METHODS FOR DETECTING ZEROVALENT NANOPARTICLE-MEDIATED OXIDATIVE STRESS IN CYANOBACTERIA

Abstract

Zero-valent iron nanoparticle-mediated oxidative stress was evaluated in F. diplosiphon as a measure of lipid peroxidation using malondialdehyde and thiobarbituric acid-reactivity (MDA-TBA) assay and the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorogenic probe. Enzymatic response of nZVI-treated cultures was determined as a measure of FeSOD (iron superoxide dismutase) using western blotting and immunodetection. In addition, the distribution and surface assimilation of nZVIs was detected using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS).

Claims

1. A method for detecting and/or quantifying nZVI-mediated oxidative stress in cyanobacterium, the method comprising: a. growing a first culture of said cyanobacterium in the presence of a concentration of zero-valent iron nanoparticles b. growing a control culture of said cyanobacterium in the absence of zero-valent iron nanoparticles; c. assaying said first culture and said control culture for lipid peroxidation; d. comparing a level of lipid peroxidation in said first culture and a level of lipid peroxidation in said control culture; e. making a determination that said concentration of zero-valent iron nanoparticles in said first culture of said cyanobacterium is causing oxidative stress in said first culture of said cyanobacterium when said level of lipid peroxidation in said first culture of said cyanobacterium is statistically higher than said level of lipid peroxidation in said control culture of said cyanobacterium.

2. The method of claim 1, wherein said assaying of said first culture and said control culture of lipid peroxidation comprises a determination of malondialdehyde content in said first culture and said control culture.

3. The method of claim 1, wherein said cyanobacterium is F. diplosiphon.

4. The method of claim 1, wherein said cyanobacterium is B481-WT F. diplosiphon.

5. The method of claim 1, wherein said cyanobacterium is B481-SD F. diplosiphon.

6. The method according to claim 1, further comprising carrying out steps a and c-e with a second culture of said cyanobacterium in the presence of a second concentration of zero-valent iron nanoparticles.

7. A method for detecting and/or quantifying nZVI-mediated oxidative stress in cyanobacterium, the method comprising: a. growing a first culture of said cyanobacterium in the presence of a concentration of zero-valent iron nanoparticles b. growing a control culture of said cyanobacterium in the absence of zero-valent iron nanoparticles; c. adding a fluorometric probe to each of said first culture and said control culture; d. measuring a fluorescence of said first culture and said control culture e. comparing a level of fluorescence of said first culture and said control culture; f. making a determination that said concentration of zero-valent iron nanoparticles in said first culture of said cyanobacterium is causing oxidative stress in said first culture of said cyanobacterium when said level of fluorescence of said first culture of said cyanobacterium is statistically higher than said level of fluorescence in said control culture of said cyanobacterium.

8. The method of claim 7, wherein said fluorometric probe is 2′,7′-dichlorodihydrofluorescein diacetate fluorometric probe.

9. The method of claim 7, wherein said cyanobacterium is F. diplosiphon.

10. The method of claim 7, wherein said cyanobacterium is B481-WT F. diplosiphon.

11. The method of claim 7, wherein said cyanobacterium is B481-SD F. diplosiphon.

12. The method according to claim 7, further comprising carrying out steps a and c-f with a second culture of said cyanobacterium in the presence of a second concentration of zero-valent iron nanoparticles.

13. A method for detecting and/or quantifying nZVI-mediated oxidative stress in cyanobacterium, the method comprising: a. growing a first culture of said cyanobacterium in the presence of a concentration of zero-valent iron nanoparticles b. growing a control culture of said cyanobacterium in the absence of zero-valent iron nanoparticles; c. carrying out a densitometric analysis of superoxide dismutase (“SOD”) in said first culture and said control culture d. comparing a level of SOD in said first culture and said control culture; e. making a determination that said concentration of zero-valent iron nanoparticles in said first culture of said cyanobacterium is causing oxidative stress in said first culture of said cyanobacterium when said level of SOD in said first culture of said cyanobacterium is statistically higher than said level of SOD in said control culture of said cyanobacterium.

14. The method of claim 13, wherein said SOD is Fe-SOD.

15. The method of claim 13, wherein said cyanobacterium is F. diplosiphon.

16. The method of claim 13, wherein said cyanobacterium is B481-WT F. diplosiphon.

17. The method of claim 13, wherein said cyanobacterium is B481-SD F. diplosiphon.

18. The method according to claim 13, further comprising carrying out steps a and c-e with a second culture of said cyanobacterium in the presence of a second concentration of zero-valent iron nanoparticles.

19. A method for detecting and/or quantifying cellular update and/or surface adsorption of nZVI by cyanobacterium, the method comprising: a. growing a first culture of said cyanobacterium in the presence of a concentration of zero-valent iron nanoparticles b. growing a control culture of said cyanobacterium in the absence of zero-valent iron nanoparticles; c. conducting transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS) analysis on each of said first culture and said control culture over a period of days; d. analyzing changes in TEM and EDS for said first culture over time, and comparing said changes to TEM and EDS for said control culture over time.

20. The method of claim 16, wherein said cyanobacterium is F. diplosiphon.

21. The method of claim 16, wherein said cyanobacterium is B481-WT F. diplosiphon.

22. The method of claim 16, wherein said cyanobacterium is B481-SD F. diplosiphon.

23. The method according to claim 16, further comprising carrying out steps a and c and d with a second culture of said cyanobacterium in the presence of a second concentration of zero-valent iron nanoparticles.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1. Quantification of malondialdehyde as a measure of lipid peroxidation in F. diplosiphon B481-WT and B481-SD strains treated with nZVIs at concentrations ranging from 0.8 to 102.4 mg L.sup.−1. Average MDA concentration (±standard error) of three biological replicates for each strain is shown. Different letters above the final concentration point indicate significance among treatments (p<0.01).

[0012] FIG. 2. Intracellular reactive oxygen species validated using highly fluorescent DCF in F. diplosiphon B481-WT and B481-SD strains grown in BG-11/HEPES medium supplemented with 1.6, 3.2, 6.4, 12.8, and 25.6 mg L.sup.−1 nZVIs. Average DCF fluorescence (±standard error) of three biological replicates for each sample is shown. Different letters above the error bars indicate significance among treatments (p<0.01).

[0013] FIG. 3. Non-denaturing PAGE analysis of FeSOD in F. diplosiphon B481-WT and B481-SD strains grown in BG-11/HEPES medium containing 3.2, 6.4, and 51.2 mg L.sup.−1 nZVIs. Different letters above the final concentration point indicate significance among treatments (p<0.05).

[0014] FIG. 4A. EDS spectrum of bare nZVIs nanoparticles.

[0015] FIG. 4B. Histogram showing size distribution of nZVIs used in the present study.

[0016] FIG. 5A. EDS of F. diplosiphon treated with 3.2 mg L.sup.−1 nZVIs after exposure to nZVIs on day 4

[0017] FIG. 5B, EDS of F. diplosiphon treated with 3.2 mg L.sup.−1 nZVIs after exposure to nZVIs on day 12.

[0018] FIG. 5C. EDS of F. diplosiphon treated with 3.2 mg L.sup.−1 nZVIs after exposure to nZVIs on day 20.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The invention summarized above may be better understood by referring to the following description, claims, and accompanying drawings. This description of an embodiment, set out below to enable one to practice an implementation of the invention, is not intended to limit the preferred embodiment, but to serve as a particular example thereof. Those skilled in the art should appreciate that they may readily use the conception and specific embodiments disclosed as a basis for modifying or designing other methods and systems for carrying out the same purposes of the present invention. Those skilled in the art should also realize that such equivalent assemblies do not depart from the spirit and scope of the invention in its broadest form.

Experimental Section

[0020] Strains and Culture Conditions. F. diplosiphon strains, B481-WT obtained from the UTEX algal repository (Austin, TX, USA) and B481-SD (overexpressed strain with the sterol desaturase gene; accession: MH329183), were used in this study. The strains were grown in liquid BG-11 medium containing 20 mM HEPES (hereafter termed as BG-11/HEPES) to an exponential growth phase (˜optical density 750 nm of 0.8). Cultures were grown in vented 40 mL tissue culture flasks with continuous shaking at 70 rpm and 28° C. in an Innova 44R incubator shaker (Eppendorf, Hamburg, Germany). The spectrum of photosynthetic light in the shaker had peak wavelengths at 437 and 600-650 nm with an intensity adjusted to 30 μmol m−2 s.sup.−1 using the model LI-190SA quantum sensor (Li-Cor, Lincoln, NE, USA). Nanofer 25 s zero-valent iron nanoparticles (nZVIs) coated with polyacrylic acid were obtained from Nano iron company (Rajhrad, Czech Republic) and adjusted to final concentrations of 1.6, 3.2, 6.4, 12.8, 25.6, 51.2, and 102.4 mg L.sup.−1.

[0021] Nanoscale Zero-Valent Nanoparticle-Induced Lipid Peroxidation in F. diplosiphon. Quantification of oxidative stress in F. diplosiphon strains treated with nZVIs was performed using the thiobarbituric acid-malondialdehyde (TBA-MDA) adduct formation according to the method described by Bhandari and Sharma.sup.28 with modifications. Cultures were grown in BG-11 media containing nZVIs at concentrations of 1.6, 3.2, 6.4, 12.8, 51.2, and 102.4 mg L.sup.−1 for a period of 15 days under culture conditions mentioned above. Cultures not treated with nZVIs served as the control. Three replicates were maintained for each treatment, and the experiment was repeated once. A total of 5 mL cells from each treatment was centrifuged at 18,000 rpm for 15 min at 4° C. The pellet was weighed and homogenized in 1 mL of 0.5% trichloroacetic acid (TCA), brought to 5 mL final volume with TCA, and centrifuged at 14,000 rpm for 15 min at 4° C. To a test tube containing 2.5 mL of 0.5% TBA in 20% TCA, 1 mL of the supernatant was added and incubated for 30 min at 90° C. in a water bath. The mixture was centrifuged for 2 min at 3000 rpm to remove the nonspecific precipitate. Absorption was measured at 532-600 nm using a microplate reader (BioTek Synergy H1 Microplate Reader, Agilent, USA). Peroxidation of lipids was calculated using an extinction coefficient of ε=155 mM.sup.−1 cm.sup.−1.

[0022] Detection of Intracellular ROS in Fremyella diplosiphon Treated with nZVIs.

[0023] Intracellular ROS in nZVI-treated F. diplosiphon was measured using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorometric probe (EMD Chemicals, USA) according to the method described by Pattanaik et al..sup.29 B481-WT and B481-SD strains were grown in nZVIs at 1.6, 3.2, 6.4, 12.8, and 25.6 mg L.sup.−1 under culture conditions as mentioned above. As cultures treated with 51.2 mg L.sup.−1 nZVIs resulted in elevated oxidative stress as observed in the lipid peroxidation assay, this concentration was not used to detect intracellular ROS.

[0024] Three biological replicates were maintained, and the experiment was repeated once. Cultures not treated with nZVIs served as the control. At 15 days after treatment, 1 ml of each culture was incubated in 10 mM DCFH-DA for 1 h at room temperature in the dark and under shaking at 70 rpm. Fluorescence of DCF-DA converted from the nonfluorescent DCFH-DA was detected by loading 200 μl of each sample in a 96 well-plate and read at an excitation wavelength of 485 nm and an emission of 520 nm in a microplate reader (BioTek Synergy H1 Microplate Reader, USA). Three technical replicates on a 96-well plate were measured for each treatment to account for plating bias. A negative control of bare nZVIs was used to account for any potential interference of autofluorescence that may interfere with the DCFH-DA probe signal.

[0025] Enzymatic Response of Fremyella diplosiphon to Nanoscale Zero-Valent Nanoparticle-Mediated Oxidative Stress.

[0026] Protein Extraction. F. diplosiphon B481-WT and B481-SD strains were grown in BG11 media/HEPES media supplemented with 3.2, 6.4, and 51.2 mg L.sup.−1 nZVIs. Cultures were grown in 250 mL conical flasks with continuous shaking at 170 rpm, 28° C., and ambient CO2 (350-1000 mg L.sup.−1) in an Innova 44R incubator shaker (Eppendorf, Hamburg, Germany). Cultures not treated with nZVIs served as the control. After 9 days, cells were pelleted at 8000 rpm for 15 min at 4° C. and total proteins extracted. For each sample, two protease inhibitor tablets (Roche) were dissolved in 30 mL of binding buffer containing 50 μM tris and 500 μM NaCl. Lysozyme (0.03 g) was dissolved in 3 mL of binding buffer, added to each sample, and incubated in ice for 1 h. The pellet was sonicated in ice at 100% ampere for 10 s with 1 min intervals, and the process was repeated nine times. The lysate was spun down at 10,000 rpm for 15 min at 4° C. and lyophilized, and the protein concentration of each sample determined using the BCA assay Smith et al..sup.30 Samples were diluted to 2.0 mg mL.sup.−1 in SDS boiling buffer with reducing agents and heated in a digital dry bath at 95° C. for 10 min before loading in a SDS gel.

[0027] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. SDS slab gel electrophoresis was carried out under reducing conditions according to the method of Laemmli,.sup.31 as modified by O'Farrell..sup.32 The samples were loaded in 10% acrylamide slab gels (0.75 mm thick) and SDS slab gel electrophoresis was carried out for about 4 h at 15 mA/gel. Myosin (220,000), phosphorylase A (94,000), catalase (60,000), actin (43,000), carbonic anhydrase (29,000), and lysozyme (14,000) were used as molecular weight standards. Following slab gel electrophoresis, the gels for blotting were placed in a transfer buffer (10 mM Caps, pH 11.0, 10% methanol) and transblotted onto PVDF membranes overnight at 200 mA and approximately 100 V/two gels. The blots were stained with Coomassie Brilliant Blue R-250 and scanned with GE Imagescanner III.

[0028] Western Blot and Densitometric Analysis. The blots were destained in 100% methanol, rinsed in Tween-20 tris buffer saline (TTBS), and blocked for 2 h in 5% nonfat Dry Milk (NFDM) in TTBS. The blots were then incubated overnight in primary antibody (anti-FeSOD [Agrisera, Cat #AS06125] diluted 1:20,000 in 2% NFDM TTBS) and rinsed for 3×10 min in TTBS. The blots were placed in the secondary antibody (anti-Rabbit IgG-HRP [SeraCare, Cat #5220-0337] diluted 1:20,000 in 2% NFDM TTBS) for 2 h, rinsed as above, treated with enhanced chemiluminescence, and exposed to Kodak BioMax MR X-ray film. Western blot films (K05401 #1-3: Kodak BioMax MR 3-min exposures) were scanned with a laser densitometer (Model PDSI, Molecular Dynamics Inc, Sunnyvale, CA). The scanner was checked for linearity prior to scanning with a calibrated Neutral Density Filter Set (Melles Griot, Irvine, CA). Band volume above background (manual marking) was quantified using Phoretix 1D software (version 11.2) with a Windows 10 compatible computer.

[0029] Distribution and Validation of nZVIs in Fremyella diplosiphon. F. diplosiphon strains grown in 0.8 mg L.sup.−1 nZVIs for 4, 12, and 20 days were tested for distribution and adsorption of nanoparticles using TEM. Samples were prepared by aliquoting 10 μL onto TEM carbon-coated 200-mesh copper grids and dried overnight at room temperature. The sizes of one hundred nZVIs were measured and their average with associated standard deviation calculated. The TEM was equipped with an EDS detector for spectral and elemental mapping.

[0030] TEM images of F. diplosiphon-treated with nZVIs, untreated cells, and bare nanoparticles were acquired using the JEM-1400 PLUS (JEOL USA, Massachusetts, USA) equipped with an energy-dispersive X-ray microanalysis system (Oxford Instruments plc, Abingdon, United Kingdom). The images were viewed using Digital Micrograph software from GATAN (GATAN Inc., CA, USA). In addition, samples were fixed to a glass microscope slide and visualized using a Wolfe DigiVu DVM 9.0 digital microscope (Carolina Biological Supply Co., Burlington, NC, USA), and images were captured using a Moticam digital microscopy camera.

[0031] Statistical Analysis. Results were reported as cumulative treatment mean±standard error. Statistical significance was determined using one-way analysis of variance and Tukey's honest significant differences post hoc test at 95% confidence intervals (p<0.05). The single-factor, fixed-effect ANOVA model, Y.sub.ij=μ+αNi.sub.+εij, was used where Y represents a single, distinct parameter (growth rate or ROS content) in nZVI treatment i and biological replicate j. The p represents the overall mean adjusted by effects associated with the nZVI treatment (αN) and εij the experimental error from nZVI treatment i and biological replicate j.

[0032] Results

[0033] Determination of Lipid Peroxidation as a Measure of ROS. The level of malondialdehyde production, which serves as an indicator for ROS in nZVI-treated F. diplosiphon, indicated increased lipid peroxidation and intracellular ROS in a dose-dependent manner. Both B481-WT and B481-SD strains treated with 3.2, 12.8, and 51.2 mg L.sup.−1 nZVIs exhibited significant increases (p<0.01) in MDA content compared to the untreated control (FIG. 1). While the MDA content of the nZVI-treated B481-WT strain ranged from 86.73±9.7 to 165.50±5.8, that of the B481-SD strain ranged from 128.46±24 to 267.20±26.23 nmol mL.sup.−1. However, we did not observe any significant differences in MDA concentration in both strains treated with 0.8, 1.6, and 102.4 mg L.sup.−1 nZVIs. Cultures treated with 12.8 and 51.2 mg L−1 nZVIs exhibited significantly higher ROS compared to the untreated control, 1.6, and 3.2 mg L.sup.−1 nZVIs (FIG. 1). Comparison of MDA content between strains in each treatment revealed significant differences from 1.6 to 102.4 mg L.sup.−1 nZVIs (data not shown).

[0034] Increased ROS Detected by the 2′,7′-Dichlor-odihydrofluorescein Diacetate Assay. Intracellular ROS that oxidizes DCFH to highly fluorescent 2′,7′-dichlorofluorescein (DCF) ranged from 0.0710±0.001 to 0.1063±0.0006 in B481-WT, while that of the B481-SD ranged from 0.0647±0.0016 to 0.1±0.002 nmol mL.sup.−1. Both strains treated with nZVIs ranging from 3.2 to 25.6 mg L.sup.−1 exhibited significantly increased levels of ROS (p<0.001) compared to the untreated control (FIG. 2). While the wild-type strain exhibited significantly increased ROS levels when treated with 1.6-25.6 mg L.sup.−1 (p<0.01) nZVIs, the B481-SD strain showed significantly higher levels of oxidative stress ranging from 3.2 to 25.6 mg L−.sup.1 (p<0.01) nZVIs (FIG. 2). In addition, we observed significant differences between the B481-WT and B481-SD strains grown in 1.6, 6.4, 12.8, and 25.6 (p<0.05) mg L.sup.−1 nZVIs (data not shown). The negative control of bare nanoparticles did not exhibit DCF signal interference.

[0035] Immunodetection Reveals Increased SOD Levels in nZVI-Treated F. diplosiphon. SOD enzymes known for scavenging ROS in cells were quantified to determine oxidative stress in nZVI-treated F. diplosiphon. Densitometric analysis of anti-FeSOD revealed significantly higher FeSOD levels in both B481-WT (p<0.05) and B481-SD (p<0.001) strains treated with 51.2 mg L.sup.−1 when compared to the control. We also observed significantly higher levels of FeSOD (731.97±89.6, p<0.01) in the B481-SD strain treated with 6.4 mg L.sup.−1 nZVIs compared to 3.2 mg L.sup.−1 (401.37±67.1, p<0.001); however, it was significantly lower than that in 51.2 mg L.sup.−1 nZVIs. Additionally, the B481-SD strain treated with 51.2 mg L−1 exhibited significantly higher SOD levels compared to that of 3.2 mg L.sup.−1 nZVIs. We did not observe significant differences in SOD levels of the B481-WT strain treated with 3.2 and 6.4 mg L−1 nZVIs compared to the untreated control (FIG. 3). Further, FeSOD activity was significantly higher in the B481-WT strain treated with 3.2 and 6.4 mg L.sup.−1 nZVIs (p<0.05) compared to the B481-SD strain grown under similar concentrations (data not shown).

[0036] Particle Size Determination and F. diplosiphon-Nanoparticle Interaction. Optical imaging revealed adsorption of nZVIs on the surface of F. diplosiphon cells. TEM equipped with EDS confirmed the composition and distribution of nZVIs through elemental mapping and revealed the adsorption of nZVIs on F. diplosiphon cells. A strong iron peak in the EDS spectra confirmed the presence of elemental iron in nanofer 25 s nZVIs (FIG. 4a). As shown in the differential particle size histogram (FIG. 4b), nZVIs used in the study ranged from a size of 24.68 to 82.72 nm, with an average of 55.17 nm. Comparison of EDS spectra on days 4, 12, and 20 in both F. diplosiphon strains treated with 3.2 mg L.sup.−1 nZVIs showed a gradual reduction of the iron signal over the period tested. While the spectral analysis of both B481-WT and B481-SD cultures treated with nZVIs indicated a strong signal for iron at day 4 (FIG. 5a) and day 12 (FIG. 5b), a significant decrease in the intensity of the iron peak was observed at day 20 (FIG. 5c). Control cultures that were not treated with nZVIs did not show the presence of the iron peak in EDS spectral analysis.

CONCLUSIONS

[0037] The exposure of F. diplosiphon to nZVIs increases intracellular ROS production, leading to oxidative stress and membrane lipid peroxidation. In addition, the data show increased SOD enzyme activities as a defense mechanism to alleviate oxidative stress. TEM imaging with EDS analysis showed strong signals for iron at 4 and 12 days after treatment.

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