Multimodal Analysis of Microplastics in Drinking Water using a Silicon Nanomembrane Analysis Pipeline

Our research goals are to improve the analysis of microplastics present in multiple sample types, as well as improve the data generated from those particles of interest. Current microplastics analytical methods are prone to the introduction of extrinsic contamination. The methods we describe here eliminate transfer steps and alleviate this contamination problem.

We are providing a protocol utilizing a silicon nanomembrane that allows researchers to conduct multimodal analyses of their particles of interest with increased efficiency and less contamination. The analysis of microplastics is only as effective as the methods utilized, combining optical, electron, and spectroscopic imaging techniques allow for the fullest picture. Silicon nanomembranes enable these multiple analyses.

[Narrator] To begin, don a 100% cotton laboratory coat and nitrile gloves. Using 99% isopropyl alcohol spray the nitrile gloves, rub the hands together thoroughly and rinse with approximately 18 megohm 0.22 micrometer filtered water. Fold a natural fiber delicate task wipe into quarters, then spray with 70% isopropyl alcohol. Wipe the hood surface from back to front and long strokes. Refolding the delicate task wipe to an unused surface every two strokes. Now roll a silicone mat across the surface of the hood to pick up any remaining particles. Spray the silicone roller with 99% isopropyl alcohol and scrub using a gloved hand. Rinse the roller with filtered water. After repeating the cleaning process three times, allow the roller to air dry inside the hood. To generate ultrapure water and isopropyl alcohol, fill a one liter beaker with the 18 megohm water under the hood. Prime a 60 millimeter syringe and attach 0.22 micrometer cutoff syringe filter by pushing at least 200 milliliters of filtered water through the syringe and filter assembly. Then rinse a glass screw cap container three times with filtered water, and fill the container with syringe filtered 18 megohm 0.22 micrometer filtered water. Repeat the beaker filling syringe priming and container rinsing steps using the desired percentage concentration of isopropyl alcohol instead of water to generate ultrapure isopropyl alcohol. Don personal protective equipment and nitrile gloves. Spray a silicone gasket with ultrapure 99% isopropyl alcohol and scrub the gasket with gloved fingers. Then rinse the gasket with ultrapure water. First, generate the process blank. Utilizing the primed syringe uptake 30 milliliters of ultrapure water and 30 milliliters of air into the 60 milliliter syringe. Screw on a syringe filter. Shake the syringe vigorously and dispense the liquid and air through the filter. After rinsing three times, assemble the filtration apparatus according to the visual assembly graphic. Turn on the vacuum to the filtration apparatus to create a negative flow through the filter disc stack. To measure the background contamination of the process blank, dispense 50 milliliters of ultrapure water slowly over the nanomembrane in the center of the top disc using the rinsed syringe. Allow the ultrapure water to filter through. Once the sample is dry, turn off the vacuum. Using clean tweezers carefully remove the filter discs from the gaskets and place them into a clean labeled container, such as a glass Petri dish or a darkened box. Image the filter discs under microscopy for optical analysis and particle counting. For experimental liquid samples, repeat the syringe rinsing process with an additional cleaned gasket and syringe filter unit. Next, uptake the desired amount of the new sample and dispense the sample slowly over the nanomembrane in the center of the top disc. Once sample filtration is complete, rinse the membrane three times with one milliliter of ultrapure water. Rinse two glass screw cap containers three times with ultrapure water. Prepare a 0.1 milligram per milliliter solution of Nile red in ultrapure 99% isopropyl alcohol in a clean glass container. Gently invert the container 10 times to mix the solution. Filter the Nile red solution into the second glass screw cap container. Place the filter disc to be stained onto the support frit of the vacuum collection flask, and pipette 20 microliters of the 0.1 milligram per milliliter Nile red solution onto the nanomembrane at the center of the filter disc. Incubate the stain on the nanomembrane for five minutes and then vacuum filter the stain. Rinse the filter disc three times with one milliliter of ultrapure 99% isopropyl alcohol. To remove excess Nile red stain. Allow the filter disc to sit on the support frit with the vacuum on for two minutes to filter and dry any residual liquid. If it still does not dry after two minutes, transfer it to a 70 degree Celsius oven for two to five minutes using a clean glass Petri dish. For particle quantification, immobilize the filter disc on a microscope slide using a silicone gasket and move it onto the microscope stage. Image the nanomembrane using bright field illumination so that the maximum detected counts are approximately 90% of the detector camera's maximum range. Image the nanomembrane using fluorescent illumination so that the maximum pixel intensities are around 25% of the detector camera's maximum range. Finally, save the acquired images as a 16 bit composite TIFF file. Bare silicon nitride and gold coated silicon nitride nanomembranes were suitable for specific analysis types. Bare silicon nitride was suitable for transmission based optical techniques as well as spectroscopy, while gold coated silicon nanomembranes were suitable for reflection based techniques. An ideal cascade of data is shown that was generated off a single silicon nanomembrane. Suspected microplastic particles stained with Nile red indicated that the tested tap water samples had a significantly higher count of particles greater than 20 microns compared to the 8 to 20 micron subfraction. Raman spectra collected with an 830 nanometer laser had a high correlation coefficient on the same particle analyzed with optical microscopy. Spectra revealed that the particle was comprised of polyethylene. Scanning electron microscopy revealed detailed morphological features of particles captured on the silicon nanomembrane. Energy dispersive X-ray spectroscopy analysis showed that the main particle composition was primarily carbon and nitrogen. This along with a trypan blue stain uptake suggests that the particle is likely organic in origin. Suboptimal sample preparation yielded unclear data. Improperly rinsed Nile red stain makes particle identification difficult and suboptimal Raman spectra with a low correlation coefficient was obtained, suggesting that the particle's chemical identity cannot be reliably confirmed.