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WO2024236575A1 - Procédé électrochimique et capteur de détection de pfas - Google Patents

Procédé électrochimique et capteur de détection de pfas Download PDF

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Publication number
WO2024236575A1
WO2024236575A1 PCT/IL2024/050478 IL2024050478W WO2024236575A1 WO 2024236575 A1 WO2024236575 A1 WO 2024236575A1 IL 2024050478 W IL2024050478 W IL 2024050478W WO 2024236575 A1 WO2024236575 A1 WO 2024236575A1
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Prior art keywords
amino acid
electrode
fluorinated
electrodes
polypeptide
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Hadar Shmuel BEN YOAV
Miriam Amiram
Avner Ronen
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BG Negev Technologies and Applications Ltd
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BG Negev Technologies and Applications Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • 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
    • G01N33/18—Water
    • G01N33/1826—Organic contamination in water
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier

Definitions

  • PFAS Poly-and perfluoroalkyl substances
  • PFOA perfluorooctanoic acid
  • PFOS perfluorooctane sulfonic acid
  • PFAS have harmful and long-lasting effects on the environment, and can build up inside the human body, leading to increased cholesterol, diabetes, and kidney cancer.
  • the U.S. Environmental Protection Agency recommends a 70 ppt concentration limit for PFOA and PFOS (PFAS Fact Sheets, 2024) , and therefore high sensitivity and selectivity are needed for determination of PFAS in various environmental matrices.
  • the standard techniques for detecting PFAS which include gas or liquid chromatography and mass spectrometry, albeit fairly accurate, require advanced instrumentation facilities, with samples to be sent to a lab for analysis .
  • Electrochemical detectability of PFAS depends on several factors, including an effective surface modification of the working electrode, that can respond to the presence of varying levels of PFAS in solution by creating an electrochemical signal in a concentration dependent manner, and a selection of an appropriate electrochemical technique for the measurement, showing acceptable limit of detection and sensitivity.
  • an effective surface modification of the working electrode that can respond to the presence of varying levels of PFAS in solution by creating an electrochemical signal in a concentration dependent manner
  • an appropriate electrochemical technique for the measurement showing acceptable limit of detection and sensitivity.
  • US 2023/0125201 shows immobilization of a redox indicator (Meldola Blue; MDB) by electrodeposition on an electrode surface.
  • MDB redox indicator
  • US 2024/0035998 provides a sensing electrode with a coating formed by electrochemical polymerization of a monomer and the analyte (PFAS) on the electrode surface, followed by removal of the analyte from the coating, thus leaving cavities behind, which during testing, can be filled with, or occupied by, PFAS in the test solution.
  • PFAS analyte
  • US 2024/0068971 relates to application of a polymer layer on the surface of the working electrode with affinity sites to bind (specifically or non-specif ically) various PFAS. Voltammetry techniques (cyclic voltammetry and differential pulse voltammetry) were reported to detect PFOA and PFOS .
  • the invention
  • voltammograms recorded by cyclic voltammetry (CV) acquired with a working electrode coated with a fluorinated polypeptide, immersed in buffer solutions containing the ferrocyanide/ ferricyanide redox couple [Fe(CN)6 3 ⁇ Fe (CN) 6 4 ⁇ ] , show decreased oxidation and reduction peak currents in the presence of PFAS, compared with voltammograms generated by the bare electrode.
  • electrochemical impedance spectroscopy EIS
  • EIS electrochemical impedance spectroscopy
  • FIG. 1 shows the major components of the invention.
  • a sample collected from the environment e.g., groundwater sample
  • a surface modified electrode e.g., a surface modified electrode.
  • Effective surface modification was achieved by deposition of elastin like peptides (ELPs) , as described in detail below.
  • a redox couple (marked by the green star) is added to the solution, which is analyzed in an electrochemical cell connected to a potentiostat, to perform an electrochemical measurement with the surface modified electrode acting as the working electrode.
  • EIS can sense the presence of PFAS in the solution.
  • the invention is primarily directed to a method for detecting and optionally quantifying one or more poly-and perfluoroalkyl substances (PFAS) in an aqueous sample, comprising adding a redox couple to the sample, electrochemically analyzing the sample with at least one surface-modified working electrode having fluorinated intrinsically disordered polypeptide deposited on its surface, by measuring current, voltage and/or calculating impedance as a PFAS-related signal, and determining the presence of PFAS and optionally the level of PFAS, in the s amp 1 e .
  • PFAS poly-and perfluoroalkyl substances
  • Another aspect of the invention is an electrochemical sensor, comprising the fluorinated intrinsically disordered polypeptide- coated electrode as a working electrode.
  • the fluorinated intrinsically disordered polypeptide-coated electrode is usually incorporated in an array of working electrodes, alongside at least one electrode selected from the group consisting of bare electrode and electrodes coated with non-f luorinated intrinsically disordered polypeptides showing varying degree of hydrophobicity (e.g., polypeptides consisting mostly of hydrophobic natural and/or unnatural amino acids versus mostly hydrophilic natural and/or unnatural amino acids, or controlling hydrophobicity by incorporation of modified amino acids bearing hydrophilic/hydrophobic substituents at specific positions) .
  • hydrophobicity e.g., polypeptides consisting mostly of hydrophobic natural and/or unnatural amino acids versus mostly hydrophilic natural and/or unnatural amino acids, or controlling hydrophobicity by incorporation of modified amino acids bearing hydrophilic/hydrophobic substituent
  • PFAS that can be detected are represented by the formulas R-C n F2n+i or R'-C n F2 n -R' ' where n is in integer in the range from 3 to 15, e.g., from 5 to 15, and R, R', R" are functional groups devoid of carbon or C-F bonds, such as -COOH and -SO3H.
  • PFAS may be divided into long and short chain derivatives.
  • PFNA perf luorononanoic acid
  • PFDA perfluorodecanoic acid
  • PFBA perfluorobutanoic acid
  • PFOS was tested, either alone or in admixture with PFOA, PFHxS, PFHxA, PFBA and PFBS .
  • the sample may be collected from the environment (a waterbody, groundwater with up to 200 ppt PFAS) , from industrial and municipal streams (wastewaters with up to 300 ppb PFAS and even higher, effluents for irrigation with up to 300 ppb PFAS) and is usually conducted onsite, in a field-deployable manner, with the aid of portable sensors the design of which is described below.
  • the working electrode is preferably made of a noble metal, e.g., gold, platinum, rhodium, and iridium.
  • a carbon-based electrode e.g., a glassy carbon electrode
  • the sensor of the invention is not limited to specific design.
  • One useful configuration is a chip configuration with one or more electrodes deployed on a substrate, e.g., a glass wafer, optionally atop of a titanium adhesion layer attached to the substrate.
  • Figure 2A shows an individual gold electrode (1) , created by photolithography and lift-off techniques described below.
  • the electrodes may be disc-shaped, with surface area in the range of 5 to 20 mm 2 .
  • the electrodes are protruding from the surface of the substrate (2) , with each electrode being encircled by a wall made of an epoxy polymer (3) .
  • the electrode is joined by a thin wire (4) to a contact pad (5) , to be connected to a potentiostat or a galvanostat.
  • another aspect of the invention is a process for preparing an electrochemical sensor by photolithography and liftoff techniques, comprising: depositing on a substrate an electrode material, optionally atop of an adhesion layer (e.g., made of titanium) attached to the substrate, to create electrodes that are spaced apart from each other, the electrodes are protruding from the surface of the substrate, wherein each electrode is encircled by a wall made of a polymer (e.g., epoxy) ; cleaning the electrode surface; and coating at least one electrode with a fluorinated intrinsically disordered polypeptide, and optionally other electrodes with nonfluorinated polypeptides that are described in more detail below.
  • an adhesion layer e.g., made of titanium
  • Coating and immobilization of the fluorinated intrinsically disordered polypeptide onto a noble metal can be achieved by incorporation of cysteine (N-terminal, C-terminal, internal) into the fluorinated polypeptide, to benefit from the strong interaction between the thiol (-SH) end group and the metal.
  • cysteine N-terminal, C-terminal, internal
  • thiol thiol
  • application of a terminal cysteine-f luorinated polypeptide onto the electrode surface by employing coating techniques such as drop-casting, spin-coating and spray coating, results in the deposition of a thin film consisting of the fluorinated polypeptide on the electrode surface.
  • the preferred method of coating is drop-casting, and such an approach is shown schematically in Figure 2B.
  • a solution of fluorinated intrinsically disordered polypeptide with a cysteine end group [in PBS, at concentration from 15 to 50 pM] is spread over a noble metal, e.g., gold electrode (1) patterned on the SiO2 wafer.
  • a noble metal e.g., gold electrode (1) patterned on the SiO2 wafer.
  • the volume of the coating solution is from 1 to 10 pL .
  • Figure 2B shows the gold-sulfur coupling.
  • the f luorine-containing functional groups embedded in the polypeptide applied on the gold surface are labeled by circles with the letter F inside.
  • the film is dried under controlled conditions, e.g., incubation for a few hours (from 1 to 12h) at 4° degrees or at room temperature, achieving stable immobilization of the polypeptides on the electrode surface in the form of thin solid films.
  • the film is washed with PBS and DDW (whereby presumably a monolayer is formed) .
  • the chip is cleaned, e.g., with acetone, methanol, isopropanol, followed by washing with DDW to remove the organic (photoresist) residues from the top of the electrodes.
  • the electrode is cleaned to improve electrochemical detectability.
  • cleaning protocols can be used for this purpose, for example, treatment with ultraviolet ozone, sulfuric acid and potential cycling, aqua regia (nitric acid/hydrochloric acid) , piranha solution (sulfuric acid + hydrogen peroxide) and potassium hydroxide + hydrogen peroxide solution.
  • the invention provides a process for preparing a sensor as defined above, wherein the cleaning step comprises treating the electrode with a solution of alkali (e.g., potassium) hydroxide and hydrogen peroxide .
  • alkali e.g., potassium
  • carbon-based electrodes e.g., glassy carbon
  • immobilization of the polypeptides onto the surface requires other type of linkages, so as to strongly bind the polypeptides chains to the surface.
  • Acceptable surface attachment chemistries are detailed for example in (https://doi.org/10.1142/S1793048006000Q45, https : //doi .org/10.1007/s00604-015-1623-4)
  • the arrays of working electrodes for example, with the following composition of bare and coated electrodes: n fluorinated polypeptide, n bare, n non-fluorinated polypeptide
  • n the number of electrodes of each type, may be independently a number from 1 to 5; n>l for repetitions
  • n may be arranged in different designs and geometries, including a simple and straightforward configuration based on the use of an electrochemical measurement cup to hold the sample, fitted with a perforated cover; the holes in the cover correspond in number and size to the electrodes, such that individual working electrodes can be inserted into the measurement cup through the holes to be immersed in the sample.
  • Commercial counter electrode e.g., commercial Pt wire
  • commercial reference electrode Ag/AgCl
  • WO 2018/225058 e.g., a cylindrical body made of silicon, polyvinyl alcohol or polydimethylsiloxane, which is a few cm long and with diameter is in the range from 2 to 3 cm.
  • the accessible surfaces of the electrodes were deployed on one base of the tubular body: a discshaped reference electrode positioned concentrically and coaxially in respect to the cylindrical body, a counter electrode at the vicinity of the reference electrode and multiple surface modified working electrodes positioned in radial direction from the reference and counter electrodes and evenly distributed along the perimeter of the base of the cylindrical body.
  • Alternative designs are based on fabrication of electrodes on silicon wafers using lithography and liftoff techniques as mentioned above in reference to Figure 2A and shown in more detail in reference to Figure 3, e.g., for a portable device.
  • a substrate (2) is cleaned, a first photoresist (6) is applied (either negative, positive or image reversal resist) , e.g., by spin coating, spray coating or dip coating, to produce a thin uniform layer on the substrate, followed by soft baking.
  • a first mask is aligned, to transfer the pattern corresponding to electrodes' sites onto the surface of the substrate.
  • the photoresist is exposed through the pattern on the mask with UV light (7) followed by a development step (8) .
  • bare electrodes are deposited in the intended sites, e.g., first titanium which serves as an adhesion layer (9) and then gold (1) followed by lift off procedure that results in a plurality of individual, spaced apart, gold electrodes protruding from the glass substrate.
  • the gold electrodes need to be encircled by a wall, to define the active surface area of the electrode. This is accomplished again by photolithography, as shown by the sequence of steps depicted in the right column of Figure 3B.
  • the gold electrode is encircled by a wall made of epoxy polymer, e.g., bisphenol A Novolac epoxy such as SU-8 photoresist, which is 5-7 pm thick.
  • Figures 3A-3B show just one approach to the structuring of electrodes on a wafer.
  • the application of positive, negative or image reversal photoresists is often a matter of choice and convenience.
  • structuring via etching i.e., wet etching/dry is also possible.
  • the desired coatings are applied, e.g., by drop-casting as described above.
  • the electrochemical sensor of the invention may be combined with commercial counter and reference electrodes to be connected to a potentiostat or galvanostat to run the electroanalytical method, e.g., voltammetry or EIS.
  • a disc-shaped counter microelectrode, and Ag/AgCl electroplated reference microelectrode may be applied on the base substrate adjacent to the working electrode.
  • Silver/silver chloride miniature electrodes are prepared as described in WO 2022/137238, Example 6.
  • fluorinated and non-f luorinated polypeptides that are used to modify the surface of the electrodes, in their most general form, these are intrinsically disordered proteins such as elastin-like polypeptides (ELPs) , resilin-like polypeptides, or other artificial polypeptides such as Proline/alanine-rich sequence (PAS) and the unstructured polypeptide Xten, that may or may not demonstrate temperature responsive phase transition behavior, having fluorinated residues (and corresponding non-f luorinated polypeptides) .
  • ELPs elastin-like polypeptides
  • PAS Proline/alanine-rich sequence
  • unstructured polypeptide Xten that may or may not demonstrate temperature responsive phase transition behavior, having fluorinated residues (and corresponding non-f luorinated polypeptides) .
  • ELPs comprising a repeat unit having the amino acid sequence VPGXG (Val-Pro-Gly-X-Gly denoted by SEQ ID NO. 1, i.e., the 'VPGXG' pentapeptide motif) wherein X, termed the guest-residue position, is any natural amino acid (except proline) or synthetically modified amino acid (also known as "unnatural” or "non-canonical amino acid", ncAAs) .
  • the number of repeat units i.e., the pentapeptide motif
  • Exemplary fluorinated modified (synthetic) amino acid residues which can serve as X 1 units are derived from the natural amino acid residues Tyrosine (Tyr, Y) , Phenylalanine (Phe, F) , Tryptophan (Trp, W) , Alanine (Ala, A) , Valine (Vai, V) , Isoleucine (lie, I) , Leucine (Leu, L) and Methionine (Met, M) .
  • the fluorinated hydrophobic modified (synthetic) amino acid residues are derived from aromatic amino acid residues, i.e., from Phe, Tyr or Trp.
  • Fluorinated functionality is one (or more) fluorine atoms covalently bonded to, e.g., a carbon ring, or fluorinated alkyl such as C1-C5 alkyl, covalently bonded to a carbon ring.
  • a sufficient level of fluorine atoms in the polypeptide is not less than 5 atoms per polypeptide, e.g., not less than 10, e.g., from 15 to 45, e.g., from 25 to 35; or from
  • X 1 is L-phenylalanine substituted at positions ortho (o) , meta (m) , or para(p) with fluorine atom(s) or fluorinated CIGS alkyl (s)
  • X 2 is 4 -hydroxy-L-phenylalanine (tyrosine)
  • X 3 is L-phenylalanine substituted at positions o, m, or p with an CIGS alkoxy, alkenyloxy or alkynyloxy group.
  • X 1 is trif luoromethyl-substituted L-phenylalanine and X 3 is alkynyloxy (e.g., propargyloxy) -substituted L- phenylalanine, e.g. X 1 and X 3 are:
  • the following compounds can be incorporated into the polypeptides as the fluorinated unit: m-trif luoromethyl-L- phenylalanine, m-f luoro-L-phenylalanine, p-fluoro-L- phenylalanine, 2 , 6-dif luoro-L-phenylalanine, 2 , 4-dif luoro-L- phenylalanine, 3, 4, 5-trif luoro-L-phenylalanine, 2-fluoro-L- phenylalanine and 3-f luoro-L-tyrosine, all of which are commercially available (e.g., from Chemimpex) .
  • the fluorinated polypeptide exemplified herein comprises amino acid sequence shown in SEQ ID. 6, the first nonfluorinated polypeptide has the sequence shown in SEQ ID. 5 and the second non-f luorinated comprises amino acid sequence in SEQ ID. 7.
  • polypeptides for use in the present disclosure may be prepared by any method known in the art, according to the desired amino acid sequence.
  • ELPs may be prepared by bioengineering methods known to the skilled artisan, which include inserting a nucleic acid sequence encoding the polypeptide into a suitable host (e.g., a bacterial strain) , expressing the encoded polypeptide and purifying it from the resulting culture.
  • a suitable host e.g., a bacterial strain
  • the polypeptide may be prepared by following the procedures described by Gueta, 0.
  • polypeptides for use in the present disclosure may further comprise additional amino acid sequences, optionally modified amino acids, such as short linkers positioned internally or at the N terminus or the C terminus of the polypeptide.
  • a redox couple e.g., at a concentration in the range from ImM to 10mM.
  • Suitable redox couples include the ferrocyanide/ ferricyanide couple and other iron-based couples, e.g., ferrocene carboxylic acid/FCA ⁇ .
  • the electrochemical cell is assembled by connecting the electrodes to e.g., potentiostat, optionally with the aid of a multiplexer, to run the electrochemical analysis, such as voltammetry where a potential sweep takes place and the current is measured as the analytic signal ( cyclic voltammetry ( CV) , or di f ferential pulse voltammetry ( DPV) ) ; or electrochemical impedance spectroscopy (EIS ) where a sinusoidal potential over a frequency range is applied and current is measured, and resistance and/or capacitance are usually calculated .
  • Figure 4 shows the Randles equivalent circuit that was used for fitting the impedance spectra produced by the EIS , i . e .
  • the invention provides a method of quanti fying one or more poly-and perfluoroalkyl substances (PEAS ) in an aqueous sample with the aid of the electrodes described above , comprising analyzing the sample by EIS to generate impedance spectra, fitting the impedance spectra with Randles circuit , and calculating one or more of the following components of the Randles circuit : the charge trans fer resistance , the solution resistance , the double layer capacitance and the Warburg impedance . As shown below, the charge trans fer resistance correlates strongly with the concentration of the PEAS in solutions .
  • a Partial Linear Regression Model can be used to determine the PEAS concentration .
  • the Origin Pro program can be used to organi ze the Bode plot data into the cell format .
  • the raw impedance spectra are assembled . Sequential placement of the signals was done for individual sensors .
  • the approach used was partial least square regression ( PLSR) , which is a combination of Principal Component Analysis ( PCA) and Multiple Linear Regression (MLR) .
  • PCA Principal Component Analysis
  • MLR Multiple Linear Regression
  • the independent variable chosen is the impedance values at varying frequencies .
  • the dependent variable chosen is the analyte solution ' s concentration .
  • the identi fied independent variables were used .
  • the components were extracted using the Singular Value Decomposition ( SVD) technique of computation .
  • a cross-validation procedure is then used to confirm the anticipated model .
  • Origin Pro software the ideal number of components is calculated using the predicted residual sum of squares ( PRESS ) and its root mean .
  • the number of factors involved is known as the "optimal number of factors" when the smallest root mean is attained .
  • the quality of the model is then assessed by the various combination of root mean PRESS between the actual concentration and predicted concentration .
  • the ef ficiency of the model was further evaluated by calculating ( limit of detection) LOD, Pearson' s Co-relation Co-ef ficient ( PCC ) , and PRESS values .
  • the invention further provides a method comprising analyzing the sample by EIS to generate impedance spectra and using a Partial Linear Regression Model (PLSR) to determine the PEA concentration based on the measured impedance spectra, wherein the PLSR model was created using as independent variable impedance values at varying frequencies , and the dependent variable is the PEAS concentration in solution .
  • PLSR Partial Linear Regression Model
  • Model predictability was assessed by testing with unseen samples .
  • Additional aspect of the invention is an electrochemical sensing system comprising the sensor described above, with the working electrodes, counter electrode and reference electrode connected to a potentiostat or a galvanostat, and a processor configured to determine, based on a chemometric model, the concentration of PEAS in a sample.
  • Acetone, isopropanol, methanol, hydrochloric acid, sulfuric acid and hydrogen peroxide were used without further purification.
  • Simulated ground water was prepared using the Humic acid (Thermo Fisher Alfa Aesar) , Potassium Chloride (KC1) from Acros Organics and Sodium Sulphate (Na2SO4) from Alfa Aesar, Deionized water (>18 M ⁇ ) was obtained from a Super Q water system (Millipore) .
  • the redox couple 5 mM ferrocyanide/ ferrocyanide (Fisher Scientific) dissolved in 10 mM phosphate buffer saline (PBS) solution prepared from PBS tablet (Acros Organics) was used.
  • the simulated ground water was prepared by adding salts 5mM (KOI , Na2SO4 ) , l OuM humic acid and ferrocyanide/ ferricyanide in DDW under constant stirring on a magnetic stirrer .
  • the concentration of the salts and humic acid used to prepare the simulated solution is similar to the concentration of salts existing in real ground water samples .
  • the real groundwater samples were collected from the groundwater drill located near LoD, I srael .
  • the samples were collected in a 50mL falcon tubes and stored at room temperature .
  • the real groundwater samples were spiked with PFOS and further diluted at various concentration ranging from 5nM to 500nM along with the redox mediator .
  • FIG. 2A An individual di sc-shaped gold electrode deposited on a silicon wafer is shown in Figure 2A, with diameter of 2 mm, encircled by SU8-3005 wall which is 5-7 ⁇ m thick .
  • Individual gold electrode chips like the one shown in Figure 2A were prepared using photolithography and li ft-of f techniques described below .
  • the wafer was cleaned with acetone , isopropanol , and N2-gun .
  • the wafer was dehydrated at a hot plate temperature of 110 ° C for a period of 10 minutes .
  • the spin coating included spinning at sequential steps , starting from spinning at 500 rpm with acceleration rate of 100 rpm/ s for 20 seconds , following through spinning at 3000 rpm with acceleration rate of 300rpm/ s for 40 seconds and finally spinning at zero rpm with acceleration rate of 100rpm/ s for 1 second using a four-inch holder .
  • the deposition of Ti/Au 20/200nm using E-beam VST process takes place .
  • the NMP solution was added into the glass bowl .
  • the bowl was agitated .
  • the wafer was dipped in warm NMP for about 20 minutes before removing it away .
  • the wafer is rinsed with isopropanol and DDW respectively and finally dry it using N2 gun .
  • Quality of li ft-off process is done under a microscope . Start cleaning the wafer by applying Acetone , I sopropanol and N2 -Gun to clean it thoroughly .
  • the wafer is dehydrated at 95 degrees for 5 minutes on a hot plate .
  • the mask aligner should meanwhile be switched of f as well as warmed up for five minutes . Thereafter, the hot plate is activated during this waiting interval and set its temperature to 150 degrees , and spinning protocol is repeated as mentioned earlier . Once the wafer is placed on the spinner the vacuum is started . 7ml of photoresist was added to the center of the wafer then spun it for 5 minutes . At the time the plate was switched on and raised the temperature to 95 degrees . Next baking the wafer was done at 95 degrees for 8 minutes . The wafer was placed in the mask aligner and wait for around 5 minutes to ensure that the hot plate reaches a temperature of 95 degrees . The other plate was heated to the temperature up to 150 degrees . Conduct an exposure bake at 95 degrees for 4 minutes .
  • the glass bowl was filled with AZ EBR liquid and another with I sopropanol .
  • the wafer was placed in the AZ EBR glass bowl for 6 minutes rotating it gently .
  • the wafer is rinsed in the I sopropanol bowl for 10 seconds followed by cleaning with DDW and N2 -Gun .
  • a hard bake is performed by baking the wafer at 150 degrees for 5 minutes . The process quality was evaluated under the microscope and profilometer .
  • the chips Prior to coating with the ELPs as described below, the chips were cleaned by rinsing with acetone , methanol , isopropanol , followed by washing with DDW to remove the organic (photoresist ) residues from the top of the electrodes .
  • the chip was immersed in KOH/H2O2 solution ( 16 ml hydrogen peroxide , 3 . 2 ml of KOH) for ten minutes , and finally dried using a nitrogen gun .
  • ELPs Elastin like peptides
  • ELPs have the same amino acid sequence as ELPs-tyr, however, their amino acid sequences include the non-canonical amino acid residues p-Tri f luoromethyl-L-phenylalanine ( CAS No . 114926-38-4 , Chemimpex ) or 4-propargyloxy-L-phenylalanine ( CAS No . 1080496- 42- 9 , Chemimpex ) at each one of the positions of the amino acid residues Tyrosine (Y) at the sequence denoted by SEQ ID NO . 5 , resulting in ELPs termed, respectively, fluorinated ELPs (ELPs- tFF) and non- f luorinated ELP ( ELPs-pPr ) .
  • ELPs- tFF fluorinated ELPs
  • ELPs-pPr non- f luorinated ELP
  • tFF indicates the positions of p-Tri f luoromethyl-L- phenylalanine in the amino acid sequence , which is denoted herein by SEQ ID NO . 6 .
  • aaRSs engineered aminoacyl tRNA synthetases
  • E. coll strains (the genomically recoded C321.AA strain, Addgene # 73,581, which is used for efficient multi-site incorporation of multiple uAAs per protein) , harboring plasmids encoding the aminoacyl tRNA synthetases M. janaschii tyrosyl-tRNA synthetase (MjTyrRS, which is the native aaRSs) for tyrosine incorporation, Mutl-RS for pPR incorporation, or the pAcFRS.2.tl for tFF incorporation and the ELP polypeptide (the ELP polypeptide was encoded by the nucleic acid sequence denoted by SEQ ID NO.
  • ncAA In the case of native MjTyrRS, no ncAA was added, as the enzyme uses tyrosine available in the growth media. Plasmids encoding the aminoacyl tRNA synthetases and the ncAAs detailed above are commercially available, e.g., plasmids can be obtained from Addgene and ncAAs from Chem-impax.
  • the cells were harvested 24 hours after inoculation by centrifugation at 4,000 g for 30 minutes at 4°C.
  • the cell pellet was then resuspended by vortex in milli-Q water ( ⁇ 4 ml) and either stored at -80°C or purified immediately.
  • Protein purification was performed via a process known as inverse-transit ion cycling, in which phase separation of ELPs is induced via heat or the addition of salt, followed by centrifugation to separate the ELP pellet. Then, the pellet is re-dissolved in cold water or buffer, and irreversibly precipitated E.coli protein contaminants are centrifuged and removed. This process is repeated 3-5 times until the ELP protein is sufficiently pure.
  • resuspended pellets were lysed by ultrasonic disruption (18 cycles of 10 seconds sonication, separated by 40 seconds intervals of rest) .
  • Poly (ethyleneimine) was added (0.2 ml of a 10% solution) to each lysed suspension before centrifugation at 4,000 rpm for 15 minutes at 4°C to separate cell debris from the soluble cell lysate containing the polypeptides.
  • All ELP constructs were purified by a modified inverse transition cycling (ITC) protocol (Hassouneh et al., 2010) consisting of multiple "hot” and "cold” spins by using sodium chloride to trigger the phase transition.
  • ITC inverse transition cycling
  • the soluble cell lysate was incubated for up to 2 minutes at 42-65°C to denature the native E. coll proteins.
  • the cell lysate was then cooled on ice, centrifuged for 2 min at ⁇ 14,000 rpm, and the pellet was discarded.
  • the ELP phase transition was triggered by adding sodium chloride to the cell lysate (from which the native E. coll proteins pellet was discarded) or to the product of a previous cycle of ITC at a final concentration of up to ⁇ 5 M.
  • the microscopic images for a bare and an ELPs modified electrode were taken by optical microscopy (MX-50A, Olympus) , and are shown in Figure 5A (i) and (ii) , respectively.
  • the images show a difference between the surface of the bare gold electrode and ELPs film-coated gold electrode .
  • the elemental analysis was performed by X-Ray Photoelectron Spectroscopy (XPS) . (ESCALAB Xi+ Thermo-Fisher Scientific) .
  • XPS X-Ray Photoelectron Spectroscopy
  • Figure 5C and the corresponding data tabulated in Figure 5D attest to the successful immobilization of the ELPs on the gold macroelectrode: the elemental analysis through XPS indicates the presence of C, N and 0 (fluorine in the case of ELPs-tFF was not detected apparently due to disordered conformation and low concentration of ELPs deposited on the electrodes surface) .
  • the bare electrode was analyzed electrochemically in 5mM ferrocyanide/ ferrocyanide solution in PBS using cyclic voltammetry (CV) . Three cycles were performed across the potential window from -0.2 to 0.65 V (vs Ag/AgCl reference electrode) , at a scan rate of lOOmV/s.
  • ELPs-tyr (least hydrophobic- red) shows a slight decrease in the overall peak current followed by ELPs-tFF (hydrophobic due to -CF3 moiety - blue) , ELPs-pPr (most hydrophobic-green) exhibits the largest decrease in the peak current which indicates the formation of insulating layer inhibiting the transfer of electrons from the redox couple.
  • hydrophobicity of the ELPs increased, it attained the collapsed state which covers the surface area of the electrode thereby blocking the transfer of electrons.
  • the bare gold electrode and the ELPs modified electrodes functioned as the working electrode, a commercial platinum electrode served as the counter electrode and Ag/AgCl as the reference electrode.
  • the CV electrochemical measurements were carried out simultaneously using MuX16 integrated with Palmsens4 instrument, in 5mM ferrocyanide/ ferricyanide 10mM PBS solutions with 100 ppm (200pM) PFOS.
  • MuX16 integrated with Palmsens4 potentiostat is used to record the EIS spectra .
  • the electrodes were thoroughly washed with DDW and dried with nitrogen prior to the electrochemical measurement .
  • the four electrodes (bare gold, ELPs-tyr-modi f led gold electrode , ELPs-tFF-modi f led gold electrode and ELPs-pPr-modi f led electrode ) were investigated by EIS in solutions with PFOS concentration ranging from 500nM-5nM ( 500nM, 400nM, 300nM, 200nM, l O OnMm, 5nM) and also PFOS- free solution, in the presence of 5 mM ferrocyanide/ ferricyanide acting as the redox couple .
  • the parameters of the EIS measurement were as previously described in Example 2 .
  • the linear regression curves for the bare and ELPs-tyr (control) electrodes are plotted in Figure 7E, and the linear regression curves for the ELPs-tFF (fluorinated) and ELPs-pPr (hydrophobic) electrodes are plotted in Figure 7F.
  • the Limit of detection for the fluorinated and hydrophobic ELPs- modified electrode towards PEGS detection are 85.4 ⁇ 5.74 nM and 67.8 ⁇ 8.08 nM respectively.
  • the sensitivity of fluorinated ELPs (ELPs-tFF) and hydrophobic ELPs (ELPs-pPr) are 8.06 ⁇ 0.5 Ohm/nM and 45.4 ⁇ 5.4 Ohm/nM respectively .
  • PCA Principal Component Analysis
  • EIS was conducted in the electrochemical cell described above , testing the four electrodes (bare gold, ELPs-tyr-modi f led gold electrode , ELPs-tFF-modi f led gold electrode and ELPs-pPr-modi f led electrode ) as working electrodes in solutions with PFOS concentration ranging from 200nM-5nM ( 5nM, l O OnM, 200nM) and also PFOS- free solution, in the presence of 5 mM ferrocyanide/ ferricyanide acting as the redox couple , and the added chemicals , in DDW .
  • the parameters of the EIS measurement were as previously described in Example 2 ( electrodes immersed in the electrochemical cup at the same time , working consecutively) .
  • the Nyquist plots are shown in Figures 9A- 9D for the four electrodes mentioned above , respectively .
  • the charge trans fer resistance values (Ret ) were calculated from the Nyquist plots and plotted against the concentration of the PFOS in the solution, as shown collectively in Figure 9E for all four electrodes .
  • the results indicate a drop in charge trans fer resistance values with increasing PFOS concentrations , suggesting lesser interaction between the modi fied electrodes and PFOS in the presence of interf erents interf erant .
  • the specificity of the electrodes was evaluated in the presence humic acid (HA) as an interferant that might possibly affect the signals generated by PFOS moiety.
  • humic acid HA
  • the typical concentration range of humic acid in groundwater is from 20 pg/L to 30 mg/L.
  • the array of electrodes was exposed to an analyte solution with and without PFOS in the presence of humic acid.
  • Figure 10 shows the interferant molecule (humic acid) effect on different sensing electrodes in 5nM PFOS solution: (A) Variation of Charge Transfer Resistance (Rct) with and without HA for bare gold and ELPS-tyr modified electrode and (B) Variation of Charge Transfer Resistance (Rct) with and without HA for ELPS-tFF and ELPS-pPr modified electrode.
  • Rct Charge Transfer Resistance
  • Rct Charge Transfer Resistance
  • Statistical analysis Welch's ANOVA test was performed in the Origin Pro software. The P value was found to be greater than 0.05 thus demonstrating no significant difference with and without HA at 5nM concentration of PFOS (the response of the fluorinated ELPs-tFF and hydrophobic ELPs-pPr coated-electrodes sensors) .
  • Groundwater samples were spiked with PFOS as described in Preparation 1 and analyzed by EIS in the electrochemical cell described above, testing the four electrodes (bare gold, ELPs- tyr-modified gold electrode, ELPs-tFF-modif led gold electrode and ELPs-pPr-modif led electrode) as working electrodes in solutions with PFOS concentration ranging from 200nM-5nM (5nM, lOOnM,
  • the Nyquist plots are shown in Figures 11A-11D for the four electrodes mentioned above, respectively.
  • the charge transfer resistance values (Rct) were calculated from the Nyquist plots, and plotted against the concentration of the PEGS in the solution, as shown collectively in Figure HE for all four electrodes.
  • the Ret values was found to decrease with the increasing concentration of PEGS, suggesting that PEGS interact with a range of molecules present in groundwater matrices, and consequently have less chance to approach the modified electrodes.
  • chemometric model i.e., the PLSR model ( Figure HE) to study the predict the PEGS levels in Groundwater samples.
  • PCC 0.99
  • PRESS 106.
  • InM 0.87 ⁇ 0.91nM respectively.
  • the computed values for LOD suggested the prediction of chemometric model from the groundwater directly to quantify PEGS levels.
  • the sequences include three types of pentapeptide repeating units, having a variable amino acid residue "X" inserted at position 4, denoted by SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. They all have the amino acid sequence of VPGXG, however, they differ in the definition of the variable amino acid residue X, as detailed in the sequence listing appended herewith and shown in Table 1 below.
  • the variable amino acid residue "X” means any natural amino acid, apart from proline, or a modified amino acid in SEQ ID NO.
  • amino acid sequence of the non-f luorinated ELP described in the Examples is denoted by SEQ ID NO. 5, and the amino acid sequences of the ELPs described in the Examples in which p- Trif luoromethyl-L-phenylalanine (tFF) or propargyloxy-L- phenylalanine (pPr) was inserted at positions 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 of the amino acid sequence are denoted by SEQ ID NO. 6, and SEQ ID NO. 7, respectively.
  • the nucleic acid used for encoding the polypeptides defined by SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 (while using the relevant specialized aminoacyl tRNA synthetases) is denoted by SEQ ID NO. 4.

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Abstract

L'invention concerne un procédé de détection et éventuellement de quantification d'une ou de plusieurs substances poly- et perfluoroalkyle (PEAS) dans un échantillon aqueux, comprenant l'ajout d'un couple redox à l'échantillon, l'analyse électrochimique de l'échantillon avec au moins une électrode de travail à surface modifiée ayant un polypeptide à désordre intrinsèque fluoré déposé sur sa surface, par mesure du courant, de la tension et/ou du calcul d'impédance en tant que signal associé au PFAS, et la détermination de la présence et éventuellement du niveau de PFAS, dans l'échantillon. L'invention concerne également un capteur électrochimique, un processus de fabrication du capteur et un système de détection électrochimique comprenant le capteur.
PCT/IL2024/050478 2023-05-16 2024-05-16 Procédé électrochimique et capteur de détection de pfas Pending WO2024236575A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2018225058A1 (fr) * 2017-06-04 2018-12-13 B.G. Negev Technologies And Applications Ltd., At Ben-Gurion University Dispositif et procédé de détection électrochimique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018225058A1 (fr) * 2017-06-04 2018-12-13 B.G. Negev Technologies And Applications Ltd., At Ben-Gurion University Dispositif et procédé de détection électrochimique

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Title
BELL ERIN M., DE GUISE SYLVAIN, MCCUTCHEON JEFFREY R., LEI YU, LEVIN MILTON, LI BAIKUN, RUSLING JAMES F., LAWRENCE DAVID A., CAVAL: "Exposure, health effects, sensing, and remediation of the emerging PFAS contaminants – Scientific challenges and potential research directions", SCIENCE OF THE TOTAL ENVIRONMENT, ELSEVIER, AMSTERDAM, NL, vol. 780, 1 August 2021 (2021-08-01), AMSTERDAM, NL , pages 146399, XP093240727, ISSN: 0048-9697, DOI: 10.1016/j.scitotenv.2021.146399 *
CLARK REBECCA B., DICK JEFFREY E.: "Electrochemical Sensing of Perfluorooctanesulfonate (PFOS) Using Ambient Oxygen in River Water", ACS SENSORS, AMERICAN CHEMICAL SOCIETY, US, vol. 5, no. 11, 25 November 2020 (2020-11-25), US, pages 3591 - 3598, XP093240723, ISSN: 2379-3694, DOI: 10.1021/acssensors.0c01894 *
CLARK REBECCA B., DICK JEFFREY E.: "Towards deployable electrochemical sensors for per- and polyfluoroalkyl substances (PFAS)", CHEMICAL COMMUNICATIONS, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 57, no. 66, 17 August 2021 (2021-08-17), UK , pages 8121 - 8130, XP093240725, ISSN: 1359-7345, DOI: 10.1039/D1CC02641K *
RODRIGUEZ KELSEY L., HWANG JAE-HOON, ESFAHANI AMIRSALAR R., SADMANI A H M ANWAR, LEE WOO HYOUNG: "Recent Developments of PFAS-Detecting Sensors and Future Direction: A Review", MICROMACHINES, vol. 11, no. 7, pages 667, XP055812923, DOI: 10.3390/mi11070667 *
ZHANG MINGYU, ZHAO YANAN, BUI BRIAN, TANG LIMING, XUE JIAJIA, CHEN MINGLI, CHEN WEI: "The Latest Sensor Detection Methods for per- and Polyfluoroalkyl Substances", CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY., CRC PRESS INC., BOCA RATON, FL., US, US , pages 1 - 17, XP093240728, ISSN: 1040-8347, DOI: 10.1080/10408347.2023.2299233 *

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