On-Field Detection of Helicoverpa armigera Nuclear Polyhedrosis

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On-Field Detection of Helicoverpa Armigera Nuclear Polyhedrosis Virus using Luminescent Amphiphilic probe: Screening of Agricultural Crops and Commercial Formulations Nilanjan Dey, Deepa Bhagat, and Santanu Bhattacharya ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06152 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019

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ACS Sustainable Chemistry & Engineering

On-Field Detection of Helicoverpa Armigera Nuclear Polyhedrosis Virus using Luminescent Amphiphilic probe: Screening of Agricultural Crops and Commercial Formulations Nilanjan Dey, [a] Deepa Bhagat,[b] and Santanu Bhattacharya*[a], [c] a

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India

b

Indian Council of Agricultural Research, National Bureau of Agriculturally Insect Resources, Bangalore 560 024, India c

Present Address: Indian Association of Cultivation of Science, Kolkata 700032, India Email: [email protected], Fax: (+91) 080-2360-0529

KEYWORDS. Biopesticide, Fluorimetric sensing, Commercial formulations, Agricultural Crops, Paper discs.

ABSTRACT. An easy to synthesize luminescent, amphiphilic probe has been designed for the first time in optical sensing of a biopesticide, Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV). The compound showed formation of a pH-sensitive, thermoreversible nanoaggregate in the aqueous medium. The addition of HaNPV resulted in the rapid change in emission color from blue to cyan at pH 7.4. Till date, no optical assay is known in the literature, which can achieve fast, on-field detection of HaNPV as low as ~103 POBs/mL. Most importantly, due to naked-eye response, this method does not need the involvement of trained personnel or any sophisticated

ACS Paragon Plus Environment

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visualizing instrument. In addition, the distinct optical response also allows us to distinguish the freshly prepared pesticide solution from its storage-old analog. The quantification of HaNPV is achieved in a wide-range of agricultural crop extracts, known to be infected by Helicoverpa armigera. Inexpensive reusable paper strips are developed for on-location detection purpose. Moreover, the presence of residual HaNPV can also be traced on leaf-surfaces. Thus, studies of such kind will be beneficial for quality verification of HaNPV and will surely add a new dimension to the better management of Helicoverpa armigera and minimize the extent of crop loss.

INTRODUCTION The economic growth (13.7% of total GDP) of India mostly depends on the prospect of its agriculture. Apart from fulfilling the food requirement, it also provides employment to more than 60% of Indian population.1 However, every year loss of agricultural crops due to pest attack is one of the major challenges faced by the farming community in India. Among different insect pests, Helicoverpa armigera (Hubner) is one of the most harmful and polyphagous pest species, known to affect more than 300 plant species including tomato, cotton, legume sunflower, groundnut, wheat, tobacco, corn and a range of vegetables, fruit crops and tree species. 2 In the Indian subcontinent, the insect shows high reproductive rate (i.e., 8-11 generations per year) with excellent adaptability towards the adverse weather conditions. 3,4 Moreover, H. Armigera is also reported to develop resistance against several commercial insecticides, like endosulfan, profenphos, thiodicarb, bifentrin, alphacypermethrin and cyfluthrin etc.5,6 Thus, as the part of integrated pest management program (IPM), people are now interested in using microbial pathogens (such as, bacteria, virus etc.) as the environmentally friendly alternatives to control these hazardous pests. Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV) is one of such biopesticides (belongs to the class baculovirilae) known to be effective against H. armigera. The


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main advantage of using this biopesticide over chemical pesticides is that it is inherently non-toxic and can result in minimum environmental pollution. Unlike chemical pesticides, this virus is highly host-specific and non-pathogenic to other beneficial insects as well as other non-target organisms such as human, domestic animals, fishes etc. 7,8 The structure of HaNPV is more complicated than most of the other viruses, the viral particles are generally encapsulated by a polyhedrin protein layer. This outermost protein overcoat and everything within it, is called “occlusion body” (Fig. 1a). As the occlusion bodies are the actual structural units which transmit during larval infection, EPA has registered the occlusion bodies of HaNPV as the pesticide active ingredient.9-12 For the large-scale commercial production of HaNPV, it is necessary to avoid the contamination of final product by other foreign insect pathogens, which often reduces the productivity of HaNPV and poses a hazard to the production staff or end users.13,14 Additionally, the quality of HaNPV production also depends on the procedure followed during their culture, nature of the water (ionic strength, pH) used during preparation etc.15.16 Thus before actual bioassay or on-field applications, it is essential to know whether the desired virus (HaNPV) is being produced in the sufficient amount or not. Till now, the counting of virus particles (OBs) can only be achieved using a hemocytometer with improved neubauer. However, this light microscopy-based method has several disadvantages.17 For example; the use of poor-quality microscopes with non-phase optics can add errors in counting. The virus suspension should be pure for microscopic analysis to minimize the false positive results due to the presence of other polyhedra like particles (for example dust or tiny oil droplets). Moreover, the suspension should be homogeneous for analysis (no clumping or aggregated particles). On the other hand, expensive caring facilities and skilled personnel are required for the maintenance of the microscopy instrument. Thus, there exists a huge urge among the scientist to


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(a)

3.0

Split pigeon peas Red lentils Split chickpeas 2.5 methodsWheat for rapid Cloves

(b) 3.0

and

Mango Banana cost-effective2.5quantification Cucumber Apple

F0/F at 394 nm

F0/F at 395 nm

design new optical

2.0 commercial formulations.

of HaNPV in the

-SLNPV -SLNPV-SLNPV-SLNPV-SLNPV (b)

1.5

1.5

Occlusion Occlusion Body BodySolution phase 1.0 detection -SLNPV

5.0x105

0.0

1.0x106

1.5x106

F. I. (a. u.)

Cabbage 60 Oat Cardamom 2.5 50 Cumin Black Pepper 5 40 Okra 2.0 Brinjal Helicoverpa armigera 30

F0/F at 395 nm

Pest

3.0

1.5

Blank 3.3 x 105 POBs/mL 6.6 9.9 13.2 16.5 19.8 23.1 26.4 x 105 POBs/mL

60 60 60 1.0

60

1.5x106

Compound 1

0.6

Flexible conformation I 410 nm/I 512 nm

0.6

Ratiometric Sensing

0.5

0.4

On-site rapid detection Blank 0.2With 1 0.4

20 MICE: Motion-Induced Change in Emission

60

6 1.0x10 0.8

HaNPV added (POBs /mL)

HaNPV (d) MICE µM

0.3

0.0 350

400

Equation

y = a + b*x

Pearson's r

0.1

0.97809 Value

Figure 1. (a) Schematic0.0diagram release1.5x10 of Helicoverpa armigera Nuclear Polyhedrosis 5.0x10shows 1.0x10 0 5

6

6

400

450

500

550

600

0.0

6

0.0056

0.99037

106

105

550

No Weighting

Adj. R-Square

104

500

Wavelength (nm)

Residual Sum of Squares

H. Armigera

450

Restricted conformation 0.2 Weight

10

Probe 1 + HaNPV

+SLNPV

5.0x105

0.0

HaNPV Added (POBs /mL)

(c)

1.0

Normalized F. I.

1.0

+SLNPV

2.0

F. I. (a. u.)

(a)

D

Intercept

D

Slope

Standard Error

0.09584

0.01739

2.09481E-7

1.10695E-8

6.0x105 1.2x106 1.8x106 2.4x106

/mL) Virus (HaNPV) occlusion HaNPV bodies added (OBs)(POBs fromWavelength deceased(nm) pests. (b) Structure of amphiphilic

Detection of HaNPV on leaf surface Conc. of SLNPV (POBs/mL)

I 410 nm/II512410I 410nmnmnm/I/I512512410 Blank Blank Blank BlankBlank I /I 0.6ometri 0.6c Sensi0.6Rati 0.6ng ometriRatiRatcoiSensi 5 5 5 0.6 5 5 Rati ometri metRari Considering this, herein we have employed an easy-to-synthesize carbazole-based fluorescent 3.3 x 10 POBs/mL 3.3 x 10 3.3 POBs/mL x 10 3.3 POBs/mL x 3. 10 3 x 10 POBs/mL POBs/ m L 50 50 50 50 50 probe for ratiometric optical sensing6.6 of HaNPV both6.6at physiological 6.6 6.6 6.pH6 in water and in different commercial formulations (Fig. 1b). The (1) possesses benzimidazole moieties 9.9 compound 9.9 9.9 9.9flexible 9. 9 0.5 0.5 0.5 0.50.5 at the 3,6-positions of the carbazole unit and hydrophilic piperazine unit at the nitrogen end. To 13.2 13.213.2 40the best of our40knowledge, 40 40this is the13.240first attempt13.2of quantifying in the aqueous 16.5 16.5 16.5 16.5a 16.biopesticide 5 medium by involving a small molecule-based optical probe. Differentiation of freshly prepared 19.8 19.8 19.8 19.80.419.8 0.4 0.4 0.40.4 HaNPV from its storage-old counterpart was achieved as well. The low-cost, reusable paper strips 23.130 detection23.1purpose. 23.1 Moreover, 23.123.1 the presence of HaNPV 30were also developed 30 30for rapid, 30 on-location 5 5 5 5 5 26.4 x 10 POBs/mL 26.4 x 10 26.4 POBs/mL x 10 26.4 POBs/mL 26. x 10 4 x 10POBs/mL POBs/extracts. mL was also detected on the surface of various leafy materials and in their aqueous


0.2

F. I. (a. u.)

F. I. (a. u.)

20

F. I. (a. u.)

20 20 20

0.3

F. I. (a. u.)

20

F. I. (a. u.)

EXPERIMENTAL SECTION:

F. I. (a. u.)

F. I. (a. u.)

F. I. (a. u.)

F. I. (a. u.)

compound 1 involved in the present study (MICE = motion induced changes in emission). 410 nm 512 nm

F. I. (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.3 0.3 0.30.3 0.2 0.2 0.20.2 4

Equation

y = a + b*x

Weight

No Weight


0.0

Pearson's r

0.99

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Material and methods: All reagents, solvents, and silica gel for TLC and column chromatography were obtained from the best-known commercial sources and were used without further purification. FTIR spectra were recorded on a Perkin-Elmer FT-IR Spectrum BX system and were reported in wave numbers (cm−1). 1H-NMR and

13

C-NMR spectra were recorded with a Bruker

Advance DRX 400 spectrometer operating at 400 and 100 MHz for 1H and 13C NMR spectroscopy, respectively. Chemical shifts were reported in ppm downfield from the internal standard, tetramethylsilane (TMS). Mass spectra were recorded on Micro mass Q-TOF Micro TM spectrometer. Fodder was cultivated on the farm at the ICAR-National Institute of Animal Nutrition and Physiology (ICAR-NIANP) without involving any pesticides. The vegetables, fruits, and cereal samples were collected from organically grown farms. UV-visible and fluorescence Experiment: The UV−vis and fluorescence spectra were recorded on a Shimadzu model 2100 UV-vis spectrometer and Cary Eclipse spectrofluorimeter respectively. In the emission experiments, the slit widths for both the excitation and emission channel were kept 5 nm and the excitation wavelength was 350 nm. To monitored the effect of pH, sensing experiment were performed in buffered media of different pH (HCO 2Na/ HCl buffer for pH 2, Tris/HCl for pH 7 and Na2B4O7·10H2O/NaOH for pH 12). Again, during temperature dependent experiments, 5 min of incubation was given in each case for thermal equilibrium. Every measurement was done in replicates to check the reproducibility of the system. Fluorescence Decay Experiment: Fluorescence lifetime values were measured by using a timecorrelated single photon counting fluorimeter (Horiba Jobin Yvon). The system was excited with nano-LED of Horiba - Jobin Yvon with pulse duration of 1.2 ns. Average fluorescence lifetimes (τav) for the exponential iterative fitting were calculated from the decay times (τ i) and the relative amplitudes (ai) using the following relation,


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τav = (a1τ12+a2τ22 +a3τ32)/(a1τ1+a2τ2+a3τ3) Where a1, a2 and a3 are the relative amplitudes and τ 1, τ2, and τ3 are the lifetime values, respectively. For data fitting, a DAS6 analysis software version 6.2 was used. Circular Dichroism (CD): All the CD spectra were recorded on a JASCO instrument, Model J815-150S. Experiments were performed by purging dry N2 gas continuously. Data were collected in a quartz cuvette of 1 mm path length. Dynamic Light Scattering and Zeta Potential: Measurement DLS measurements were performed at room temperature using a Malvern Zetasizer Nano ZS particle sizer (Malvern Instruments Inc., Westborough, MA). Samples were prepared and examined under dust-free conditions. Mean hydrodynamic diameters reported were obtained from the Gaussian analysis of the intensity-weighted particle size distributions. TEM Analysis: TEM samples were made using the drop coating method from compound 1 (10 µM) in pH 6.5 buffer medium. Also, TEM images of HaNPV and 1 + HaNPV conjugate were recorded in pH 7.4 buffer medium. TEM images were collected using a JEOL field emission Transmission-Electron-Microscope JEM-2100F under 80KV working voltage. Atomic Force Microscopy: Solution of compound 1 (10 µM in water pH 6.5) were drop-coated on mica and then carefully air-dried. Each sample was analyzed using a Bruker diInnova SPM instrument: Tapping mode, 10 nm tip radius, silicon tip, 292 KHz resonant frequency, 0.7-1 Hz scan speed, 256 × 256 and 512 × 512 – pixels. Real-life sample analysis: The agricultural crops were thoroughly washed with deionized water to remove any dirt and chopped into pieces (especially the vegetables, fruits, and fodders). In almost every case, about 1 g of the processed samples (cereals were weighed as they were) were mixed with pH 7.4 buffer (10 mL) and sonicated at 50 oC for 1 h. Then, the mixtures were subjected


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to centrifugation at 1000 rpm for 10 min and the clear supernatants were used for fluorimetric analysis. During studies, the diluted solution of each supernatant (20% in pH 7.4 buffer) was mixed with the 1. The emission spectra were recorded both with and without the addition of HaNPV. The detection limit in each case was calculated by using blank variation method. Preparation of paper discs for sensing: To prepare the compound-coated paper strips, 40 µL methanolic solution of 1 (0.02 mM) was drop cast onto the filter paper using micropipette to form a spherical luminescent spot of diameter ~1.5 cm. The concentration of 1 in the solution (pH 7.4) as well as immersion time were crucially maintained in order to avoid aggregation (cyan emission) to begin with. The solution was completely absorbed in filter paper within 15 minutes and then the filter papers were kept overnight for air dry. Finally, the air-dried paper strips were ready for sensing studies. RESULTS AND DISCUSSION Preparation as well as Characterization of Nanoaggregates: Since the carbazole-based amphiphilic probes are known in the literature for exhibiting excellent solid-state luminescence, we were intrigued to investigate the nanoclustering behavior of compound 1 in the buffered medium (pH 6.5). In non-polar/aprotic solvents like THF or dioxane, the probe exhibited moderate emission with highly-structured monomer bands at ~395 nm. However, in the water-rich environment (Tris-HCl buffer, pH 6.5), a ~70 nm redshift in the emission maxima was observed due to the formation of close-contact nanoaggregates (Fig. 2a). The aggregate formation was also evidenced by both dynamic light scattering experiment as well as conventional microscopic imaging techniques (Fig. 2a & S1). Emission studies at different pH condition indicates that the extent of inter-chromophore association is pH-dependent and most prominent at the pH range 4.56.5 (Fig. 2b). The protonation states of benzimidazole as well as piperazine residues probably


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Fluorescence (a. u.)

F.I. 460 nm/F.I. 395 nm


Intensity (counts)

F. I. (a. u.)

1 2 3 dictate the overall amphiphilic nature of the compound, which eventually control the extent of self4 5 aggregation. For instance, below pH 4.5, both benzimidazole and piperazine units exist in the 6 7 protonated state, which probably enhance the hydrophilic nature of the compound and reduce the 8 9 10 extent of agglomeration. Similarly, the extent of aggregation was found to be less at alkaline pH 11 12 (even at physiological pH 7.4) due to shift in the protonation equilibrium in the reverse direction. 13 14 Further, to verify the above conjecture, we have also determined critical aggregation concentration 15 16 17 of 1 at various pH (Table S1). 18 19 (a) 400 (b) 1.0 20 1 in THF 21 Water THF 300 0.8 22 Compound in water in THF 23 0.6 Water THF 200 600 24 0.4 Compound in water 25 in THF 500 TEM and AFM images of 6.5 1 100 26 1 at pH 0.2 27 400 0.0 28 0 4 6 8 10 350 400 450 500 TEM 550and AFM 600 images 650 of 1 2 29 300 pH of the medium 30 Wavelength (nm) 200 31 (c) 150 (d) 10000 40 480 520 560 600 32 oC velength (nm) 100 At 85 33 In THF 120 1000 34 0 In water (pH 6.5) 360 400 440 480 520 560 600 35 90 Wavelength (nm) 36 100 At 15 oC 37 60 38 10 39 30 40 1 41 20 40 60 80 100 350 400 450 500 550 600 42 Time (ns) Wavelength (nm) 43 44 Figure 2. (a) Fluorescence spectra of 1 (10 µM, ex = 350 nm) in buffered medium (Inset shows 45 46 TEM images of 1). (b) Effect of pH on the emission response (I460 nm/I 395 nm) of 1. (c) Effect 47 48 of temperature on the ratiometric variation of emission intensity (I460 nm/I 395 nm) of 1 (each 49 50 spectrum was recorded at 10 oC interval). (d) Time-dependent fluorescence spectra of 1 in THF 51 and buffered medium (pH 6.5). 52 53 54 55 56 57 58 59 ACS Paragon Plus Environment 60

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In addition, the temperature variation studies at pH 6.5 suggested that the nanoassembly formation is highly thermo-reversible in nature (Fig. 2c). On rising the solution temperature from 15 to 85 o

C, a decrease in fluorescence intensity was observed at ‘aggregate’ emission band (em = 472

nm) with concomitant increment in the ‘monomer’ fluorescence (em = 395 nm). Similarly, regenration of aggregate emission band was noticed when the solution temperature was brought back to 15 oC. Since cooling is also known for the retardation of intramolecular rotation process, we recorded 1H-NMR spectra of 1 at two different temperatures (10 and 80oC) (Fig. S2). At high temperature, the fast-conformational exchange caused by the rapid intramolecular rotation resulted in the formation of sharp peaks, whereas slow conformational exchange at low temperature led to broadening of NMR signals. The time-dependent fluorescence studies indicated that the presence of monomer species in THF medium with faster decay rate, while in the aqueous medium (at pH 6.5), self-aggregation restricts the twisting motion of the flexible terminal groups and stabilizes the compounds at excited state (Fig. 2d). This resulted in the formation of long-lived emissive species with multi-exponential decay pathways.18,19 Similarly, formation of redshifted emission maxima was also observed in presence of glycerol, which is however due to motion-induced fluorescence enhancement (Fig. S3a).20,21 Again, the effect of glycerol on fluorescence intensity of 1 was also checked over a wide range of pH (Fig. S3b). Interaction of compound with biopesticide: After thoroughly characterizing the aggregation property of 1 in the buffered medium, we proceeded to explore its biopesticide (HaNPV) sensing efficiency at physiological pH (pH 7.4) in PBS buffer. The fluorescence spectrum of 1 at pH 7.4 was mostly dominated by monomeric emission bands (max = 395 nm) with blue fluorescence. The addition of HaNPV in this condition resulted in an immediate change in emission color from blue to cyan (observed under 365 nm UV lamp) (Fig. 3a). Titration studies showed a concentration-


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F/F0 at 395 nm

410 nm

F. I. (a. u.)

5

F. I. (a. u.)


1 2 3 dependent (0 – 7.35 x 105 POBs/mL) quenching of fluorescence intensity at 395 nm band with 4 5 concomitant increment at 470 nm. Moreover, it also indicates that detectable signal to noise ratio 6 7 can be obtained in presence of HaNPV as low as 2.82 x 103 POBs/mL (Fig. 3b).22,23 Since 8 9 10 reversibility is also an important criterion for an ideal sensory system, we have checked whether 11 12 the same probe solution can be used multiple times for the detection of HaNPV or not. For this, 13 14 we first allowed the compound to interact with HaNPV at pH 7.4 (a change in emission color from 15 16 17 blue to cyan). Then the pH of probe-HaNPV mixture solution was lowered to 4.5 (cyan emission). 18 19 At this condition, the preformed HaNPV-conjugate was found to be unstable in the solution and 20 21 HaNPV came out as white precipitate (presence of free probe was determined in the supernatant). 22 23 24 Then the precipitate was separated by centrifugation and the pH of the probe solution was adjusted 25 26 again to 7.4 (blue emission) for further HaNPV sensing (Fig. 3c & S4). 27 -SLNPV +SLNPV 28 (a) 250 (b) 1.0 29 30 0.8 200 31 60 0.6 32 I /I Blank 0 POBs/mL 0.6 15050 Ratiometric Sensing 3.3 x 10 POBs/mL 33 6.6 0.4 9.9 0.5 34 10040 13.2 16.5 35 0.2 19.8 0.4 23.1 7.35 x 105 POBs/mL 36 5030 26.4 x 10 POBs/mL 0.3 0.0 37 20 0 1 2 3 4 5 6 0 38 0.2 5 350 400 450 500 550 600 650 Conc. of HaNPV (x 10 POBs/mL) 10 39 Wavelength (nm)-SLNPV 0.1 40 -SLNPV -SLNPV +SLNPV +SLNPV +SLNPV 0 (d) 0.0 1.26.0x10 1.2x10 1.8x10 2.4x10 400 450 500 550 600 41 (c) 4 Wavelength (nm) 42 Conc. of SLNPV (POBs/mL) 0.9 43 3 44 0.6 60 60 60 45 -SLNPV -SLNPV -SLNPV2 +SLNPV +SLNPV +SLNPV I /I I /I I /I Blank Blank Blank 46 0.6 0.6 0.6 Ratiometric Sensing Ratiometric Sensing Ratiometric Sensing 3.3 x 10 POBs/mL 3.3 x 10 POBs/mL 3.3 x 10 POBs/mL 50 0.3 50 50 6.6 6.6 6.6 47 9.9 9.9 9.9 0.5 1 0.5 0.5 48 13.2 13.2 13.2 40 40 40 0.0 16.5 16.5 16.5 49 19.8 19.8 19.8 0.4 60 60 0.4 0.4 50 23.1 23.1 23.1 30 30 I 30 /I I /I I /I Blank Blank Blank 26.4 x 10 POBs/mL x 10 26.4 x 10 POBs/mL 51 0.6 0.6POBs/mL 0.6 26.4 Ratiometric Sensing Ratiometric Sensing Ratiometric Sensing 0.3 3.3 x 10 POBs/mL 3.3 x 10 POBs/mL 3.3 x 10 POBs/mL 0.3 0.3 50 50 6.6 6.6 6.6 52 20 20 20 Sequential Addition of Analytes 9.9 9.9 9.9 0.5 0.5 0.5 0.2 0.2 0.2 13.2 13.2 13.2 40 4053 16.5 16.5 16.5 10 54 10 10 Figure 3.19.8(a) Fluorescence titration of 1 (10 µM, ex = 350 nm) with HaNPV in buffered medium 19.8 19.8 0.4 0.4 0.4 0.1 0.1 0.1 23.1 23.1 23.1 30 3055 26.4 x 10 7.4). POBs/mL 26.4 26.4 x 10 Effect POBs/mL 0 x 10 POBs/mL 0(b) 0 (pH concentration on the emission intensity (I6 395 56 nm) of6 1 in buffered 0.3of HaNPV 0.3 6 6 5 6 6 6 0.05 1.2x10 400 0.3500 450 500 550 600 0.0 0.051.8x10 400 450 400 550 450 600 500 550 6.0x10 600 6.0x10 1.2x10 1.8x10 2.4x10 6.0x10 2.4x10 1.2x10 1.8x106 2.4x106 20 2057 (nm) (nm)0.2Wavelength (nm) Conc. of SLNPV 0.2Wavelength 58 0.2Wavelength Conc. of(POBs/mL) SLNPVof(POBs/mL) Conc. SLNPV (POBs/mL) 10 1059 0.1 0.1Paragon Plus Environment 0.1 ACS 60 5

512 nm

Equation

y = a + b*x

Weight

No Weighting

0.0056

Residual Sum of Squares Pearson's r

0.99037

Adj. R-Square

0.97809

5

450 400

0 500 400 550 450 600 500 450 500 550

0.0 550 600

512 nm

Slope

5

6

410 nm

512 nm

Standard Error

0.09584

0.01739

2.09481E-7

1.10695E-8

6

6

410 nm

512 nm

410 nm

5

Equation

y = a + b*x

Equation

y = a + b*x

Equation

y = a + b*x

Weight

No Weighting

Weight

No Weighting

Weight

No Weighting

0.0056

Pearson's r

0.99037

Adj. R-Square

0.97809

D

Intercept

D

Slope


512 nm

5

Equation

y = a + b*x

Equation

y = a + b*x

Equation

y = a + b*x

Weight

No Weighting

Weight

No Weighting

Weight

No Weighting

0.0056



0

Fluorescence at 395 nm

410 5 nm

512 nm

Intercept

D

C ha B rc la oa nk l0 F. I.Su (a..5%u.) ga r1 0% H R a an N i p PV al u.) F. I. (a. 0 N .5% a G Cl ly ce 10% ro l1 U 0% C re a 3 (P a O 2% 4) M 2 10 gC % l2 10 %

410 5nm

512 nm

F. I. (a. u.)

u.)

Pr ob e + F. I.H (a. aN PV Pr ob e + H aN PV Pr ob e 410 nm

5

F. I. (a. u.)

5

H aN PV

Pr

ob F.+ I. (a. e u.)

5

F. I. (a. u.)

5

5

5

F. I. (a. u.)

F. I. (a. u.)

5

F. I. (a. u.)

F0/F at 395 nm

Value

D

0.0056

0.99037



0.0056

Pearson's r

0.99037

Pearson's r

0.99037

Adj. R-Square

0.97809

Adj. R-Square

0.97809

D

Intercept

D

Slope

Value Standard Error Intercept D0.09584 0.01739 Slope D 2.09481E-7 1.10695E-8

0.0056


0.99037

0.97809 Adj. R-Square Value Standard Error Value Standard Error 0.01739 Intercept 0.09584 0.01739 D 0.09584 2.09481E-7 1.10695E-8 Slope 2.09481E-7 1.10695E-8 D

0.0056

0.99037

0.97809 Adj. R-Square 0.97809 Adj. R-Square Value Standard Error Value Standard Error Value Standard Error 0.01739 Intercept 0.09584 0.01739 D0.09584 Intercept 0.01739 D0.09584 2.09481E-7 1.10695E-8 Slope 2.09481E-7 1.10695E-8 D Slope 2.09481E-7 1.10695E-8 D

5 6 5 6 6 5 6 6 6 6 6 0.01.8x10 600 0.0 6.0x10 1.2x10 6.0x10 2.4x10 1.2x10 1.8x10 6.0x10 1.2x10 1.8x10 2.4x10

2.4x106

10

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medium (pH 7.4). (c) Reversible interaction of 1 with HaNPV in buffered medium. (d) Effect of different analytes on the emission intensity (at 395 nm) of 1 in buffered medium (pH 7.4). Interference study: Interaction with HaNPV in control environment: Realizing the high sensitivity of the present system towards HaNPV, further, it was also utilized for the estimation of HaNPV content in the commercial formulation, such as Bio Kill-H®. The insecticidal activity of HaNPV was measured by quantifying the number of occlusion bodies present in the commercial formulation. However, in order to quantify the amount of HaNPV present in the commercial formulations, it is essential to check the response of 1 towards the other adjuvants, as they may pose serious interference in HaNPV sensing (Fig. 3d).24 Thankfully, none of these control analytes showed detectable alteration in the emission signal under similar condition. Since, surfactant solutions are sometimes used as dispersants for spraying pesticides in fields, we have also included various surfactants in the sensing studies as control. As expected, compound showed no detectable change in fluorescence response in presence of CTAB (positively charged) or Brij-58 (neutral). However, a small redshift was observed in presence of SDS (negatively charged), probably due to dye-surfactant assembly formation owing to the electrostatic interaction (Fig. S5). Thus, it can be presumed that the changes in emission signal observed in presence of commercially available biopesticide formulations can directly be correlated to their HaNPV contents. When the commercially available virus suspension was spiked (0-5 µL) to the aqueous solution of 1 (PBS buffer, pH 7.4), a concentration-dependent linear change in fluorescence intensity was noticed at 395 nm band (Fig. S6a). Following the extent of emission quenching, the amounts of HaNPV in the spiked samples were determined using the regressive equation, Y = 0.9577 - 0.193x (r2 = 0.998) (Fig. S6b). The recovery values were found to be varied from 100.5% to 107.0% with RSDs in the range of 2.0-4.5%. The sufficiently low RSD values (

On-Field Detection of Helicoverpa armigera Nuclear Polyhedrosis

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