Capture and Protection of Environmental DNA in a Metal-Organic Framework

Abstract

Environmental DNA (eDNA) is released by organisms into their surroundings, enabling non-invasive species detection and biodiversity assessments without the need for direct observation. However, collection poses challenges due to the generally low abundance of eDNA and the presence of degradation agents, including enzymes, UV radiation, and microorganisms, rendering samples unstable. Active filtration, which is frequently used to capture eDNA in water, can be time-consuming and cumbersome in field conditions. Herein, a filter-free one-pot procedure for capturing eDNA with the metal-organic framework (MOF), zeolitic imidazolate framework 8 (ZIF-8), is examined. The method is evaluated on 15 mL water samples from diverse sources (aquarium, river, and sea). ZIF-8 forms in all with high capture efficiency (>98%) using spiked salmon DNA to represent eDNA. The DNA is resistant to degradation by endonucleases and UV light. In addition, it remains stable over time as a species-specific salmon quantitative polymerase chain reaction detected genomic DNA in all samples captured with the MOF to a maximum of 28 days at 37 °C while the untreated control samples were below the assay detection limit by day 6. These results highlight the efficacy of ZIF-8 capture in overcoming challenges associated with the preservation of eDNA obtained from aquatic environments.

Keywords: biodiversity; capture; environmental DNA; metal‐organic framework; quantitative polymerase chain reaction; zeolitic imidazolate framework 8.

Figure 1

Schematic representation showing the capture of environmental DNA (eDNA) with a metal‐organic framework (MOF). Addition of salt precursors trigger formation of the MOF around the eDNA, allowing it to be concentrated and protected. The eDNA can later be released using a chelating agent.

Figure 2

Characterization of ZIF‐8 capturing 25 μg salmon gDNA in Milli‐Q water. SEM images at a) 5000×, b) 15 000×, and c) 30 000× magnification with asterisks (*) highlighting a 200 nm subpopulation, demonstrating the mixed sizes of the sample. d) EDS elemental mapping images for carbon (C), nitrogen (N), oxygen (O), and zinc (Zn). e) FTIR spectra comparing the synthesized material to the precursors and pure ZIF‐8 without DNA. f) Simulated XRD pattern of pure ZIF‐8 compared with synthesized material with and without added gDNA.

Figure 3

Capture of salmon gDNA in ZIF‐8 in Milli‐Q water. 275 μL samples containing final concentrations of 86 mg mL⁻¹ 2‐methylimidazole, 8.7 mg mL⁻¹ zinc nitrate hexahydrate, and 25 μg gDNA. Samples were incubated for 15 min before centrifuging at 10 000 g for 5 min and removing 2.2 μL of the supernatant for the SYBR Gold assay. a) Percentage capture relative to free DNA diluted to 90.9 μg mL⁻¹ in Milli‐Q (MQ) and b) release following incubation of the pellet in 275 μL 100 mm EDTA for 15 min. The mean percentage (a) captured or (b) released amount of gDNA is plotted with error bars representing the standard deviation of three independent experiments (N = 3). c) Fluorescence spectra of free gDNA, captured gDNA, or ZIF‐8 labeled with SYBR gold. d) Orthogonal view of a confocal microscopy image showing gDNA labeled with SYBR gold in ZIF‐8 particles synthesized in Milli‐Q water. Scale bar = 30 μm.

Figure 4

Protection provided by the MOF against endonuclease and UV degradation. a) Lanes containing 0.75 μg of 1 kb DNA ladder with (+) or without (−) DNase treatment. Lanes 1 and 2 contain free ladder while Lanes 3–6 have been captured with the ZIF‐8 MOF. Lanes 5 and 6 show the captured ladder that has been subsequently released with 100 mm EDTA. b) Lanes containing 1 μg of salmon gDNA with or without UV treatment. Lane 1 contains 0.25 μg of a 1 kb ladder. Lanes 2 and 4 contain free gDNA while 3 and 5 have been captured in the ZIF‐8 MOF. Lanes 2 and 3 were kept in the dark while 4 and 5 were subjected to 254 nm UV light for 2 h. ZIF‐8 samples were released with EDTA before loading. Both gels are 0.75% agarose containing SYBR Safe and were run at 80 V for 1 h. The ladder in (a) is from Sigma‐Aldrich while (b) is from New England Biolabs.

Figure 5

Capture and release in environmental water sources. a) Capture efficiency of 25 μg salmon gDNA spiked into 275 μL tank, river, or seawater samples containing final concentrations of 2‐methylimidazole at 86 mg mL⁻¹ and zinc nitrate at 8.7 mg⁻¹. SYBR Gold was added to the supernatant to determine remaining free DNA and infer precipitated quantity. b) Release of salmon gDNA with 100 mm EDTA for 15 min at room temperature compared to gDNA at the same concentration without capture. The mean in panels (a) and (b) is plotted with error bars representing the standard deviation of three independent experiments (N = 3).

Figure 6

ZIF‐8 provides long‐term protection of DNA in water over time. Total DNA amount following ethanol precipitation and extraction from free or MOF‐captured water samples spiked with 10 μg mL⁻¹ salmon gDNA in a) tank water incubated at 37 °C for 6 and 28 days or b) 7 days at room temperature in river or sea water measured via Nanodrop. The mean amount is plotted with error bars representing the standard deviation of three extracted samples (N = 3). Species‐specific salmon qPCR for the cytochrome oxidase subunit 1 gene assayed from the same c) tank or d) river and sea samples. The mean amount is plotted with error bars representing the standard deviation of three water samples analyzed in quadruplicate wells across triplicate qPCR runs (N = 12).