Reusable and Field-Deployable Nanobiocatalysts for Detection of Pesticides and Herbicides
Primary Information
Domain
Nano-Technology Hardware
Project No.
6349
Sanction and Project Initiation
Sanction No: 6349
Sanction Date: 10/01/2017
Project Initiation date: 10/01/2017
Project Duration: 36
Partner Ministry/Agency/Industry
Department of Science and Technology
Role of partner:Funding partner
Support from partner:Partner will provide funding to the tune of 50% of total budget (not realized so far).
Principal Investigator
Dr. Bhaskar Datta
Indian Institute of Technology Gandhinagar
Host Institute
Co-PIs
Dr. Arnab Dutta
Indian Institute of Technology Gandhinagar
Dr. Sharad Gupta
Indian Institute of Technology Gandhinagar
Scope and Objectives
To develop and produce nanomaterials with immobilized biocatalysts that are: (1) capable of generating distinctive and direct colour changes in presence of commonly used amino acid-derivative pesticides and herbicides, and (2) reusable across multiple samples separated by space and time, either in reagent or in strip-based formats, and (3) field-deployable in a simple format of use without constraints of specialized handling or need of special skill/ knowledge.
Deliverables
Colour change effected by nanobiocatalyst strip in response to presence of specific pesticides and herbicides can be read by the naked eye without visual aids or special sources of illumination. Colour development is within a few minutes, less than 2 mins, of sample application, and persists for a few hours afterwards. The nanobiocatalyst strip can operate at ambient temperatures spanning 5 to 45 degrees C. Mediator solutions necessary for the colorimetric assay are provided separately in pre packed containers. The nanobiocatalyst strips are reusable across at least 10 different samples or trials without any restriction on the down time between those analyses.
Scientific Output
Colorimetric determination of commonly used herbicides and pesticides is not widely accessible. We have designed and developed reusable magnetic nanoparticle with immobilized enzyme for simple colorimetric assessment of glyphosate. Glyphosate is the active component in RoundUp, the most widely used herbicide in the world. We have developed protocols for the synthesis of nanoparticles and immobilization with enzyme that provide consistent results in terms of the resultant nanobiocatalyst function. Three main aspects of the colorimetric scheme have been successfully optimized. These include (1) identification of suitable nanoparticle composition or identity and linker lengths for immobilization of enzyme, (2) examination of effects of pH and optimization of colorimetric assay based on this examination, and (3) comparison of the assay for selectivity and sensitivity against target compounds. We have filed a patent that covers various parts of our effort to identify presence of specific herbicides and pesticides such as glyphosate in a visual assay format. A variety of nanoparticles and nanoconjugates have been prepared and tested till now; a specific linker length and linker have been identified as being optimum for enabling immobilized enzyme activity. Work performed in the project towards optimization of nanobiocatalysts has been submitted and is currently under review in BMC Journal of Nanobiotechnology. We have pursued a detailed investigation of effects of pH and other experimental variables in the enzyme-mediated assay for glyphosate. The key principles being used in our work include (a) potential range of the enzyme laccase, and (b) ability of specific mediator small molecules to tune this potential range. To this end, we have investigated and identified specific mediator that is capable of reacting with glyphosate bringing about a colour change in the process. This effect is specific not only to the glyphosate (as analyte being tested) but also in terms of the mediator being used as accessory reagent in the process. We are currently in the process of using other mediators which can effect similar changes on other herbicides and pesticides. Finally, a comparison of our visual assay has been performed with respect to different analytes and potentially interfering components. Optimized protocols that preclude interference in the colorimetric results have been developed. This work is currently being written up for submission to Scientific Reports.
Results and outcome till date
We have now firmly validated the ability of our immobilized laccase to not only detect but provide quantitatively accurate estimation of amount of glyphosate present in samples. In this regard, we modified a reported approach for chemical derivatization of glyphosate followed by chromatographic separation and quantification. This method of Catrinck et al. has been adopted with some modifications. The method relies on derivatization of glyphosate with the commonly used protecting agent Fmoc-Cl. Briefly, three samples were prepared. Sample A corresponded to Fmoc-Cl in the reaction medium without glyphosate. Sample B corresponded to Glyphosate in the reaction medium without Fmoc-Cl. Sample C corresponded to Glyphosate in the reaction medium with Fmoc-Cl. Samples A-C were transferred to round bottom flasks and homogenized by vortexing for 30 minutes. Subsequently, they were washed with diethyl ether (twice). 750 uL of the derivatized compound was diluted into different concentration (10 ppm, 20 ppm, 50 ppm, 100 ppm and 200 ppm) in borate buffer and analyzed by HPLC using the method of Wang et al. Briefly, mobile phase components were 5 mM ammonium acetate (adjusted with aqueous NH3 to pH 9.0) and methanol. The percentage of methanol was changed linearly as follows for gradient elution: 0 min, 20%; 3 min, 20%; 6 min, 70%; 18 min, 70%; 23 min, 20%. The column was re-equilibrated for 2 min, resulting in a total run time of 25 min at a flow rate of 0.5 mL per min. RP-C18 column was used, injection volume was 20 uL and the column was maintained at room temperature. Calibration plots were created using peak area in case of chemical derivatization-HPLC method and absorbance value in case of our approach. One unknown concentration of glyphosate was determined by both the approach. Our laccase mediated system based visual detection of glyphosate was validated by use of the reported method of glyphosate detection by HPLC via Fmoc-derivatization. While the HPLC method provides a linear calibration for glyphosate concentration, the laccase mediator system bears an exponential calibration with respect to glyphosate concentration (Figure 1A, B and C). Based on these, an unknown glyphosate concentration could be determined with 98% accuracy using the visual detection method.
Societal benefit and impact anticipated
Unfettered use of pesticides and herbicides has sought to address increasing demands on agri-cultural production. However, their impact on the environment and on human health has pro-gressively emerged as a matter of grave concern. The complex nature of food storage and distribution necessitates ease-of-use in techniques for monitoring and detection of contaminants. Colorimetric sensor strips are at the forefront of simple and sensitive assays adaptable into commercially viable products. Colorimetric determination of commonly used herbicides and pesticides is not widely accessible. We have designed and developed reusable magnetic nanoparticle with immobilized enzyme for simple colorimetric assessment of glyphosate. Glyphosate is the active component in RoundUp, the most widely used herbicide in the world.
Next steps
Field trials of the strategy adopted are in full swing; analysis of real samples has begun and these tests are being scrutinized thoroughly so as to troubleshoot interfering outcomes and false positives. The main points of optimization in field trial thus far is the use of reagents in kit bottles, need for addition of precise amounts of reagents, time taken for colorimetric test completion and reusability of developed reagents. It is expected that a field trial prototype in the form of paper or membrane strip will be able to avoid several disadvantages associated with use of reagent bottles and kits; work is under way currently to fulfill this objective.
Publications and reports
Kumar, S.; Maurya, V.; Gadhavi, J. and Datta, B. (2018) Nanoparticle and linker optimization for immobilized enzyme activity. BMC Journal of Nanobiotechnology (under review); Kumar, S.; Maurya, V. and Datta, B. (2018) Laccase mediated detection of glyphosate. Scientific Reports (under review).
Patents
Kumar, S.; Sharma, P. and Datta, B. (2017) Nanobiocatalyst for Detection of Organophosphorus Herbicide, Application No. 201621013049, Complete Specification: 12/04/2017, Indian Patent Office.
Scholars and Project Staff
2 PhD + 1 Post-Doctoral fellow
Challenges faced
Awaiting fund release to expedite work on remaining objectives
Other information
Delay in receipt of funds is likely to delay thorough field assessment of strategy and conclusion of project.
Financial Information
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Total sanction: Rs. 5640000
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Amount received: Rs. 3776422
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Amount utilised for Equipment: Rs. 0
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Amount utilised for Manpower: Rs. 400339
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Amount utilised for Consumables: Rs. 2300279
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Amount utilised for Contingency: Rs. 92311
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Amount utilised for Travel: Rs. 25473
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Amount utilised for Other Expenses: 0
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Amount utilised for Overheads: Rs. 640000
Equipment and facilities
N/A