Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/06—Investigating concentration of particle suspensions
  • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
  • G01N15/0612—Optical scan of the deposits
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/06—Investigating concentration of particle suspensions
  • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
  • G01N15/0618—Investigating concentration of particle suspensions by collecting particles on a support of the filter type
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/44—Sample treatment involving radiation, e.g. heat
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
  • G01N2001/2866—Grinding or homogeneising
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N2015/0042—Investigating dispersion of solids
  • G01N2015/0053—Investigating dispersion of solids in liquids, e.g. trouble

Definitions

• the invention relates to detection and analysis of plastic contamination in for example food and beverages.
• Microplastics defined in the art as plastic particles in the 5mm-l pm size range
• nanoplastics defined in the art as plastic particles in the 1 nm -1000 nm size range
• plastic particles are described in literature as a significant environmental and health concern. Since 1950, more than 9 billion tons of plastics have been produced, among which more than 5 billion tons have been disposed as waste. It takes more than 300 years for plastics to degrade in the natural environment. During degradation, plastic waste fragments into smaller and smaller pieces and leaches out hazardous chemicals. Besides the unintentionally made microplastics, plastic microbeads are widely applied to personal care and house-hold products. Eventually, they enter the sewage system and then into the water system. These minuscule plastic fragments have infiltrated ecosystems and found their way into the food chain.
• Microplastics can enter human gastrointestinal (GI) tract via ingestion. According to a recent report by WWF and the University of Newcastle, an average adult ingests 5 grams of microplastics weekly, equivalent to the weight of a credit card, or 250 grams in a year. So far, microplastics have been reported in food and water, and recently in human placenta. Although their impact on human health is not yet fully understood, microplastics are found to be associated with serious diseases such as cancer, immune system disruption, childbirth defects, diabetes, digestive system dysfunction, inflammation. The prevalence and persistence of microplastics have raised alarms about their long-term impact on human health.
• Microplastic particles smaller than 150pm are the most concerning for food safety reasons, as they are small enough to be ingested unintentionally and are known to trigger inflammation and immune responses in the human GI tract.
• Microplastic particles smaller than 10pm can cross the biological barriers and can translocate into the edible parts of plants and animals for food consumption. The particles smaller than 300nm could enter the blood and organs of human body posing a health risk.
• micro-FTIR and micro-Raman have been widely applied to analyse microplastics down to 10 microns.
• Scanning electron microscopy could be applied to image particles down to the nanometre sizes, but with limited capability for chemical composition analysis.
• Identification of microplastics using SEM often relies on EDS to distinguish carbon-rich particles from particles made of other elements.
• the method could not exclude any other carbonaceous micropollutants such as black-carbon or brown-carbon particles from the analysis.
• fluorescence microscopy has not been widely adopted for detecting microplastics due to the uncertain staining selectivity for plastic particles. Presence of other organic matters such as proteins, lipids could introduce artifacts and background signals that will interfere with those from stained particles.
• Microplastic analysis in food is further complicated by the complicity of food itself.
• digestion treatments Besides the potential artifacts introduced by the harsh chemical treatment at elevated temperatures, the different digestion protocols used for different types of food make the data not directly comparable.
• the present invention comprises of a method for separating and analysing plastic particles from food or beverage products.
• the method comprising the general steps of (1) an optional sample homogenization depending on the type of sample, (2) an optional filtration to remove coarse particles or food structures, (3) an optional digestion, (4) filtration to isolate particles with a desired size range for analysis, (5) staining, (6) imaging and analysis, (7) plastic content confirmation.
• biological and chemical agents may be used.
• Biological agents such as enzymes or chemical agents (e.g. acids or bases) may be used to remove biological materials under these conditions, thereby avoiding any artifacts resulting from harsh treatments.
• the samples can go through one or several filtration steps to select particles of the targeted size ranges (>10pm, 10-lpm, 1-0. lpm).
• Particles residing on the filters are stained with a one or more fluorescent dyes that have a high affinity to plastics.
• fluorescent staining one can image and analyse the quantity and sizes of the isolated particles using fluorescence microscopy.
• fluorescence emission spectra and lifetime different types of plastics can be identified.
• the sample on the filter undergoes a gradual thermal heating treatment.
• thermal heating treatment By monitoring the morphological evolution and heat decomposition of the particles, one can distinguish plastic particles from non-plastic particles in the previous fluorescence analysis and identify different plastic types based of their different melting points. The identification of different types of plastics can be carried out based on their melting points even without staining with a dye.
• the filter being used is resistant to temperatures up to 400, 450 or 500 °C.
• suitable filter material are silicon based filters, aluminium or other metal based filters, and ceramic filters.
• the invention relates particularly to the isolation and staining of plastic particles in food and food ingredients, and in beverages, including water, packaged in plastic bottles, as well as in glass bottles.
• the methods of the present invention are also applicable for other types of samples such as plastic particle detection in environmental monitoring, wastewater treatment, biomedical research, detection in tissue samples or body samples such urine or blood.
• samples such as plastic particle detection in environmental monitoring, wastewater treatment, biomedical research, detection in tissue samples or body samples such urine or blood.
• the same principle can be applied wherein the sample is processed such that isolate particles with a diameter below 100 pm are isolated, stained for the presence of plastic labels, and subsequently heated to detect the background signal by nonspecific staining of non-plastic particles.
• Heating particles after analysis and repeating the analysis allows to determine nonplastic particles in the sample.
• a method of determining plastic particles in a food or beverage sample comprising the steps of: a. Providing a food or beverage sample free from packaging material, b. Optionally homogenizing the food or beverage sample, c. Optionally performing a digestion on the food or beverage sample, d. Filtering the food or beverage sample to isolate particles with a diameter below 100 pm, e. Staining isolated particles of step d, f. Identifying particles stained in step e), g. Heating the stained particles of step e) thereby deforming or decomposing the plastic particles , h. Staining particles remaining after step g) i. Identifying stained particles of step h), j. Based on the identification in step f) and i) determining the number of plastic particles.
• the method allows identifying the number of plastic particles in the method allowing to determine the concentration in the food or beverage sample.
• the method allows to identify the type of plastic.
• the homogenizing step and digestion step can be omitted in case of for example bottled water and other liquids.
• Sample can refer to a food or beverage ready for consumption, or can refer to an ingredient to be used in a food or beverage.
• the method can be performed in a continuous setting whereby the steps or performed consecutively and in the same place.
• the homogenization and digestion, the coarse filtration of step d, optional further fractionation and the staining and analyses can be done at separate locations.
• a filter is made of a thermally stable material (i.e. silicone, ceramic, or of aluminium oxide).
• Filters with a well-defined and homogeneous pore diameter and resistant against a temperature up to 400 or 500 °C can be used as well.
• a device for analysing plastic particles in a food or beverage sample comprising :
• An optional unit for mechanically homogenizing a food or beverage sample is an optional unit for mechanically homogenizing a food or beverage sample
• An optional unit for chemical or enzymatic digestion of a food or beverage sample A filtration unit for retaining particles with a diameter below 100 pm, 16. A unit for staining and detecting said particles on a support,
• a unit for heating said particles on said support to a temperature modifying or decomposing plastic particles is provided.
• Figure 1 Flowchart of an embodiment of the present invention.
• Figure 2 Fluorescence from NR-stained plastics can be used to distinguish different plastic types based on (a) emission spectra and (b) fluorescence lifetime.
• FIG. 1 gives a general overview of the methods of the invention.
• a food or beverages sample is homogenised, if necessary.
• a filtration step is performed to remove larger particles while allowing the passage of microplastics.
• an chemical or enzymatic treatment can be performed to e.g. further reduce the particle size of non-plastic material, reduce the viscosity of the sample or to degrade lipids or carbohydrates.
• the fraction with particle size below 100 pM is further fractionated by filtration over filters with decreasing pore size, where plastic particles and other small particles (organic and/or non-organic) remain on the filter.
• Staining with a dye already allows to identify the plastic particles which reside on the filter.
• the optical signal from a fluorescent dye can be used for determining fluorescence intensity, spectrum and lifetime.
• heating plastic particles can be identified and classified by their glass transition temperature, melting temperature, and differences in properties of the dye upon heating.
• Non-plastic heat resistant particles which falls-positively stained with the dye will not degrade.
• Poly particles in the context of the present invention relates to particles with a diameter less than 100 pm.
• a specific embodiment hereof are particles with a diameter between 100 pm and 0.1 pm.
• fractions can be isolated with a diameter below 100 pm, below 10 pm, below 1 pm; with a diameter between 100 pm and 10 pm, between 100 pm and 1 pm, between 100 pm and 100 nm; with a diameter between 10 pm and 1 pm, between 10 pm and 100 nm; and with a diameter between 1 pm and 100 nm.
• Homogenization steps This breaks a food or beverage sample comprising solid parts into smaller pieces for analysis and comprises in a specific embodiment of the following steps: i. Collecting the food or beverage samples and remove any packaging materials or non-edible parts from the sample.
• This step is to remove the coarse particles that may enter the next digestion step. As larger particles are more difficult to be digested by enzymes or would require longer times for the enzymes to fully digest them. By removing the coarse particles, we expect to shorten the time duration and quantity of enzymes required in the digestion step.
• (3) digestion of organic matter in food or beverage samples may be done using biological and/or chemical agents. Biological agents such as enzymes may be used to perform enzymatic digestion of lipids and proteins may be done using lipase and proteinase-K. Enzymatic digestion take place under mild conditions and do not cause any artifacts on the plastic particles. The quantity and types of enzymes will depend on the organic (nutrition) content such as lipids, proteins, cellulose, carbohydrates, etc.
• This step is to remove the organic (nutrition) content that may interfere with the staining and fluorescence imaging steps, particularly to remove proteins and lipids that may be stained by fluorescent dyes.
• chemical digestion may be performed using i.e. alkali (NaOH, KOH) or oxidizing agents (H2O2) to remove any remaining organic matter from the food or beverage samples.
• alkali NaOH, KOH
• H2O2 oxidizing agents
• An example of a digestion method comprises the following steps: i. OAdd lOOpL of digestion buffer (Triton X-100 2.5%) to the homogenized filtered food or beverage sample (1 mL) to create the desired enzymatic conditions.
• Enzymatic digestion conditions may be optimized depending on the sample type. Additionally, samples may also be treated with 10 to 30 % H2O2 from room temperature to 70 ° C to oxidize organic matter.
• extra steps are used.
• a calcium chelating agent such as EDTA.
• micro-fabricated silicon filter allows to guarantee a robust analysis outcome.
• Silicon filters further have a flat surface, and good contract in transmission microscopy.
• these filters also do not get stained with fluorescent dye, resulting in lower background signal and provides better sensitivity.
• micro-fabricated silicon filters that have well-defined and homogeneous pore diameter are used to collect plastic particles in the defined size ranges.
• the size ranges can be configured by using filters with corresponding pore diameter.
• aluminium oxide filters may be used.
• Filters can be purchased from SmartMembranes GmbH. However, it is not necessary to utilize silicon or alumina filters. Filters made of other materials with flat surface and uniform pore size can be employed.
• the samples after the filtration step and before the staining step can also be suitable for alternative analysis methods such as FTIR, Raman, and Scanning Electro Microscopy.
• Numerous dyes can be used for fluorescent tagging of custom-made plastic particles, enhancing the range of observable plastic shapes and polymer compositions.
• these dyes are Nile Red and its derivatives, 4-Dimethylamino-4'-nitrostilbene (DANS), oil red EGN, Rose Bengal, Neutral Red, Trypan Blue, and a selection of textile dyes available in the market. Although these dyes have demonstrated their effectiveness in staining plastic particles, their affinities for distinct polymers differ. Notably, Nile Red stands out as the most commonly employed dye for plastic staining.
• a combination of a mixture of various fluorescent dyes may be used to ensure different types of plastics are labelled with a reasonable signal-to- background ratio
• analysis may be performed wherein multiple fluorescence signals are analysed such as intensity, emission spectra, and lifetime to distinguish different plastic types, this would work both in the single-dye settings and the multidye settings.
• An example of a staining comprises the steps of: i. Prepare a solution of a one or more fluorescent dyes such as Nile Red (Sigma Aldrich) by dissolving the dye in an appropriate solvent DMSO/PBS (50/50). The concentration of the solution is 10 pg/mL
• Fluorescence microscopy imaging and analysis images of (i) fluorescence intensity, or (ii) fluorescence emission spectra, or (iii) fluorescence lifetime may be acquired for analysis.
• Fluorescence imaging comprises the following steps: i. After placing the sample on the microscope stage focus on the samples using an objective lens (i.e. 40x air objective (NA 0.60)).
• an objective lens i.e. 40x air objective (NA 0.60)
• the temporal response of single particles is also recorded using fluorescence lifetime imaging (FLIM).
• FLIM fluorescence lifetime imaging
• the acquisition of images is carried out for 50 frames per measurement. This system is integrated with a phasor analysis software, which allows the representation of decay lifetime components on a 2D vector plot.
• an additional imaging may be performed to exclude possible contamination with carbonaceous particles:
• An example hereof comprises steps of: i. Turn on the multiphoton laser and set the wavelength at 810 nm.
• the entire food or beverage sample under analysis undergoes a thermal heating process, wherein the environmental temperature is increased from room temperature to temperatures up to 400 Celsius degrees. As different materials have different melting temperatures, the morphological changes of the particles while elevating the temperature can be monitored.
• the heating is done using a thermal stage for microscopes, detection can be performed by optical microscopes as well as electron microscopes. The upper temperature is selected to destroy certain types of plastics for analysis.
• thermal heating and staining comprising steps of: i. After microscopy analysis, keep the silicon membrane filters in the sample holder.
• re-staining may be required, repeat step (5).
• Steps (7) to (9) may be repeated multiple times with different heating upper temperatures to confirm different types of plastic particles.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Immunology (AREA)
• Analytical Chemistry (AREA)
• Physics & Mathematics (AREA)
• General Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Dispersion Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Food Science & Technology (AREA)
• Signal Processing (AREA)
• Medicinal Chemistry (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=87930247

Family Applications (1)

Country Status (2)

Families Citing this family (1)

• 2024
  • 2024-08-30 EP EP24762323.4A patent/EP4771355A1/de active Pending
  • 2024-08-30 WO PCT/EP2024/074277 patent/WO2025046069A1/en not_active Ceased

Also Published As

Legal Events