CLIAMS:We claim:
1. An optical nanosensor for early stage detection of Bactocera oleae infestation, the optical nanosensor comprising,
a functionalized surface;
a mount for embedding the functionalized
surface; and
a perforated housing for replacably retaining
the mount.
2. The optical nanosensor of claim 1, wherein the functionalized surface is formed on an oxide surface having free hydroxyl group.
3. The optical nanosensor of claim 2, wherein the oxide surface having free hydroxyl group is selected from a group comprising silicon dioxide, zinc oxide, titanium dioxide, cerium dioxide, aluminum dioxide and iron oxide.
4. The optical nanosensor of claim 1, wherein the functionalized surface is a ß-cyclodextrin unit further wherein the functionalized surface is configured to capture a pheromone specific to Bactocera oleae.
5. The optical nanosensor of claim 1, wherein the functionalized surface is embedded on a microstructure or a nanostructure wherein the structure is atleast one of a cantilever, a fixed- fixed beam, a commercially available surface and a combination thereof.
6. The optical nanosensor of claim 1, wherein the perforated housing is a box, a bottle, a can or a canister.
7. The optical nanosensor of claim 1, wherein the material of the perforated housing is a metal, a plastic, a glass, an acrylic, a polycarbonate, or a combination thereof.
8. The optical nanosensor of claim 1, wherein the detection is achieved by measuring the change in the resonance frequency.
9. The optical nanosensor of claim 8, wherein the change in the resonance frequency is due to change in the mass of the embedded functionalized surface.
10. The optical nanosensor of claim 1, wherein the detector is placed proximal to an agricultural and/or a horticultural produce having high risk of infestation.

Bangalore NARENDRA BHATTA HL
June 15, 2015 (INTELLOCOPIA IP SERVICES)
AGENT FOR APPLICANT
,TagSPECI:OPTICAL NANOSENSOR FOR EARLY STAGE DETECTION OF BACTOCERA OLEA INFESTATION

FIELD OF INVENTION
The present disclosure relates to the field of pest management and particularly to an optical nanosensor for early detection of Bactocera olea pest infestation in fields growing horticultural produce, namely olea europaea.

BACKGROUND
Olea europaea, commonly referred to as Olive, is one of the most important and widespread fruit trees cultivated in the Mediterranean basin, where the fruit has versatile impact on the society, environment and economy. The entire fruit and the oil extracted from the fruit are exploited commercially worldwide. The oil, specifically, possesses organoleptic and antioxidant properties. However, the quality of oil extracted depends on the quality of the olive fruit harvested. One significant factor that could affect the quality of olive fruit harvested is infestation. The predominant cause of infestation, in the Mediterranean region including some areas in India, is through olive fruit fly, referred to as, Bactocera oleae (Rossi) (Diptera: Tephritidae).
Various analytical techniques including but not limited to electro-antennogram, single sensillum, electronic nose and computer based monitoring are known for the detection of Bactocera oleae infestation. An ATR-FTIR-PLS based non-destructive method is also known wherein the strategy developed analyzes the quality of olive fruits post infestation by Bactocera oleae. Near IR Spectroscopy is also used to detect the level of infestation in olive fruits. However, one significant disadvantage of the aforementioned techniques is that the techniques are able to detect the pheromones only subsequent to the infestation. Another disadvantage is that the aforementioned techniques cannot measure the concentration of the pheromone excreted by the fruit pests in an olive orchard.
Farmers also use various protein based food attractants, mixed with pesticides, for monitoring and mass trapping of olive pest population, but aging of the food attractants weakens the attractiveness of these bait traps. One significant disadvantage of the aforementioned method is the development of resistance due to overuse of pesticides or insecticides together with the changes in climate. There are also devices known that utilize pheromone traps for the management of these olive flies. The spiroketal pheromone together with (-)-a -pinene, n-nonanal and ethyl dodecanoate are used in a particular ratio of 3:1:0.3:1 as the primary pheromone extract to attract these flies. A major disadvantage of the trap is the need for frequent replacement of the trap. Thus, there is need for an efficient sensor that is capable of rapidly detecting low levels of pheromones that are released by pest during early infestation.

BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG. 1 shows a schematic representation of the surface functionalization of silicon dioxide based cantilevers and fixed-fixed beams, according to an embodiment of the invention.
FIG. 2 shows reduction in resonance frequency and increase in pheromone mass over the functionalized surfaces, according to an embodiment of the invention.
FIG. 3 shows the scanning electron micrographs (SEM) of the functionalized surfaces before and after functionalization, according to an embodiment of the invention.
FIG. 4 shows XPS analyses of the functionalized surfaces, according to an embodiment of the invention.
FIG. 5 shows change in frequency of the functionalized surfaces in response to various semiochemicals, according to an embodiment of the invention.
FIG. 6 shows change in resonance frequency and effective number of pheromone molecules attached to the functionalized surfaces, according to an embodiment of the invention.
FIG. 7 shows a plot for calculation of limit of detection of the functionalized cantilever, according to an embodiment of the invention.
FIG. 8 shows schematic representation of an optical nanosensor, according to an embodiment of the invention.

SUMMARY OF THE INVENTION
The invention provides an optical nanosensor for an early stage detection of Bactocera oleae infestation in a field. The optical nanosensor includes a functionalized surface, a mount for embedding the functionalized surface and a perforated housing for replacably retaining the mount. The invention also provides a convenient and energy efficient technique to detect the pheromones.

DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide an optical nanosensor for early detection of Bactocera oleae infestation in a field. Pheromones are a class of semiochemicals that insects release to communicate with other members of the same species. The pheromones leave the body of the first organism, pass through the air (or water) and reach the second organism, where they are detected by the receiver. In insects, these pheromones are detected by the antennae on the head. The signals are effective in attracting faraway mates, and also allow marking of food sources. The levels of pheromone remain at a low level at that point of time. The optical nanosensor provided in the invention detects very low levels of pheromones enabling an early detection of impending pest attack in a horticultural field.
One embodiment of the invention provides an optical nanosensor for early stage detection of Bactocera oleae pheromones in a field. The optical nanosensor includes a functionalized surface, a mount for embedding the functionalized surface and a perforated housing for replacably retaining the mount. The functionalized devices detect the pheromones at an early stage of pest infestation in a rapid and energy efficient way. The functionalized devices include but are not limited to functionalized cantilevers, fixed-fixed beams and other such microstructures known to a person skilled in the art and the combination thereof.
In one embodiment of the invention functionalized cantilevers are used as chemically functionalized surface. In an alternate embodiment of the invention fixed-fixed beams are used as chemically functionalized surfaces. In another embodiment of the invention both cantilever and fixed-fixed beams are used as functionalized surfaces. The functionalized surfaces are obtained either by the surface functionalization of the cantilevers and fixed-fixed beams, or by other methods available in the art or commercially available functionalized surfaces are used.
In one embodiment of the invention covalent surface functionalization is chosen as the method for obtaining functionalized surfaces (FIG 1). In one embodiment of the invention, the functionalized surface is formed on an oxide surface having free hydroxyl group. The oxide surface having free hydroxyl group is selected from a group comprising silicon dioxide, zinc oxide, titanium dioxide, cerium dioxide, aluminum dioxide and iron oxide.
FIG. 1 shows a schematic representation of the surface functionalization of silicon dioxide based cantilevers and fixed-fixed beams, according to an embodiment of the invention. In one embodiment, for surface functionalization, a silicon wafer of 100 mm diameter and approximately 500-550 µm thickness (type P, dopant boron, orientation <100>, resistivity 0-100 ohms) is taken and cleaned well with piranha solution (H2SO4:H2O2 = 9:1) for 5 min and washed twice with distilled water to remove organic and metallic contaminants from the surface. A layer of silicon dioxide approximately 1 µm is then thermally grown over the silicon wafer and the resultant surface is again cleaned with piranha solution.
The cleaned wafer is dried at 250 ºC for 10 min and then spin-coated with a photoresist (AZ5214E), of nearly 1.5 µm thickness at 6000 rpm for 40 sec. The spin-coated wafer is then baked at about 110 ºC for 2 min to evaporate the solvents of the photoresist. The spin-coated wafer is then kept under UV exposure for 2 sec in a double-sided mask aligner (50 mill joules/cm2). The mask is developed by dipping the spin coated wafer in a developer solution of AZ351B:H2O in a ratio of 1:4 for 30 sec. The wafer is then washed with distilled water, dried under nitrogen and finally heated at 110 ºC for 4 min to get desired pattern of the microstructures.
The patterned structures are then dry etched to release the microstructures. For etching, formalin oil is spread over the wafer to prepare sticky base which is then loaded inside a reactive ion etching chlorine (RIE-Cl) chamber and is followed by three steps i.e. i) anisotropic plasma etch, ii) isotropic Si etch and iii) oxygen etch.
The completely released cantilevers and fixed-fixed beams are then carefully diced from the wafer. Four different cantilevers and fixed-fixed beams each of which is distinguishable from the other based upon its length are fabricated. The cantilevers 1, 2, 3 and 4 have lengths of 86.6, 36.5, 28.1 and 19.8 µm respectively. Similarly, the fixed-fixed beams 1, 2, 3 and 4 have lengths of 53.4, 35.5, 27.1 and 21.3 µm respectively. The cantilevers have uniform width of 5.1 µm, whereas the fixed beams have uniform width of 4.7 µm. Each structure has a uniform thickness of 1.04 µm.
The microstructures are then cleaned to remove contamination from the silicon dioxide surfaces by dipping in piranha solution at 85 ºC for 10 min, then rinsed twice with sterilized-filtered water and once with deionized water and finally dried under a nitrogen flow. The cleaned microstructures are then functionalized with 4% silane in organic solvent at 25 ºC. Functionalization with the silane reagent results in the formation of active anchor sites on the surface of the microstructures. The microstructures are then reacted with 10% glutaraldehyde for 4 hr followed by ß-CD-ONH2 in 5 mg/mL concentration for 6hr to achieve at least one ß-cyclodextrin unit at each anchor site.
Characterization: These functionalized microstructures are then exposed to the major volatile pheromone component of the olive fruit pests particularly Bactocera oleae at room temperature and then dried under a nitrogen flow. The microstructures are then characterized using the techniques including but not limited to laser doppler vibrometry, scanning electron microscopy and X-Ray photoelectron spectroscopy.
FIG. 2 shows reduction in resonance frequency and increase in pheromone mass over the functionalized surfaces, according to an embodiment of the invention, according to an embodiment of the invention. The figure shows that the cantilevers are more efficient is sensing the pheromone of the olive fruit fly than the fixed-fixed beams. The sensitivity depends on the length of the functionalized surfaces; the longer the functionalized surface greater the sensitivity. The cantilever having the length of 36.53 µm is found to be the most sensitive.
FIG. 3 shows the scanning electron micrographs (SEM) of the functionalized surfaces before and after functinalization, according to an embodiment of the invention. The figure shows that the functionalized surfaces are fairly stable after the covalent functionalization procedures.
FIG. 4 shows XPS analyses of the functionalized surfaces, according to an embodiment of the invention. The XPS data indicates the appearance of C-1s and N-1s peaks after the successful covalent functionalization by the silane reagents to the silicon dioxide surfaces. Also the intensity of C-1s peak gets increased after the physical trapping of the pheromone of the olive fruit fly.
Specificity and Reversibility: The functionalized surfaces are specific towards the pheromone of the olive fruit fly Bactocera oleae. FIG. 5 shows change in frequency of the functionalized surfaces in response to various semiochemicals, according to an embodiment of the invention. The decrease in frequency of the functionalized surfaces only after the physical trapping with the pheromone shows specificity of the functionalized surfaces towards Bactocera oleae pheromone. Again the regeneration of the resonant frequency after keeping the devices for 24 h at room temperature specified the reversible use of the devices. The slow release of the pheromone from the ß-cyclodextrin cavity leads to the reversibility of the devices.
FIG. 6 shows change in resonance frequency and effective number of pheromone molecules attached to the functionalized surfaces, according to an embodiment of the invention. The functionalized surfaces show change in resonance frequency in response to the added mass due to attachment of pheromone molecules. The relationship between the natural frequency of functionalized surfaces and the added mass is as follows:

where k1 is the spring constant of the microstructures without any treatment, m1 is the mass of the microstructures without any added mass, f1 is the resonant frequency without the added mass and f2 is the resonant frequency with the added mass, ?m. This relationship is employed to detect the mass of the pheromones attached to the functionalized surfaces by measuring the change in the first order resonant frequency of the microstructures. It is clearly shown that increasing concentration of pheromone is very well sensed by the proportionate change in the frequency of the functionalized surface and the extent of functionalization is measured by the drop in the first mode of vibrational frequency. This is evident that with increasing concentration of pheromone, the resonant frequency decreased significantly as recorded by laser doppler vibrometry. It has also been confirmed that the longer the device, the higher is its sensitivity and higher the effective number of pheromone molecules attached to the functionalized devices.
FIG. 7 shows a plot for calculation of limit of detection of the functionalized cantilever, according to an embodiment of the invention. The limit of detection of these functionalized devices is found to be 0.25 femtogram of pheromone mass captured which is much below the concentration of the Bactocera oleae pheromones found at the time of pest infestation in an olive orchard.
The functionalized surfaces are embedded in electro-mechanical systems devices. One example of embedding the functionalized surface is a micro electro-mechanical systems device, hereinafter referred to as a MEMS device. Other examples include but are not limited to nanoelectromechanical systems (NEMS) devices. The MEMS device can be housed in a container. Examples of container include but are not limited to boxes, bottles, cans and canisters. The material from which holder is made includes but is not limited to metal, plastic, glass, acrylic and polycarbonate sheets.
FIG. 8 shows a schematic representation of an optical nanosensor, according to an embodiment of the invention. In one embodiment of the invention the MEMS device containing the surface functionalized surface, is housed in a rectangular box 1. The top wall 3 of the box 1 is provided with a means 5 for hanging. The side walls are provided with perforations 7(mesh size of 0.2 mm) so that air can circulate through the box 1. The functionalized surface 9 is detachably mounted to the inside surface 3a of the top wall 3. Air carrying pheromones enters the box 1 through the perforations 7 on the side walls. The functionalized surfaces have active anchor sites where the pheromones are physically trapped. The capturing of pheromones to the functionalized surface is reversible in nature. The capturing of the pheromones results in increase in mass of the functionalized surfaces. This increase in mass is sensed by a proportionate change in the frequency which is measured and monitored continuously.

INDUSTRIAL APPLICABILITY:
The MEMS devices housed in a container can be used in horticulture fields for early detection of pests. Timely use of the functionalized microstructures provided by the invention, help in the early detection of the pheromones and prompts early action against pests before major infestation. On the field each of the devices can be used several times over and over again after the detection. The common advantages of the optical nanosensor are:
• No harmful effects to beneficial insects, non-target organism or on environment.
• Help in monitoring and early detection of pests (at moth stage only).
• Helps in scheduling pest control measures.
• Localized treatment of infested area instead of applying pesticides/ insecticide all over field. This will reduce exposure of pesticides to the workers in the agricultural field as well as cost of application of pesticides.
• Simple to operate as no requirement of specialized training of the workers in the agricultural field for the use of the devices.
The invention provides a nanosensor which can selectively detect the presence of olive fruit flies in an olive fruit orchard and hence can be used as an alerting system to the farmers to take necessary actions only before infestation. This methodology will not only reduce the cost of farming, but also drastically diminish the chemical burden from the environment as the farmers will also be capable of applying the remedies in a localized manner. The invention specifically target the major pheromone component of the olive fruit flies for detection of them prior the infestation and hence covalently functionalize the MEMS devices with ß-cyclodextrin moieties which can form stable inclusion complexes with the particular pheromone. Pests are predominantly responsible for the loss of agricultural and horticultural produce. The invention provides for detection of pheromones at very low levels, thus providing an early and rapid detection of pheromones.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.