Biophotonic Ring Resonator for Ultrasensitive Detection of DMMP As

Article pubs.acs.org/ac

Biophotonic Ring Resonator for Ultrasensitive Detection of DMMP As a Simulant for Organophosphorus Agents Karine Bonnot,*,† Francisco Cuesta-Soto,‡ Manuel Rodrigo,‡ Antonio Varriale,§ Nuria Sanchez,⊥ Sabato D’Auria,§ Denis Spitzer,† and Francisco Lopez-Royo⊥ †

Nanomatériaux pour les Systèmes Sous Sollicitations Extrêmes (NS3E), French-German Research Institute of Saint-Louis, UMR 3208 CNRS/ISL/UDS, 68301 Saint-Louis, France ‡ DAS Photonics, Ciudad Politécnica de la Innovación, 46022 Valencia, Spain § Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, 80131 Napoli, Italy ⊥ Nanophotonics Technology Center, Universitat Politècnica de València, 46022 Valencia, Spain ABSTRACT: Combining photonic integrated circuits with a biologically based sensing approach has the ability to provide a new generation of portable and low-cost sensor devices with a high specificity and sensitivity for a number of applications in environmental monitoring, defense, and homeland security. We report herein on the specific biosensing under continuous air flow of DMMP, which is commonly used as a simulant and a precursor for the synthesis of Sarin. The proposed technology is based on the selective recognition of the targeted DMMP molecule by specifically modified proteins immobilized on photonic structures. The response of the biophotonic structures shows a high stability and accuracy over 3 months, allowing for the detection in diluted air of DMMP at concentration as low as 35 μg/m3 (6.8 ppb) in less than 15 min. The performance of the developed technology satisfies most current homeland and military security requirements.

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The work presented herein exploits the capability of monitoring interactions between targeted molecules and a specific layer of proteins immobilized on a ring resonator-based chip. The high selectivity of biosensors compared with chemical sensors lies in the use of very specifically occurring biomolecules, such as enzymes, antibodies, etc. The concept of molecular recognition of a bioreceptor to a targeted analyte mimics the immunological response of some animals. In analogy with dogs’ detection of drugs and illicit substances, the proposed technology is based on the combination of bioreceptors with a photonic detection of binding molecules. Specific detection needs for defense against chemical and biological threats could be addressed with PIC biosensor-based devices for monitoring and detecting the release of toxic substances, such as nerve agents, in air in large areas. As an example, because of its high toxicity and the fact that noticeable health effects have been observed for an exposure of 30 min at concentration levels of 8.5 ppb, devices achieving a fast detection of Sarin at the parts-per-billion level within minutes are mandatory.14 DMMP is a precursor of Sarin (GB) nerve gas, and it is actually used as a simulant for many organophosphorus toxic compounds. Because of its low stability in air, it is often detected in aqueous media.15 In the

ecause of their versatility, optical sensors are powerful detection and analysis tools with many applications: environmental and industrial monitoring, defense and border security, and for the surveillance of large areas such as airports and stadiums.1 Under this category, there exist various optical detection methods, including refractive index change, optical absorption, and infrared or Raman spectroscopy.2,3 We chose to focus on the approach of the photonic measurement of changes in the refractive index (RI) of a sensitive surface derivatized with a biomolecule able to bind to a specific targeted analyte. Refractive index is widely used for the monitoring of chemical processes and implementation in photonic integrated circuits (PIC), and it holds great promise for the development of mobile, low-cost sensor arrays for real time monitoring.4 For use in such integrated systems, many resonating techniques can be employed, such as surface plasmon resonance,5 Mach−Zehnder interferometer,6 and optical waveguide and ring resonator-based biosensors.7−9 To allow selective detection of small molecules at very low concentrations (parts per billion, parts per trillion, and even parts per quadrillion), molecular recognition approaches show great potential in the capture of molecules. This approach is frequently combined with labeling molecules or fluorescent tags.10,11 A label-free approach such as the one presented in this work yields some benefits. The detection procedure is easier and less expensive to perform and permits quantitative and kinetic measurements of molecular interaction between a sensitive layer and the targeted analyte.12,13 © 2014 American Chemical Society

Received: March 11, 2014 Accepted: April 25, 2014 Published: April 25, 2014 5125

dx.doi.org/10.1021/ac500903s | Anal. Chem. 2014, 86, 5125−5130

Analytical Chemistry

Article

Figure 1. (a) Schematic showing the main fabrication steps for each layer. (b, top) SEM micrograph of the coupling region between a Si3N4 waveguide and a ring resonator. (b, bottom) Cross section of those same structures near the coupling region where the external sidewalls can also be identified.

consists of the aforementioned ring resonator critically coupled to a single bus waveguide. Critical coupling is achieved by accurately adjusting the separation of the bus waveguide. With this design approach, the extinction ratio of the resonance in the transmission of the photonic structure is optimized to help in the tracking of the resonance. High quality commercial silicon wafers with 300 nm of LPCVD silicon nitride over 5 μm of silicon oxide were used as substrates. The fabrication route consists of three processing levels. The main process steps in each level are shown in Figure 1a. After cleaning and surface dehydration, a thin film of photoresist is applied on the wafers. Following a prebake, the resist is then exposed using a pattern including the photonic circuits in a mask aligner (EVG-620) using i-line UV illumination, baked, and developed. A thin layer of chromium is then deposited over the wafer using an e-beam metal evaporator (Pfeiffer Classic 500) such that the chromium covers the resist, except in the exposed regions where chromium lies directly over the silicon nitride, thus forming the photonic circuit pattern. The next process step is lift-off, which involves a high-pressure stream of hot solvent impinging on the wafer to remove the resist, hence “lifting off” the chromium, except in regions where it lies directly on the silicon nitride, that is, the desired circuit pattern that stays as a chromium mask for future processing. After further acid cleaning, the wafers are then processed in an inductively coupled plasma reactive ion etching (ICP-RIE, from STS model “Mesc Multiplex”) tool, where a combination of chemical etching and ion bombardment removes the silicon nitride from the wafer, except in regions protected by the chromium mask, thus transferring the chromium pattern to the silicon nitride. Finally, a bath in a chromium etching chemistry (CR7) and an oxygen plasma removes the chromium mask, leaving only the desired photonic circuit (waveguides, couplers and ring resonators) in silicon nitride over the silicon oxide layer (last step in Figure 1a). The second level involves processing similar to that described above (up to the lift-off step) using a different photolithography mask, which leaves chromium square pads over the sensing ring resonators in the circuit. These protective chromium pads will serve as etch-stops in subsequent processing. A thin layer of silicon oxide (1−2 um) is then deposited with PECVD (Applied Materials P5000) over the entire wafer (not shown in Figure 1a) to protect the photonic circuit from physical damage

past decade, a few studies have referred on the detection of DMMP vapor down to 50 ppb by employing mainly acoustic wave techniques or Raman spectroscopy.16−18 Using laser photoacoustic spectroscopy, Pushkarsky et al.19 achieved a threshold of 3.5 ppb for the continuous monitoring of DIMP (another simulant of Sarin) in a reconstituted air including pollutants generally observed in towns. Despite attaining an unprecedented detection limit, this technology is currently not adapted for portable device development. Although optical methods coupled to chemical recognition systems have gained interest during the last years,20 this is the first time, to our knowledge, that biological recognition (furthermore, label-free) has been employed in combination with a photonic readout. This unique and innovative sensor configuration allows improvement in specificity, sensitivity, and accuracy of the detection resulting from the high selectivity of the recognition offered by specifically tuned odorant-binding proteins (OBP) in olfactory systems of mammalians (such as pig, bovine) or insects.21 The selectivity to one solely specific target is induced by a specific property of OBPs, which bind selectively one analyte and are not sensitive to molecules other than the one to which they are specifically tuned for selective biorecognition. Such bioreceptors, by specifically tuning their binding properties, then allow achievement of highly selective detection that is insensitive to an interfering agent and unspecific adsorption as a result of their selectivity-induced recognition properties.22,23 The optical response of the biophotonic sensor was measured with different chips functionalized with proteins either sensitive or not to DMMP and also with blank chips. It shows that selectively tuning the protein system to the biorecognition of one specific molecule allows one to perform a specific detection on each functionalized ring and also improves the detection ability of the silicon-based surface of the photonic structures. The sensitivity of the technique and its reliability over months are also described and discussed toward safety requirements.

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EXPERIMENTAL DETAILS Fabrication of the Resonating Sensor Chip. Silicon chips with silicon nitride photonic waveguides and ring resonators (1.1 μm wide and 0.3 μm high) were fabricated using standard microfabrication techniques as described by Barrios et.al.9 The design of the photonic sensing structure 5126



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a Veeco Multimode Nanoscope IV atomic force microscope (AFM) with a Bruker TESP-SS probe (tip radius = 2 nm) Generation of DMMP Vapors. The DMMP vapor at a known concentration was generated using a calibrated permeation tube (Owlstone Ltd., UK). The system allows the analyte to diffuse through tube wall microholes, following Fick first law. At the steady state, the gradient is constant, and the concentration of the DMMP vapor depends on the temperature of the permeation tube and the flow of nitrogen surrounding the tube,28 accordingly to eqs 1 and 2.

and chemical or biological modification. This layer of silicon oxide also serves as the optical upper-cladding material for the silicon nitride waveguide cores. Finally, a third photolithography level is used to expose windows on the silicon oxide on top of the sensing resonators, followed by an etch step that removes the silicon oxide in these regions (the chemistry of the etch “stops” at the chromium pads, thus leaving the silicon nitride resonators intact). A final CR7 bath removes the chromium pads, which exposes the sensing resonators to air (Figure 1), and eventually to the liquid solution by means of a microfluidics circuit. Figure 1b (left) shows the coupling region of a ring resonator and a bus waveguide, which is curved by design to better define the coupling section. Smooth and vertical sidewalls were obtained by optimizing fabrication parameters, such as adjusting the chromium thickness to improve the lift-off process and fine-tuning the etching step. The robustness of this fabrication process with large “process windows” allowed demonstration of a good reproducibility in the number of chips. It should be noted that the small size of the chips and the asdescribed fabrication method and equipment employed (such as a standard i-line mask aligner) would make it possible to mass produce these chips at relatively low cost. To allow for functionalization, the fabricated chips were then introduced in an inert atmosphere for 24h before being cleaned in a 1% hydrofluoric acid (HF) solution for 3 min. HF was removed by rinsing the chips in a 10 mM phosphate buffer solution (PBS) three times, 10 min each, followed by extensive washing with deionized water to eliminate salts on the chip surface. The chips were then dried under nitrogen flow and placed under UV light (280 nm) for 30 min before functionalization. Functionalization was then performed with bovine odorant-binding proteins (b-OBP) as a biological element. Specific b-OBPs were obtained by inserting a 6-histidine amino acid (6-His) affinity tag in the deoxyribonucleic acid (DNA) chain, which was further subcloned in the expression vector pT7−7 of the protein. The expression of the proteins was then realized in BL21-DE3 Escherichia coli24 (E. coli). The purified protein solutions were then obtained by affinity chromatography on Ni-NTA Agarose (Qiagen, Germany), followed by anion-exchange chromatography in fast protein liquid chromatography mode. The concentrations of the solutions were calculated equal to 48 000 M−1 cm−1, based on UV absorbance at 280 nm. The functionality of the OBPs and their affinity for DMMP after their immobilization onto the chip surfaces were determined using the fluorescent ligand 1amino-anthracene (AMA). Direct titrations of the formation of the AMA−OBP complex were obtained by following the fluorescent emission intensity25 at 480 nm while using a fixed excitation wavelength of 380 nm (PerkinElmer LS50). A solution of either b-OBP or mutant b-OBP (5 nm in diameter, 19 kDa weight) at a concentration of 2.0 mg/mL was deposited on the chip surface in the opened window area by manual pipetting with the help of a glass magnification. The chips with the deposited proteins were re-exposed to UV illumination for 120 min at 298 K to allow the immobilization of the proteins by binding on the silicon nitride surface.26,27 The chips were then placed in a Petri dish with pure deionized water and gently shaken for 30 min at 298 K to remove exceeding proteins not immobilized on the surface. Characterization of the protein immobilization on the ring resonator surface was performed on

log(qd1) = log(qd2) − 2950((1/T1) − (1/T2))

(1)

φDMMP = (qd1Vm/M )Q

(2)

where qd1 and qd2 are the permeation rates (ng/min) at the temperatures T1 and T2, respectively (K); φDMMP is the concentration of DMMP in the nitrogen flow in parts-perbillionv; Vm is the molar volume of nitrogen at the temperature T1 (mL/mol); M is the molar mass of the DMMP (124.08 × 109 ng/mol); and Q is the nitrogen flow surrounding the permeation tube (mL/min). The DMMP permeation tube we purchased from Owlstone for the experiments has a permeation rate of 494 ng/min at 70 °C. To ensure an accurate and tunable concentration of DMMP at 20 ppbv under a nitrogen flow of 200 mL/min, the permeation tube was placed in a Liebig column heated at a constant temperature of 24 °C, according to values calculated from eqs 1 and 2 given by Owlstone Ltd. The constant temperature of the column cooling mantle was assured with the help of a thermostatic bath with a circulation pump (Julabo, Germany) and a temperature probe in contact with the glass walls of the column mantel, which permits precise control of the temperature to about ±0.1 °C, corresponding to an error of ±0.23 ppbv (±1.15%) on the concentration value. The Liebig column is connected at its two ends to nitrogen flow and to the detection system with GL14 connectors and 1/8 in. Sulfinert tubes (Restek, US Patent 6.444.326). The Sulfinert treatment results in an inert chemically protective barrier of amorphous silicon deposited on all parts in contact with gas flow to prevent the potential adsorption of analytes in the circuit before contacting the sensor surface. Photonic Transduction Readout System. The molecular binding of the bioreceptor molecule to the target analyte is detected by means of a refractive index sensitive photonic transducer. The transducer consists of a photonic ring resonator that transduces the variations of the refractive index (due to the binding of molecules) into spectral shifts within the transfer function response of the photonic circuit. The position shift of the cavity spectral peaks is proportional to the variation of the refractive index.29 The biospecificity of the recognition molecules introduced above is the mechanism that ensures the selectivity of the sensor to the target DMMP. The optical subsystem implemented for the measurement of a detection signal is based on a transfer function measurement of the chip by using a component tester (Yenista CT400). A reference wavelength sweeping signal from a tunable laser source (Yenista Tunics plus) is measured continuously (power and wavelength), and the power transmitted from the circuit is simultaneously measured. (Figure 2) This way, a fast method is implemented to evaluate the spectral response over more than 50 nm with a high resolution (1 pm). When the biorecognition proteins bind an analyte, a wavelength shift of the spectral response of the photonic chip is produced. By tracking this shift over time, the response of the 5127



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Figure 3 shows AFM images of the proteins once immobilized on a ring resonator. AFM imaging shows a high coverage ratio of more than 96% of the ring resonator with OBP molecules. Imaging was performed on different parts of the ring resonator and on various rings to verify the reproducibility of the functionalization with OBPs. Apart from the high coverage ratio of b-OBPs on the ring resonator surface (>96%), it also shows that the chosen method for the immobilization of the OBP does not result in the presence of salts, which could lead to optical measurement disturbances. The OBP layer also shows a homogeneous rugosity of ∼5 nm on the all surface of the ring resonator (Figure 3). These two combined results indicate that the OBP molecules are good candidates to be used as probe for biophotonic sensors because we have a high coverage ratio with homogeneous repartition of the proteins on the surface, and thus, these proteins are fully active after their immobilization onto the chips, and they are still able to bind to DMMP molecules.27,30,31 Measurement of the Sensing Performances of the Biophotonic Ring Resonator. The performance tests of the biophotonic device to measure DMMP detection signals were performed at laboratory conditions and under a nitrogen flow of 200 mL/min. Pulses of 20 ppbv of DMMP (101 μg/m3) were regularly injected into the sensor ring resonator array. The system is shown in Figure 4. The wavelength shift induced by the interaction between the sensing surface and the DMMP molecules was recorded over time. The experiments were realized for several chips which were either (i) not functionalized (blank chips); (ii) covered with original proteins (b-OBP) not tuned for any specific biorecognition activity; or (iii) chips covered with mutant bOBP specifically tuned to be sensitive only to DMMP. The results are plotted in Figure 5, parts a, b, and c, respectively. At first, once a pulse of DMMP vapor in nitrogen is injected on the chip surface, a strong peak of around 50−90 pm appears, followed by a relaxation of the signal to a stable value. The wavelength shift signal over time returns to its original zero value when the chip is not functionalized (Figure 5a) or when the functionalized protein layer is not specifically designed for DMMP biorecognition (Figure 5b). Conversely, in the case of a sensor covered with mutant OBPs, the wavelength shift does not return to its zero value, stabilizing at around +30 pm higher value (Figure 5c). As the proteins bind the DMMP molecules

Figure 2. Block diagram of the characterization setup.

system to the binding can be monitored in the sensing experiments. For the interconnection of the chip to the optical measurement system, two nanopositioning stages (Thorlabs MAX301) are employed to accurately place the optical input fibers on the chip waveguides. An infrared camera is used to help in the initial exploration of the fibers’ alignment. A fiber polarization controller permits finely adjusting the TE polarization with the help of a polarization filter. All the experiments were conducted under laboratory-controlled environmental conditions: room temperature at 24 °C and 40% humidity.

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RESULTS AND DISCUSSION Characterization of the OBP Affinity to DMMP. Fluorescence emission spectra of the OBP−AMA complex were recorded between 450 and 550 nm at a fixed excitation wavelength of 290 nm. The formation of the AMA−OBP complex was followed as the increase in the fluorescence emission intensity at 480 nm due to the resonance energy transfer between the tryptophan residues of the protein and the AMA molecules that are located in Foster distance from the protein indolic residues.25 The addition of DMMP to the OBP−AMA complex results in a displacement of the AMA molecules from the protein binding site, resulting in a quenching of the fluorescence emission at 480 nm (data not shown). This phenomenon issues from the replacement of 1AMA by DMMP molecules that complex with the OBPs, resulting in the formation of a stronger OBP−DMMP complex.

Figure 3. AFM imaging of (a) a section of a ring resonator on the chip (the inserted picture shows the position of the cantilever on the ring resonator) and (b) the surface of the ring resonator with immobilized OBPs, showing a high ratio coverage of the surface with a mean rugosity of 5 nm corresponding to the diameter of proteins. 5128



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However, once DMMP molecules are bound to the proteins, flowing pure nitrogen on the sensor surface is not sufficient to clean it. (Figure 5c) This is in accordance with a chemical adsorption of the DMMP molecules on the biosensitive layer. Rinsing this chip by flowing nitrogen on its surface results in a slight decrease in the wavelength shift to +20 pm compared to the zero basis, which is assumed to be due to misalignment of the laser as explained above. A second DMMP pulse was then performed, leading to the same signal as previously obtained. To further verify the stability of the response of the detection system over months, a second set of chips was functionalized the same way as the first set and stored for 3 months inside a box in a fridge without any specific treatment and without sealing. After those three months, the second group of functionalized chips was then subjected to pulses of DMMP under the same conditions as the previous set of experiments and showed a similar response. The standard deviation of the optical signal is assumed to be equal to round ±3.4 pm, which results in a detection threshold of 6.8 ppb (35 μg/m3) in 10 to 15 min.

Figure 4. Global system includes bioreceptors immobilized on the ring resonators designed on silicon nitride chips. The chip is connected to the microfluidic system, allowing for flowing gas over the ring resonator surface. The optical fibers connected to the chip like it is shown in Figure 1 permit measurement of the system response in terms of wavelength shift when a molecule in the gas flow binds a bioreceptor.

present in the nitrogen flow, the protein-layer immobilized over the ring resonator surface undergoes physical and chemical modifications, which leads to a change in its effective refractive index. The optical transduction of this phenomenon is a shift in the wavelength of the light transmitted to the sensor chip. Figure 4c shows that this shift is only observed at a positive stable value over time, when specific binding of the targetedanalyte molecules occurs on the sensor surface. However, in any of the three studied cases−chips covered with either mutant b-OBPs or with the b-OBPs or with no proteins−a sharp peak occurs when DMMP is pulsed for the first time, which resulted in a delay of 10 to 15 min in the readable response of the detection device. The sharp peak observed while flowing DMMP for the first time can be attributed to the condensation of molecules on the surface, which is rapidly cleaned by the flow when the molecules are not bound by proteins or in the case of the blank chip (Figure 5a, b). On these figures, a drift can be observed when switching from DMMP to N2 flow. This is due to a perturbation of the laser by switching flows, which results in a slight misalignment of the optical fibers. To avoid perturbing the measure, we chose not to realign the laser during experiments. This inconvenience could be avoided in a future lab-on-chip solution by continuously comparing the response of the ring resonator measurment under flow conditions to a parallel balance reference ring acquiring signal in the absence of gaseous flow.32

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CONCLUSION The work presented herein permitted validation of the use of photonic integrated circuits as an enabling technology for the sensitive detection of DMMP molecules at trace concentrations in air. The label-free approach permitted direct measurement of DMMP from air samples without time-consuming steps, such as labeling. The proposed technology is based on a wavelength shift of propagated light, which occurs when the targeted molecules are bound by the biorecognition sites immobilized on the sensor chip. Compared with a chemical functionalization of the ring, coupling biorecognition molecules with photonic ring resonator structures resulted in several improvements in detection capacities by (i) enhancing the affinity of the sensing surface to the targeted molecules and (ii) tuning the bioreceptors for the reliable and solely detection of one specific analyte. Therefore, the developed technology allows the accurate detection of a DMMP vapor in air at concentration levels as low as 6.8 ppb (35 μg/m3) in less than 15 min. Accuracy of the measurement at the parts-per-billion level without preconcentrating is a strong improvement compared with other portable sensors for DMMP detection. Thin films of carbon nanotubes on flexible polyimide substrate showed a LoD of 232 ppb in air.33 Without

Figure 5. Mean resonance wavelength shifts (Δλ) induced by the cyclic injection of a DMMP vapor (20 ppbv concentration) on a (a) blank chip, and (b) chip covered with b-OBP not tuned for any specific biorecognition activity, and (c) chip covered with mb-OBP specifically tuned to be sensitive to DMMP-like compounds. The DMMP and N2 pulses are indicated on the graphs with an arrow. The zero base signal is indicated with a black dot line. A slight perturbation of the laser alignment could occur when switching between DMMP and N2 flows, resulting in a modification of the position of the zero baseline. Graphs a and b show that no signal is observed after 15 min, when the sensitive layer is not specifically tuned for the detection of DMMP. Graph c shows that DMMP-sensitive bioreceptors immobilized on the photonic structure bind DMMP molecules, leading to a 30 pm residual deviation of the signal from zero baseline and with a good reproducibility over cyclic DMMP pulses. 5129



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(6) Blanco, F. J.; Agirregabiria, M.; Berganzo, J.; Mayora, K.; Elizalde, J.; Calle, A.; Dominguez, C.; Lechuga, L. M. J. Micromech. Microeng. 2006, 16, 1006. (7) Claes, T.; Bogaerts, W.; Bientsman, P. Opt. Express 2010, 18, 22747. (8) De Vos, K.; Girones, J.; Claes, T.; De Koninck, Y.; Popelka, S.; Schacht, E.; Baets, R.; Bienstman, P. IEEE Photon. J. 2009, 1, 225. (9) Barrios, C. A.; Gylfason, K. B.; Sanchez, B.; Griol, A.; Sohlström, H.; Holgado, M.; Casquel, R. Opt. Lett. 2007, 32, 3080. (10) Moerner, W. E. Proc Natl. Acad. Sci. 2007, 104, 12596. (11) Cox, W. G.; Singer, V. L. BioTechniques 2004, 36, 114. (12) Chao, C. Y.; Fung, W.; Guo, L. J. IEEE J. Sel. Top. Quantum Electron. 2006, 12, 1077. (13) De Vos, K.; Bartolozzi, I.; Schacht, E.; Bienstman, P.; Baets, R. Opt. Express 2007, 15, 7610. (14) Krewski, D.; Walker, B. Acute Exposure Guideline Levels for Selected Airborne Chemicals; The National Academies Press: Washington DC, 2003; Vol. 3; pp 15−76. (15) Hou, C.; Yang, L.; Huo, D. Proc. 2nd Int. Conf. Bioinf. Biomed. Eng., Shanghai, China, 2008, p 1640. (16) Grate, J. W.; Rose-Pehrsson, S. L.; Venezky, D. L.; Klusty, M.; Wohltjen, H. Anal. Chem. 1993, 65, 1868. (17) Zimmermann, C.; Mazein, P.; Rebiere, D.; Dejous, C.; Pistre, J.; Planade, R. IEEE Sens. J. 2004, 4, 479. (18) Wang, F.; Gu, H.; Swager, T. M. J. Am. Chem. Soc. 2008, 130, 5392. (19) Pushkarsky, M. B.; Webber, M. E.; Macdonald, T.; Patel, C. K. N. Appl. Phys. Lett. 2006, 88, 044103. (20) Orghici, R.; Lützow, P.; Burgmeier, J.; Koch, J.; Heidrich, H.; Schade, W.; Welschoff, N.; Waldvogel, S. Sensors 2006, 10, 6788. (21) Paddle, B. M. Biosens. Bioelectron. 1996, 11, 1079. (22) Korndorfer, I. P.; Schlehuber, S.; Skerra, A. J. Mol. Biol. 2003, 330, 385. (23) Staiano, M.; D’Auria, S.; Varriale, A.; Rossi, M.; Marabotti, A.; Fini, C.; Stepanenko, O. V.; Kuznetsova, I. M.; Turoverov, K. K. Biochemistry 2007, 46, 11120. (24) Ramoni, R.; Vincent, F.; Ashcroft, A. E.; Accornero, P.; Grolli, S.; Valencia, C.; Tegoni, M.; Cambillau, C. Biochem. J. 2002, 365, 739. (25) D’Auria, S.; Staiano, M.; Variale, A.; Gonnelli, M.; Marabutti, A.; Rossi, M.; Strambini, G. J. Proteome Res. 2008, 7, 1151. (26) De Stefano, L.; Rossi, M.; Staiano, M.; Mamone, G.; Parracino, A.; Rotiroti, L.; Rendina, I.; Rossi, M.; D’Auria, S. J. Proteome Res. 2006, 5, 1241. (27) Di Pietrantonio, F.; Cannata, D.; Benetti, M.; Verona, E.; Varriale, A.; Staiano, M.; D’Auria, S. Biosens. Bioelectron. 2013, 41, 328. (28) Boyle, B. The complete guide to testing chemical sensors, Owlstone, Inc: Cambridge, UK, 2011. (29) McKinnon, W. R.; Xu, D. X.; Storey, C.; Post, E.; Densmore, A.; Delage, A.; Waldron, P.; Schmid, J. H.; Janz, S. Opt. Express 2009, 17, 18971. (30) Borini, S.; D’Auria, S.; Rossi, M.; Rossi, A. M. Lab Chip 2005, 5, 1048. (31) D’Auria, S.; De Champdoré, M.; Aurilia, V.; Parracino, A.; Staiano, M.; Vitale, A.; Rossi, M.; Rea, I.; Rotiroti, L.; Rossi, A. M.; Borini, S.; Rendina, I.; De Stefano, L. J. Phys.: Condens. Matter 2006, 18, S2019. (32) Xu, D.; Vachon, M.; Densmore, A.; Ma, R.; Janz, S.; Delage, A.; Lapointe, J.; Cheben, P.; Schmidt, J. H.; Post, E.; Messaoudène, S.; Fedeli, J. Opt. Express 2010, 18, 22867. (33) Wang, Y.; Yang, Z.; Hou, Z.; Xu, D.; Wei, L.; Siu-Wai Kong, E.; Zhang, Y. Sens. Actuators, B 2010, 150, 708.

preconcentrating, Grate et.al. achieved a detection of DMMP down to 120 μg/m3 using surface acoustic wave sensors, and Love acoustic waves permits detection at 900 ppb.16,17 The ability of the sensitive biostructures to detect DMMP shows a high accuracy and reliability over 3 months, as compared with the signal observed just after functionalization. The stability over 3 months of the biophotonic technology presented here allows for its use for the implementation of portable devices or network of sensors for monitoring and control of large areas. It is thus noticeable that (i) no previous concentration of air samples is needed, and measurements can be done over large volumes; and (ii) biosensor chips show a good reliability for detection over months. As a first attempt, this work proved PIC ability to be used to develop highly sensitive gas sensors for the selective detection of a chosen analyte. The main important and challenging novelty consists here in (i) employing immobilized biological recognition receptors mimicking the olfactory sensing observed in mammalians and insects, and (ii) in combining it with photonic read-out to achieve the sensitive sensing of a specific target. This work shows promising features for the development of sensitive and selective sensors. This research has led to a follow-up project in which the influence of temperature, humidity, and interfering compounds on signal drift will be assessed by using a three-parallel-ring resonator scheme: the first functionalized for detection; the second not functionalized for monitoring the drift due to condensation or unspecific adsorption; and the third, not in contact with flow, to compensate for any signal drift resulting from operational conditions of measurement.32

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AUTHOR INFORMATION

Corresponding Author

*Phone: +33 3 8969 5872. Fax: +33 3 8969 5074. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the NANOCAP project A-1084RT-GC that is coordinated by the European Defence Agency (EDA) and funded by 11 contributing members (Cyprus, France, Germany, Greece, Hungary, Italy, Norway, Poland, Slovakia, Slovenia, and Spain) in the framework of the Joint Investment Program on Innovative Concepts and Emerging Technologies (JIP-ICET).

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REFERENCES

(1) Narayanaswamy, R.; Wolfbeis, O. S. Optical Sensors: Industrial, Environmental and Diagnostic Applications; Springer: New York, 2005; p18. (2) Prasad, P. N. Introduction to Biophotonics; John Wiley &Sons: Hoboken, NJ, 1999. (3) Vo-Dinh, T. Biomedical Photonics Handbook; CRC Press: Boca Raton, FL, 2003. (4) Jokerst, N.; Royal, M.; Palit, S.; Luan, L.; Dhar, S.; Tyler, T. J. Biophotonics 2009, 2, 212. (5) Yeatman, E.; Ash, E. A. Electron. Lett. 1987, 23, 1091. 5130


Biophotonic Ring Resonator for Ultrasensitive Detection of DMMP As

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