WO2016059641A1

WO2016059641A1 - A pheromone detector

Info

Publication number: WO2016059641A1
Application number: PCT/IN2015/000084
Authority: WO
Inventors: Bhattacharya Santanu; Pratap Rudra; Moitra Parikshit; Bhagat Deepa
Original assignee: Indian Institute Of Science
Priority date: 2014-10-17
Filing date: 2015-02-13
Publication date: 2016-04-21

Abstract

The invention provides a pheromone detector for early detection of pheromones. The detector comprises of a functionalized surface, a mount for embedding the functionalized surface and a perforated housing for replacably retaining the mount. After the detection of the pheromone, the user may be alerted of an impending pest attack and the user can then take appropriate pest control measures.

Description

A PHEROMONE DETECTOR

FIELD OF INVENTION

The present disclosure relates to the field of pest management and particularly a pheromone detector for early detection of pest infestation in fields growing agricultural and horticultural produce. BACKGROUND

Pests are predominantly responsible for the loss of agricultural and horticultural produce. The loss due to pest infestation is estimated to be in the range of about 20-40% of global crop yields. Examples of methods employed for controlling insect pests include but are not limited to fumigation, pesticide spray, bait traps, biological control, mating disruption, male annihilation and pheromone traps.

Significant disadvantage of the aforementioned methods are fumigants and pesticides are carcinogenic and mutagenic and accumulate in the food cycle. The bait spray method utilizes a protein hydrolysate and insecticide, which strongly attract insects. One significant disadvantage is the short life of the protein. Biological control uses an environmentally sound and effective means of reducing or mitigating pests and pest effects through the use of natural enemies. The biological control pests including but not limited to predators, parasitoids and pathogens have very limited host range and is costly to develop and manage. In mating disruption technique, synthetic pheromones are released in the air to confuse the male so that it has difficulty in locating female for mating. One significant disadvantage is that incorrect time of releasing pheromones will result in failure to disrupt mating.

Male annihilation technique involves luring the male and killing them, thus reducing the chances of insect's mating. Pheromone traps are used to attract the insects and trap them. One common limitation of these techniques is that pheromones are released continuously thus requiring periodic replacement of pheromones. Thus, there is need for an efficient detector that is capable of rapidly detecting low levels of pheromones that are released by insects 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 is 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 schematic representation of a pheromone detector, according to an embodiment of the invention. FIG. 3 shows the scanning electron micrographs (SEM) of cantilevers and fixed-fixed beams after each of the functionalization steps, according to an embodiment of the invention.

FIG. 4 shows changes in frequency of the cantilevers and fixed- fixed beams with increasing concentration of pheromones, according to an embodiment of the invention.

FIG. 5 shows percentage change in frequency after the functionalization steps and effective number of pheromone molecules attached to the functionalized cantilevers and fixed- fixed beams, according to an embodiment of the invention.

FIG. 6 shows a plot of change in frequency with respect of change in mass of the devices, according to an embodiment of the invention.

FIG. 7 shows specificity of the functionalized devices towards the specific insect pheromone, according to an embodiment of the invention. SUMMARY OF THE INVENTION

The invention provides a detector for rapid detection of pheromones in the field. The pheromone detector comprises 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 a device for early detection of pheromones in a field. Pheromones are semiochemicals which are used as signals for communicating with the members of same species. Whenever there is a pest attack in a field, first few female pests arrive at the site and send signals to other male members of the species. The levels of pheromone remain at a low level at that point of time. The pheromone detector provided in the invention detects very low levels of pheromones and the user can be alerted of an upcoming pest attack. The user can then take appropriate pest control measures to alleviate crop losses.

One embodiment of the invention provides a pheromone detector for early detection of pheromones in a field. The detector comprises of 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 surface functionalization is chosen as the method for obtaining functionalized surfaces (FIG. 1 ). FIG. 1 is 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 m thickness (type P, dopant boron, orientation <100>, resistivity 0-100 ohms) is taken and cleaned well with piranha solution (F^SO^F C^ = 9:1 ) for 5 min and washed twice with distilled water to remove organic and metallic contaminants from the surface. A 1 μιη layer of silicon dioxide is then thermally grown over it and the resultant surface is again cleaned with piranha solution.

The cleaned wafer is first dehydrated at 250 °C for 10 min, spin- coated with a photoresist (AZ5214E), of nearly 1.5 pm thickness at 6000 rpm for 40 sec and baked at about 1 10 °C 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 AZ351 B:H₂0 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 °C 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-CI) 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 μηπ respectively. Similarly, the fixed-fixed beams 1 , 2, 3 and 4 have lengths of 53.4, 35.5, 27.1 and 21.3 μιη respectively. The cantilevers have uniform width of 5.1 pm, whereas the fixed beams have uniform width of 4.7 μπτ Each structure has a uniform thickness of 1.04 μιτι.

The microstructures obtained are then functionalized with an optimum concentration of (3-aminopropyl)triethoxysilane. Functionalization with the (3-aminopropyl)triethoxysilane reagent results in the formation of active anchor sites on the surface of the microstructures. The semiochemicals having aldehyde or ketone functionality attach covalently at these active anchor sites. The example of semiochemical includes but is not limited to pheromones, specifically released by the female insects, Helicoverpa armigera (Hubner) and Scirphophaga incertulas (Walker).

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. 2 shows a schematic representation of a pheromone detector, according to an embodiment of the invention. In one embodiment of the invention the MEMS device containing the surface functionalized specifically for the covalent capture of pheromones, having aldehyde or ketone functionality, is housed in a rectangular plastic box 201 . The top wall 203 of the box is provided with a means 205 for hanging the device. The side walls are provided with perforations 207(mesh size of 0.2 mm) so that air can circulate through the box 201. The functionalized surface 209 is detachably mounted to the inside surface 203a of the top wall 203, Air carrying pheromones enters the detector through the perforations 207 on the side walls. The functionalized surfaces have active anchor sites where the pheromones are covalently attached. The covalent attachment of pheromones to the functionalized surface is reversible in nature. The attachment 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.

FIG. 3 shows scanning electron micrographs (SEM) of cantilevers and fixed-fixed beams after each of the functionalization steps, according to an embodiment of the invention. FIG. 3(a) represents the piranha cleaned silicon dioxide surface, 3(b) represents the (3- aminopropyl)triethoxysilane functionalized silicon dioxide based cantilevers (at left) and fixed-fixed beams (at right). FIG. 3(c) shows effect of the covalent attachment of the pheromones to the active oxide surface, whereas FIG. 3d shows the control experiment with the semiochemicals having no aldehyde or ketone functionality.

The (3-aminopropyl)triethoxysilane functionalized devices are tested for their interaction with the various pheromones. FIG. 4 shows change in frequency for the cantilevers and fixed-fixed beams with increasing concentration of the specific pheromones, according to an embodiment of the invention. The figure shows the interaction of the cantilevers and fixed-fixed beams with one of the female sex pheromone of a particular insect, Helicoverpa armigera (Hubner). The covalent attachment of the pheromones with the functionalized devices is characterized by determining the change in the base frequency of the cantilever and fixed-fixed beams. The change is frequency is estimated from the general formula (1 ):

where k_t is the spring constant of the microstructures without any treatment, m-i is the mass of the microstructures without any added mass, ^ is the resonant frequency without the added mass and f₂ is the resonant frequency with the added mass, Am. This relationship is employed to detect the mass of the semiochemicals attached to the functionalized surface of the microstructures by measuring the change in the first resonant frequency of the microstructures. It is clearly shown that increasing concentration of semiochemicals is very well sensed by the proportionate change in the frequency of the microstructures 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.

Sensitivity: The sensitivity of the surface functionalized oxide surfaces is measured through laser doppler vibrometry. FIG. 5 shows the percentage change in frequency with respect to their original frequency after the functionalization steps and effective number of pheromone molecules attached to the functionalized cantilevers and fixed-fixed beams, according to an embodiment of the invention. It is observed that cantilever 2 having the length of 36.5 m is the most sensitive one among all the microstructures studied as shown in FIG. 5. The cantilever 2 also captures more number of pheromone molecules than other microstructures at the same concentration of pheromones. The possibility of larger deflection of the cantilevers, having one free end, than the fixed-fixed beams, having two attached ends, may have given greater sensitivity at the same concentration of semiochemicais.

FIG. 6 is a plot of change in frequency with respect of change in the mass of the devices, according to an embodiment of the invention. The plot shows a proportionate change in frequency with change in the mass of the functionalized surfaces of the microstructures which is independent of the length, width or thickness of the functionalized devices.

Specificity: The functionalized surfaces are specific towards the aldehyde and ketone group of insect pheromones and do not interact with other interfering moieties such as alcohol, amines, acid, simple aliphatic chains and kairomones of specific plant leaves and stems, e.g. tomato, cotton etc. FIG. 7 shows specificity of functionalized devices towards the particular insect pheromone molecule, according to an embodiment of the invention. The change in frequency is observed to be maximum with insect pheromone having free aldehyde functionality (e.g. female insect pheromone of Helicoverpa armigera) showing that the cantilevers and fixed-fixed beams are specific towards this insect pheromones.

Coverage: The sensitivity of detection is also assessed by estimating the area covered by a single pheromone detector. The limit of detection and the limit of quantification for the pheromone detector are estimated to be -2.7 fg/mL and ~8.2 fg/mL, respectively. The pheromone detector is hung through the hanging means provided on the detector. The height at which the pheromone detector is hung is approximately one foot above the crop level. The concentration of pheromone released by insects per acre per hour is estimated to be ~0.195 fg/mL. In a volume of 1 acre- foot, only fourteen pheromone detectors are needed to cover nearly one acre of the field to detect the incidence of pest infestation. Similarly in a volume of 3 acre-foots, the density of pheromone released by insect per acre per hour is estimated to be approximately 0.065 fg/mL. Hence only forty two pheromone detectors are needed in the field to detect the incidence of pest infestation.

The invention thus provides a pheromone detector which is specific towards insect pheromones having aldehyde or ketone functionality. The invention provides for detection of pheromones at very low levels, thus providing an early and rapid detection of pheromones.

INDUSTRIAL APPLICABILITY: 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. In general, ~14 devices per acre are recommended for monitoring of pest incidence in a volume of 1 acre-foot. These are arranged such that the trap is ~1 foot away from the crop level. On the field each of the devices can be used several times over and over again after the detection, with the use of simple steps involving common chemicals. The common advantages of pheromone detector 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 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.

Claims

We claim:

1. A pheromone detector for early detection of pheromones, the pheromone detector comprising,

a functionalized surface;

a mount for embedding the functionalized surface; and

a perforated housing for replacably retaining the mount.

2. The pheromone detector of claim 1 , wherein the

functionalized surface is an oxide surface having free hydroxyl group.

3. The pheromone detector 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, aluminium dioxide and iron oxide.

4. The pheromone detector 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.

5. The pheromone detector of claim 1 , wherein the

perforated housing is a box, a bottle, a can or a canister.

6. The pheromone detector 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.

7. The pheromone detector of claim 1 , wherein the detection is specific to the pheromone having a carbonyl functional group.

8. The pheromone detector of claim 1 , wherein the detection is achieved by measuring the change in the resonance frequency.

9. The pheromone detector of claim 8, wherein the change in the resonance frequency is due to change in the mass of the embedded functionalized surface.

10. The pheromone detector of claim 1 , wherein the detector is placed proximal to an agricultural and/or a horticultural produce having high risk of infestation.