Selective Sensing and Imaging of Penicillium italicum Spores and Hyphae using Carbohydrate-Lectin Interactions

Transcription

1 Selective Sensing and Imaging of Penicillium italicum Spores and Hyphae using Carbohydrate-Lectin Interactions Idris Yazgan a,, Jing Zhang a,victor Kariuki a, Ayfer Akgul b, Lauren E Cronmiller a, Ali Akgul c, Francis Osonga a, Abbey McMahon a, Yang Gao d, Gaddi Eshun a, Seokheun Choi d, Omowunmi A Sadik a * a Department of Chemistry, Center for Research in Advanced Sensing Technologies & Environmental Sustainability (CREATES), State University of New York at Binghamton, Binghamton, NY b Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, P. O. Box 6100, MS c Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Box 9820, Starkville, MS d Department of Electrical and Computer Engineering, Center for Research in Advanced Sensing Technologies & Environmental Sustainability (CREATES), State University of New York at Binghamton, Current address- Department of Biology, Faculty of Art and Science, Kastamonu University, Kastamonu-Turkiye

2 Figure S1: Growth pattern of Penicillium italicum at different inoculation conditions. Figures S1-A and -B depict the top and bottom images of P. italicum on PDA media, whose inoculum was prepared in Nutrient broth at 5-days incubation. Figures S1-C and D show the P.italicum prepared in ph 7.4 PBS buffer 3- and 7-days incubation respectively. Reproduction of Penicillium italicum typically occurs in vegetative, asexual, and sexual forms. During the vegetative growth, detached parts from hyphae called the mycelium will grow as a single fungus. Asexual growth [anamorph] is a unicellular conidia-based growth while sexual growth [teleomorph] is more complex and typically starts with the formation of young ascogonium. The colonies are typically blue or grey-green colonies and are commonly seen as bundles of conidiophores and conidial heads. When cultured in a conventional plate, the bottom side of the plate appears colourless, grey or yellowish-brown [1].

3 Figure S2: Detection of 10 4 spores/ml P.italicum at gold-coated paper electrodes. 10 mm K 3 (CN) 6 in 1 M NaNO 3 solution was used for CV experimentation. BG refers to bare-gold electrode; BG-TH refers to galactose-4-mercaptoaniline modified gold electrode surface; BG- TH-PI refers to P.italicum captured on the modified gold-electrode surface. Silver wire was used as the reference electrode while platinum electrode was used as the auxiliary electrode. The experimentation was run between V. In the case of 10 4 spores/ml of P.italicum concentration, the surface was capable of capturing the spores and provided a discernible response. However, at 10 2 spores/ml P.italicum concentration, both of the surfaces failed to recognize P.italicum. Actually, any of the sensor surface utilized in the study did not give response to 10 2 spores/ml of P.italicum. The reduction and oxidation peaks moved more positive and negative values in addition to decreases in the obtained current, which reveals the binding of P.italicum on the sugar-modified paper electrode surface. Therefore, it can be speculated that sugar-functionalized paper electrodes can be used in detection of presence of P.italicum. Detection of P.italicum on PAA Membranes

4 Figure S3: Detection of P. italicum on PAA membranes and their SEM images. The surface of the membrane was coated 2 nm Palladium for SEM imaging to visualize the capturing of P.italicum species. The membrane surface was modified with NLPDA via GA linkage on the membrane surface. P.italicum that was made up in acetate-buffer (ph 5.5) was applied to the surface at10 4 spores/ml, which was then stained with sugar-functionalized gold nanoparticles. Digital camera images of the AuNP staining on membrane where P. italicum hyphae and conidiophore were captured [Figure S3C/D]. As seen from digital image Figure S3A, only certain parts of the membrane were stained into slight pink. Figure S3B microscopy imaging revealed the presence of P.italicum hyphae. SEM images [Figure S3C] and [Figure S3D]

5 clearly showed the presence of hyphae and spores in the pinkish area of the membrane, while [Figure S3E] and [Figure S3F] images showed only artefacts seen for non-stained sections [remained yellow] of the PAA membrane. Even though hyphae and conidiophores are shown in Figure 3, spores were also captured on the ligand functionalized PAA membrane surfaces. Two characteristic results were obtained for the membrane support materials in comparison to glass-surfaces. (i) GA-mediated introduction of NLPDA on top of the PAA surface resulted in capturing of hyphae and conidiophores more preferentially while GMBS mediated NLPDA introduction to mercaptosilane activated glass-surface resulted in capturing of spores. (ii) Hyphae and conidiophores were stained by a different sugar-functionalized AuNPs than the one staining the spores preferentially. Antibacterial Characteristics of P. italicum

6 Figure S4: Antibacterial activity of P.italicum on bacteria. (A) E. coli, (B) C. freundii and (C) S. epidermidis at 10 3 cfu in 50 µl as two drops were placed on top of PDA agar. 2-day incubation allowed bacterial growth, followed by 10 3 spores of P.italicum in 50 µl as two drops spread around the grown E.coli (D), C.freundii (E) and S.epidermidis (F). 3-days incubation allowed fungal growth, and 5-days incubation allowed complete coverage of the bacterial species. At the end of the 5 th day, samples were swiped from the media and placed on the freshly prepared PDA agar, where no bacterial growth was observed. In the case of coinoculation of P.italicum and the bacteria, the size of the bacterial growth area was less than half of the area shown in Figure S4A-C. The initial growth of bacteria could not be prevented by P.italicum since bacterial growth rate was faster and P.italicum probably needed sometime to release its antibacterial molecules. Validation of the Sensor using Lemon Samples Since P. italicum is the major pest for lemons, they were used in this work to validate the sugarlectin recognition. Figure S5: Infection of P.italicum on lemon samples. At 48 th h incubation the front side (A) and back side (B) of lemon 1 and lemon 2 (C and D) showed fungal growth. At 72 nd h incubation,

7 mycelia growth was only observed for the back side (E) of lemon 1 while strong appearance of mycelia was observed within the intentionally wounded and the surrounding areas. Blue circles are highlight the grown mycelia.

8 Figure S6- Detection of P.italicum on lemon samples. The membrane surface was modified with NLPDA via GA on the rhodamine 6G membrane surface. P.italicum 10 4 spores/ml in acetatebuffer ph 5.5) was applied to the surface, which was then stained with sugar-functionalized gold nanoparticles. Freshly purchased lemons [Binghamton, NY] were infected with 100µL of 10 6 spores/ml of P. italicum. Immediately after incubation, the lemon s surfaces were smeared with a sterile blade in a sterile-glass [Figure S6A], where the final P. italicum concentration was adjusted to 10 4 spores/ml. [Figure S6B] PAA membrane surface was activated with GA, followed by the introduction of NLPDA. Residual free carbonyl groups were deactivated with glycine and 10 4 spores/ml of P.italicum in acetate buffer was introduced on top of the NLPDA functionalized PAA membrane, where 6-8 h incubation was performed. Then, unbound spores were washed away with acetate buffer. As seen from the circles, opaque areas formed, where higher amount of P.italicum was captured [Figure S6C/E]. The control showed [Figure S6D/F], no spores were captured on non-functionalized surface. When the membrane surface was treated with the same nanoparticles mentioned utilized in Figure 3 for 8h, no visible color formation was observed. Visible formation of spores was obtained by the naked eye in 8 h staining at 10 6 spores/ml treatment.