEK-293 cells were seeded and cultured in 6-well plates. When being 80% confluent, cells were transfected with 4 g of the expression plasmid and 10 L of LipofectamineTM 2000 (Invitrogen) for each well according to the LipofectamineTM 2000 instructions. After 48 h, transfected cells were collected for subsequent assays. Reverse transcription polymerase chain reaction (RT-PCR) was conducted to indicate the expression of ODR-10 at mRNA level. Total RNA was collected from transfected cells by TRIZOL and analyzed with PrimeScript® 1st Strand cDNA Synthesis Kit (Takara). The cDNAs were used as templates for PCR amplification. Forward primer: 5 -AGAGGTACCGATGCACCACCACCACCACCACATGAACGG GACCGAGGGCCCAA-3 , and reverse primer: 5 -TGTGGATCCTCATTACTACGTCGGAACTTGAGACAAATTGG-3 . The reaction products were resolved by electrophoresis. The expected length of the target product was 1120 bp. Western blot was conducted to verify the expression of His6 tagged ODR-10 at the protein level. Transfected cells were collected and lysed. The protein supernatants were loaded on a SDS-PAGE gel and transferred onto a PVDF membrane. Then the membrane was incubated with the primary antibody anti-His6 mouse IgG, followed by incubation with the secondary antibody DyLight 680 conjugated goat anti-mouse IgG. The bands were visualized with the Odyssey infrared imaging system. The expected size of target protein was about 45 kDa. The membrane protein extraction kit (Sangon Biotech, China) was used to extract the functional His6 -tagged ODR-10 from the plasma membrane of transfected HEK-293 cells. About 5 × 108 cells were collected from 6-well plates and washed with ice cold cell wash buffer for three times. After resuspended in 1 mL extraction buffer (containing 1 L protease inhibitor and 1 L DTT), cells were sonicated with a sonicator for 3–4 times and centrifuged at 14,000 rpm for 10 min at 4 ◦ C. The supernatant was transferred to a new vial and centrifuged at 13,000 rpm for 5 min at room temperature. The pellet was the total cellular membrane protein, containing proteins from both plasma membrane and cellular organelle membrane. By further centrifuging, 10 L of the plasma membrane proteins were harvested. This solution was the mixture of total plasma membrane proteins, including His6 -tagged ODR-10, and stored at −70 ◦ C for the development of biosensors. 2.2. Aptamer-assisted olfactory receptor immobilization and characterization The aptamer-assisted immobilization method was proposed to immobilize the His6 -tagged ODR-10 efficiently and specifically. Its schematic was shown in the inset of Fig. 1. Before immobilization, the gold electrode surface of QCM was cleaned thoroughly to remove inorganic and organic contaminants, and then incubated with a mixture solution for 21 h to form a self assembly monolayer (SAM) through Au S bonds, which contained 5 M of thiol-modified anti-His6 aptamer molecules (5 -HS-(CH2 )6 GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-3 ) [25] and 50 M of blocker molecules 11-mercaptoundecanoic acid (11-MUA) (Sigma, USA). 11-MUA was used to block free sites of the electrode surface and provide suitable steric spaces for the aptamers. Thereafter the gold electrode was rinsed with ethanol thoroughly and dried under the nitrogen flow. 10 L of the plasma membrane extraction protein solution, containing the His6 -tagged
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Fig. 1. Piezoelectric olfactory receptor biosensor and measurement system. The inset figure illustrated the aptamer-assisted ODR-10 immobilization mechanism.
ODR-10, was dissolved in 90 L of phosphate buffer solution (PBS) containing 0.5% Trion X-100 and incubated onto the aptamermodified electrode for 3 h in 37 ◦ C bath. Then the functionalized gold electrode was rinsed by deionized water thoroughly to remove the unspecific binding proteins. In this study, immobilized anti-His6 aptamers on the electrode were able to capture the His6 -tagged ODR-10 specifically. Other irrelative proteins without His6 -tags cannot be captured onto the electrode. Therefore this method achieved the purification, pre-concentration and specific immobilization of ODR-10 simultaneously. To compare the different immobilization methods, another piezoelectric biosensor was also developed using the physical adsorption method to immobilize His6 -tagged ODR-10 [8]. 10 L of the plasma membrane extraction protein solution was evenly spread onto the electrode of the quartz crystal for 3 h at 37 ◦ C. The aptamer-assisted immobilization process was monitored by electrochemical cyclic voltammetry (CV) and electrochemical impedance spectrum (EIS). The gold electrode modified with His6 -tagged ODR-10 was the working electrode. A Pt plate and a saturated calomel electrode (SCE) were used as the counter electrode and reference electrode, respectively. All the electrochemical measurements were performed in PBS (pH 7.0) with 5 mM K4 [Fe(CN)6 ] and 5 mM K3 [Fe(CN)6 ]. The potential range of CV was from −0.3 to +0.7 V versus SCE at a rate of 50 mV/s. EIS was performed in the frequency range from 1 Hz to 100 kHz at an open loop potential with a frequency modulation of 5 mV. All electrochemical measurements were performed in a Faraday cage. The impedance data was fitted with a commercial available software Zview/Zplot. The equivalent circuit was shown in the inset of Fig. 2b, which included four elements: (1) Rs, the ohmic resistance of the electrolyte solution; (2) Rct , the charge transfer resistance; (3) CPE, the constant phase elements. Two fitting parameters, CPE-T and CPE-P, represented the double layer capacitance and its phase angle, respectively. The impedance of CPE was expressed by ZCPE = 1/[T(i × ω)P ], i: the imaginary unit, ω: the circular frequency, T: CPE-T, P: CPE-P; and (4) Zw , the generalized finite Warburg impedance in the open circuit model. W–R, W–T, and W–P were three fitting parameters of Zw . Its impedance was given by Zw = R × ctnh([i × T × ω]P )/(i × T × ω)P .
Scanning probe microscopy (SPM) (VEECO, USA) was used to characterize the surface morphology of the ODR-10 modified electrode. An antimony (n) doped silicon tip with spring constant of 30 N/m was used. The thickness and length of the cantilever are 4 m and 130 m, respectively. The free resonance frequency is 318 kHz. The image is taken in tapping mode, which can minimize the potentially destructive shear and adhesive forces on the sample. All the experiments were performed under ambient conditions with relative humidity of 60%. The image was first-order flattened. 2.3. Piezoelectric olfactory receptor biosensor and measurement system QCM200 system (Stanford Research Systems, Inc., USA), which has 5 MHz AT-cut quartz crystals with polished gold electrodes, was used as a piezoelectric transducer. The QCM200 is a standalone instrument with a built-in detection circuit and frequency counter. The resonant frequency changes as a linear function of the mass of material deposited on the crystal surface. The schematic of measurement system was shown in Fig. 1. After immobilization process, the ODR-10 modified QCM sensor was rinsed with deionized water to remove excess proteins and dried with nitrogen. Then it was placed in a detection chamber with inlet and outlet ports. The frequency data was detected by QCM200 detection system and transmitted into a computer via the RS-232 interface. All the odorants was freshly prepared in Tedlar bags (SKC Inc., USA) by a liquid organic gas blender (MF-3B, Huanchen Instruments, China). A syringe pump associated with valves controlled the injection of odorants into the detection chamber. The fresh air was used as the reference gas to simulate real-world conditions and to refresh the detection chamber after each measurement. The flow rate of odorants kept at 1 mL/s. Gas injection time was 1 min and the minimal interval for the next injection was 5 min. All the measurements were carried out at the same environmental conditions at 25 ◦ C. Six odorants were applied to test the specificity of this ODR10 based QCM biosensor, which include diacetyl, isoamyl acetate, anisole, lavender, butanone, and 2,3-pentanedione. Isoamyl acetate, anisole, and lavender belong to three different odorant groups according to their effectiveness and their molecular
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experimental data was presented as means ± standard deviation (SD). The significant difference analysis was carried out by performing Student’s t-test and p < 0.05 was considered as significant difference.
3. Results and discussion 3.1. Preparation results of olfactory receptors Functional ORs are the most crucial sensing materials of ORbased biosensor. In this study we prepared His6 -tagged ODR-10 by extracting proteins from pcDNA 3.1(+)/His6 -tag/rho-tag/odr-10 transfected HEK-293 cells. During the expression process, RT-PCR and Western blot were performed to validate the expression of His6 -tagged ODR-10 at the mRNA and protein levels. Fig. S1 was the result of RT-PCR, in which an obvious right band can be observed above 1000 bp this result verified the expression of ODR-10 in HEK293 cells at the mRNA level. Fig. S2 showed the result of Western blot and one obvious band was found between 43 and 55 kDa. It was consistent with the theoretical value of ODR-10 (45 kDa). The negative control group (HEK-293 cells without any transfection) showed no band. All these results inferred that ORs with His6 -tags were expressed successfully and there was no other His6 -tagged proteins in the extracted protein solution.
3.2. Characterization of aptamer-assisted olfactory receptor immobilization process
Fig. 2. Electrochemical characteristions of the ODR-10 immobilization procedure. (a) CV measurements. (b) Nyquist plots of impedance spectra in the frequency range from 1 Hz to 100 kHz. The inset of (b) shows the equivalent circuit model. All the electrochemical measurements were conducted in PBS solution (pH 7.0) with 5 mM K4 [Fe(CN)6 ] and 5 mM K3 [Fe(CN)6 ].
structure in the electro-physical experiments [26,27], while the other two odorants differ from diacetyl only by the addition of a methyl group (2,3-pentanedione) or the absence of a keto group (butanone). Therefore they were selected for the specificity test. Fresh air was used as the negative control group. All the odorants were applied at the concentration of 100 ppm (v/v) for the specificity test. The sensitivity assay was performed by detecting different concentration of diacetyl ranging from 10 to 100 ppm. All the odorants were purchased from Sigma–Aldrich (USA). 2.4. Data processing and statistical analysis All the measurements were conducted after the frequency baseline stabilized at f0 Hz. When injecting one odorant, the frequency will change and stabilize at f1 Hz again. Then frequency shift (f = f0 − f1 ) was the response of biosensor to this odorant. In the specificity test, data normalization was conducted to compare visually the different responses of various odorants, namely the maximum frequency shift was normalized as 1 and other results were calculated by dividing the maximum frequency shift. The
The aptamer-assisted His6 -tagged ODR-10 immobilization was monitored by electrochemical techniques (CV and EIS). Fig. 2a showed CV results of each step, from which a good reversible cyclic voltammogram was observed in the bare electrode. After the modification of thiolized anti-His6 aptamer and blocker molecules, a mixed SAM was formed on the surface of gold electrode, accompanying by a considerable decrease in the amperometric responses of the electrode and an increase in the peak to peak separation between the cathodic and anodic waves of the redox probe. This indicates the formation of SAM resulted in an insulating surface and the electron transfer kinetics of [Fe(CN)6 ]4−/3− are perturbed. When His6 -tagged ODR-10 was captured by anti-His6 aptamers, a slight current response was observed. The current increase indicated that His6 -tagged ODR-10 was captured by anti-His6 aptamers and the stable immobilization was formed on the surface of electrode. It was probably due to the conformation changes of aptamers and structural re-arrangement on the electrode after binding with His6 -tagged ODR-10. Another possible reason was that the amino group of His6 -tagged ODR-10 interacted with the carboxylic terminal group of blocker molecules, which was negatively charged in the condition of pH 7.0 and might repulse the negative redox ions [Fe(CN)6 ]4−/3− . Nyquist plots of impedance spectra of the immobilization procedure were shown in Fig. 2b, in which significant differences between each step of immobilization were observed. Comparing with the bare gold electrode, the deposition of the mixed aptamer and block molecules produced a significant increase of the charge transfer resistance (Rct ), indicating the good insulating properties of the mixed monolayer. After the immobilization of His6 -tagged ODR-10, a decrease in polarization resistance was observed and consistent with the CV result. This indicated a recovery in the efficiency of the mass transfer phenomenon with formation of a new layer. The fitting data of the electrochemical procedure was shown in Table S1 and consistent with results of CV. All these data verified the successful immobilization of His6 -tagged ODR-10 on QCM electrode. All the results suggested this aptamer-based method has
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Fig. 3. SPM images (3 m × 3 m) of QCM surface before (a), and after (b) the immobilization of ODR-10.
high efficiency for the specific immobilization of His6 -tagged ODR10. SPM was employed to characterize the surface morphology of QCM electrode before and after the immobilization of ODR-10, as shown in Fig. 3. The area is 3 m × 3 m. Fig. 3a illustrates the rather uniform aptamer-modified sensor surface without His6 tagged ODR-10. The average height is around 2.4 nm, which is close to the size of aptamer molecules. Fig. 3b is the morphology of the sensor surface with His6 -tagged ODR-10. Evenly distributed grainy features can be observed and the sizes range from 30 nm to 100 nm. The average height is around 8 nm. These sizes are consistent with the theoretical value of membrane proteins. The thickness of the His6 -tagged ODR-10 layer is approximately equivalent to that of the cell membrane, indicating that the captured His6 -tagged ODR-10 may still fold in the cellular membrane fractions. This could provide hydrophobic environment for the immobilized ODR-10 to maintain the natural structures and functions. All these results demonstrate that His6 -tagged ODR-10 has been evenly immobilized onto the sensor surface.
ODR-10 were used as a negative control group. Isoamyl acetate, anisole, lavender, butanone, and 2,3-pentanedione were selected to test the specificity of the biosensor. From the normalized specificity result in Fig. 5, it can be found that the response of ODR-10 immobilized QCM biosensor to diacetyl was significantly higher than those of other odorants tested, while the group without ODR10 responded to each odorant without significant difference. It
3.3. Performance of piezoelectric olfactory receptor biosensor Using the aptamer-assisted immobilization method, ODR-10 based QCM biosensor has been well developed for the odorant detection. The typical response–time curve of an odorant detection cycle was presented in Fig. 4a, consisting of the odorant-puffing stage, the stabilization stage, the refreshing stage I, and the stabilization stage II. Once diacetyl was introduced into the detection chamber at t0 , the frequency changed quickly and then stabilized gradually at another frequency. The moment of 90% stabilization frequency is t1 , and the response time of this biosensor was defined as t1 − t0 . Fresh air was introduced at t2 to refresh the detection chamber, and the frequency returned to the stabilization phase at t3 . The recover time was defined as t3 − t2 . The results demonstrated that the response time and recover time of this piezoelectric ODR-10 based biosensor are about 21 ± 3.2 s and 25 ± 2.9 s (n = 4), respectively. The fluctuation of frequency in the stabilization phase is about ±1 Hz. Fig. 4b showed four diacetyl detection cycles, and the curve in the dotted rectangle was one complete detection cycle shown in Fig. 4a. In the diacetyl detection cycle, the average frequency shift between t0 and t3 is about ±3 Hz (n = 4), indicating the good reversibility of this biosensor. The specificity of immobilized ODR-10 was tested by recording responses to several odorants. Diacetyl is the only natural ligand of ODR-10, which has been identified by cellular assays [16,23]. Therefore diacetyl was the detection target of this biosensor. Fresh air was used as a background gas to detect the influence of the air flow. The extracted membrane proteins of HEK-293 without
Fig. 4. (a) One typical odorant response cycle curve of the ODR-10 based QCM biosensor; (b) QCM responses to 100 ppm of diacetyl. (a) is taken from one cycle of (b).
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Fig. 5. The selectivity test of ODR-10 based QCM biosensor to various odorants. All the data are represented by means ± SD (standard deviation), n = 4. ** significant difference, p < 0.05, Student’s t-test.
was indicated that aptamer-assisted immobilization method kept the odorant detection function of ODR-10 and this ODR-10 based QCM biosensor had good specificity to its natural ligand, diacetyl. Based on this idea, various ORs can be tagged by different tags and captured by the corresponding aptamers on the biosensor surface. Therefore various olfactory receptor molecular sensor arrays can be achieved for the high sensitive and high-throughput detection of specific odorants. The sensitivity of this ODR-10 immobilized QCM biosensor was determined by testing various concentrations of diacetyl. Fig. 6 showed the dose-dependent responses of two ODR-10 based biosensors, whose production procedures were same completely except the immobilization method of ODR-10. One was aptamer-assisted immobilization method (the upper blue line); while the other was physical adsorption method without specific covalent immobilization (the lower black dotted one). The linear response range is from 10 to 100 ppm. It can be found that the aptamer-assisted immobilization technique has improved the sensitivity of this biosensor compared to the physical adsorption group. The detection limit was estimated and calculated from 3*
the standard deviation of the blank response frequencies (fresh air). The estimated detection limit result is 1.5 ppm. Many ODR-10 based biosensors have been developed by utilizing various techniques, such as QCM [8], SAW [10,11], BRET [15] in cell-free format, nematode chemotaxis assay [23], Ca2+ imaging [16] and SPR technique [14] in whole cell. The summarized results were shown in Table S2. It can be found that the reported most sensitive one is the BRET system by inserting a bioluminescent donor and a fluorescent acceptor protein into the sequence of ODR-10 with the detection limit of 3.55 fM, which is equivalent to 0.31 ppq [15]. Other ODR-10 based mass sensitive biosensor can detect gaseous diacetyl in ppq levels with wide linear response range (approximately 8 log units) [8,10,11]. The previous work has also demonstrated that the efficient immobilization of ODR-10 will improve the sensitivity of biosensor [11]. The sensitivity of biosensor is determined by many factors, such as secondary transducers, the amount and immobilization methods of sensitive materials. This aptamer-assisted ODR-10 biosensor is relative sensitive among the same kind of biosensors [9], and its sensitivity can also been improved by increasing the oscillation frequency of QCM. For the hazard gases, the detection in low concentration range is more meaningful. Therefore we pay more attention to the low linear detection range of the biosensor (10–100 ppm) in this work, where it has a good linear relationship, not ‘S’ response curves in the wide concentration range [15,16,23]. Besides, the short response and recovery time make this biosensor very convenient and useful in food safety and direct environmental detections. The performance of reproducibility has been evaluated by analyzing the sensitivities of several biosensors fabricated by the exact same procedures. The statistical results of five biosensors were shown in Table S3. The average sensitivity and limit of detection are 0.553 ± 0.0236 Hz/ppm and 1.524 ± 0.0321 ppm. The small standard deviation means that these ODR-10 biosensors have fine reproducibility performance. Additionally, ODR-10 biosensors were stored in a dry chamber at 4 ◦ C and tested the responses to 100 ppm of diacetyl for two weeks. The results demonstrate that aptamer-assisted ODR-10 biosensors have stable responses within one week, better than the physical adsorption group (2–3 days). While after one week, the responses become much more variable. The reasons may be due to the structure changes of ODR-10 and the loss of odorant detection functions. That is still the major challenge of olfactory receptor based biosensors for the practical applications and commercialization. The potential solutions maybe focus on the mimicking natural environments of olfactory receptors on the sensor surface. All these results demonstrated that the aptamer-assisted olfactory receptor immobilization was an effective method and can improve the performance of biosensor effectively. Additionally, aptamer-assisted immobilization method, as a general protein immobilization technique, can also be applied into the development of olfactory receptor sensor array for different ORs immobilization.
4. Conclusion
Fig. 6. Concentration-dependent responses lines of two ODR-10 based QCM biosensors. All the data are represented by means ± SD (standard deviation), n = 4.
In this study, we investigated a piezoelectric olfactory receptor biosensor for the odorant detection, which was prepared with the aptamer-assisted immobilization method. The results demonstrated that His6 -tagged ODR-10 was successfully immobilized onto the sensor surface by anti-His6 aptamers with high efficiency and specificity. The sensitivity and selectivity have been greatly improved compared with the physical adsorption group. This ODR-10 based QCM biosensor has lower detection limit and good repeatability for the real-time odorant detection. Using
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this aptamer-assisted immobilization method, different olfactory receptors could be immobilized onto the sensor surface to develop biosensor arrays. Considering the fast response time and the good reversibility, this aptamer-assisted ODR-10 based QCM biosensor has great application potential in many fields, such as food safety, environment monitoring, and poison gas detection.
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Acknowledgments
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This work was supported by grants from the National Natural Science Foundation of China (grant nos. 31000448, 81027003, 31228008), the China Postdoctoral Science Foundation (grant no. 201104734), the National Public Welfare Project (no. 201305010), and the 973 Program of Ministry of Science and Technology of China (no. 2009CB320303).
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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.2013.02.009.
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Biographies Liping Du received her B.S. degree from China Medical University in 2008. She is currently a Ph.D. candidate at the Department of Biomedical Engineering in Zhejiang University. Her research interests focus on olfactory receptor-based biosensor and olfactory electrophysiology. Chunsheng Wu received the Ph.D. degree of Biomedical Engineering from Zhejiang University, Hangzhou, China in 2009. He is currently an Associate Researcher of Biosensor National Special Laboratory at Zhejiang University. He was a joint Ph.D. student of Micro Systems Laboratories at University of California, Los Angeles (UCLA) in 2008. He was a Post-doctoral Fellow of Instrument Science and Technology at Zhejiang University from 2010 to 2012. His research is focused on the development and application of cell and molecule-based biosensors, instruments, and systems. He Peng received her M.S. degree in Biochemistry and Molecular Biology from Zhejiang University, Hangzhou, China, in 2012. Her research interests focus on the heterologous expression of membrane receptors and functional assays. Ling Zou is a Ph.D. candidate of biomedical engineering in Zhejiang University. Her research is focused on the heterologous expression of receptor proteins and the development of receptor-based biosensors. Luhang Zhao received the B.S., M.S., and Ph.D. degrees in Biochemistry and Molecular Biology from Zhejiang University, Hangzhou, China, in 1985, 1998, and 2011, respectively. He is currently an Associate Professor with the Department of Biochemistry and Genetics, Zhejiang University. His current research interests include Genetic Engineering and Glycobiology. Liquan Huang received his Ph.D. degree in Molecular Biology from Yale University. His research is directed at the molecular mechanisms that underlie taste and olfaction sensations. The focus is on identifying molecules involved in the recognition and transmission of chemical stimulus. He is currently an associate member of Monell Chemical Senses Center, USA. Ping Wang received the B.S. degree, M.S. degree and Ph.D. degree from Electrical Engineering of Harbin Institute of Technology, Harbin, China in 1984, 1987 and 1992, respectively. From 1992 to 1994 he is Post-Doctoral Fellow in Biosensor National Special Lab, Dept. of Biomedical Engineering, Zhejiang University, Hangzhou, China. At present, He is Professor of Biomedical Engineering of Zhejiang University, while he is Director of Biosensor National Special Laboratory and Director of Key Lab for Biomedical Engineering of National Education Ministry of China, Zhejiang University. He is a member of The International Society for Olfaction and Chemical Sensing and a member of steering committee of Asia Chemical Sensors Society. He is also a Director of Biomedical Measurement Society of China, Vice-Director of Ion and Biological Sensing Society and Vice-Director of Biomedical Sensors Technique Society of China. Besides, he is a visiting scholar at Edison Sensors Laboratory of Case Western Reserve University, USA and Biosensor and Bioinstrumentation Laboratory in University of Arkansas, USA, in 2002 and 2005, respectively.