Recipient Organization
VERRAGLO, LLC
2029 HEATHER WAY
LEXINGTON,KY 405032639
Performing Department
(N/A)
Non Technical Summary
Collaborators at VerraGlo, LLC and the University of Kentucky have developed foundational technology for light-emitting crystals (LEC) that possess artificial peroxidase activity and emit luminescence in the visible region of the electromagnetic spectrum as a result of the peroxidase-like reaction. Luminescence emissions are proportional to the hydroperoxide content of samples (r-square > 0.98 using commercially available luminometers). Because of the combined peroxidase-like activity and luminescence emissions, LEC are very promising for use in a technique to measure peroxide value in edible fats and oils. Edible fats and oils exhibit inherent susceptibility to oxidation leading to rancidity. Throughout the food industry, from the edible oil refinery to foodservice and retail food processors, fats and oils must be continually tested to meet product and quality specifications. The industry standard for measuring levels of oxidation in edible fats and oils is the peroxide value (PV). The current American Oil Chemists' Society (AOCS) current official method for PV is an iodometric titration that requires laboratory glassware, the use of flammable and toxic solvents that are expensive to purchase and dispose after use, takes about 15 minutes to conduct, suffers from lack of sensitivity and poor endpoint determination and requires trained personnel. The goal of this project is to further develop a novel technique for measuring peroxide value in edible fats and oils using structured compound semiconductor materials that possess peroxidase-like activity and emit luminescence proportional to hydroperoxide contents. This method will be able to measure PV in about 5 minutes with an existing commercially available luminometer. The method will use no laboratory glassware, no solvents and require no cleanup (the sample vial is disposable).Implementation of this novel technology for measuring hydroperoxides in edible oil will also make a strong contribution to the sustainability of our natural resources by elimination of toxic solvents that are currently widely used in a variety of official methods that measure peroxide values (PV) in agricultural products, petrochemicals and pharmaceuticals. All these official methods are iodometric titration procedures that have several disadvantages and require the use of flammable and toxic solvents that are expensive to purchase and to dispose of after use. The waste product from conducting the "peroxide value" iodometric titration includes a mixture of isooctane, acetic acid, oils and iodine. These chemicals are ignitable, corrosive, and/or toxic. The best way to deal with hazardous waste is not to use/create it in the first place. The spillage or release of hazardous substances into the environment can have serious economic consequences. Sustainability of natural resources will also be enhanced by providing improved monitoring of oxidative conditions of an important agricultural commodity with a more convenient and less expensive technique, thus minimizing waste. This project will further develop the LEC material to provide a practical, reliable method to measure PV in the food industry that does not use any hazardous materials.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
There are three major objectives of this project. The first is to determine the structure/function relationship affecting chemically-stimulated luminescence intensity as a result of the peroxidase mechanism from the novel VerraGlo light-emitting nanocrystals. This will involve correlating the luminescence intensity from a variety of compositional and processing variations, and parameters employed during the measurement of luminescence (including type of photomultiplier tube, effects of temperature, water content, types of reaction mediums and prior exposure to pressure). Additionally, the most effective materials (based on quantum yield, ease of use, and ease and cost of manufacturing) will be examined for structural determinations using a variety of techniques (e.g., x-ray photoelectron spectroscopy, EPR spectroscopy, x-ray diffraction, FTIR spectroscopy, and scanning electron microscopy). The bulk of the products from this first objective should be completed during the period of September 1, 2020-Feburary 28, 2021.This projects second objective is to determine if the current flowing from the peroxidase reaction can be measured using a potentiometric technique. This investigation will employ a potentiostat and a variety of different electrode compositions (both standard and screen-printed electrodes), reaction mediums and co-substrates used to bind the light-emitting materials to the electrodes. If successful, this technique will provide a high level of correlation between the hydroperoxide content of edible oils and the current measured by the potentiostat, and provide the bases for a simplified disposable test strip. The products from this second objective should be completed during the period of November 1, 2020-April 30, 2021.The third objective of this project incorporate the optimal compositions and techniques as determined during accomplishment the first two objectives into a commercially practical sensor for measuring the peroxide values in edible oils. A successful outcome will be an inexpensive assay kit (ca. $6 per assay) that eliminates the need for laboratory glassware and a fume hood, uses no flammable or toxic solvents that are expensive to purchase and dispose of after use, and can be conducted in less than 15 minutes by minimally trained personnel. Additional improvements will include overcoming the lack of sensitivity and poor endpoint determination inherent in the iodometric assay used in current official methods for determining peroxide value in edible oils. The products from this third objective will be completed during the period of November 1, 2020-April 30, 2021.
Project Methods
This Phase I SBIR project is designed to further improve the composition and manufacturing of VerraGlo, LLC light-emitting crystals (LEC) prototypes so as to provide a commercially practical alternative to the existing iodometric titration methods currently available. Examples of "Official methods" currently in use that rely upon iodometric titrations to measure peroxide values (PV) in agricultural products, petrochemicals and pharmaceuticals include the American Oil Chemists Society (AOCS) (official method Cd 8b-90)(AOCS, 1989), Association of Official Analytical Chemists (AOAC) 965.33, the International Union of Pure and Applied Chemistry (IUPAC) 2.501, the International Organization for Standardization (ISO) 3960:2017 and U.S. Pharmacopeia Convention methods (USP) 401. All these official methods are iodometric titration procedures that have several disadvantages and require the use of flammable and toxic solvents that are expensive to purchase and to dispose of after use. The following is the example of the procedure for using VerraGlo LEC to measure PV in edible oils.DEFINITION: This method determines hydroperoxides in edible oils, expressed as of milliequivilents of peroxide per 1000 grams of sample, using a structured semiconductor sensor with peroxidase-like activity. The light emitting crystals (LEC) of semiconductor material generate luminescence proportional to the hydroperoxide content of oils.SCOPE: The method is applicable to all edible oils (liquid at 20-25 degrees C)APPARATUS 1. Glass/Teflon syringe (e.g., 0.5 mL) capable of accurately measuring 0.40 mL of oil, equipped with an 18 gauge x 1.5 inch needle, or a pipette capable of accurately dispensing 0.4 mL of oil.2. Luminometer (e.g., Charm Scientific Novalum) (Lawrence, MA, USA)).3. Charm Sciences LUM-T holder (part No. MT-HLDR) and threaded polypropylene tubes, 2 mL.4. Vortex mixer.REAGENTSLight emitting crystals* (LEC) comprised of structured semiconductor material (e.g., VerraGlo P35e7, VerraGlo, LLC, Lexington, KY, USA), 20 mg per assay in sealed test vial with septa (See Notes, 3).SOLUTIONS: REAGENTS - No reagent solutions are required.STANDARDS - Because of potential variations among different types of luminometers, each instrument must be calibrated. Calibrate the luminometer against AOCS Official Method Cd 8b-90; iodometric titration method using a series of edible oil samples. PROCEDURETest sample preparation - Procedure for oils liquid at 20-25 degrees C -1. Equilibrate liquid oil test samples to 20-25 degrees C.2. Using a glass/Teflon syringe, inject 0.4 mL of oil (at 20-25 degrees C) through the septum into the vial containing LEC.3. Vortex for 3 seconds to thoroughly mix the LEC and oil.4. Wait 1 minute and take a single reading on the luminometer by placing the glass vial inside a 2 mL polypropylene tube, attach the tube to the Charm Sciences sample holder, and insert the sample into the luminometer. Record the value from the luminometer.PREPARATION OF THE CALIBRATION CURVECarry out iodometric titration and plot the results against triplicate luminescence readings from the VerraGlo PV assay. Figure 1 is an example of a standard curve for liquid oils (r-square = 0.995).CALCULATIONSFor a given oil take triplicate luminescence readings and calculate the peroxide value from the standard curve equation.The following are examples of analytical techniques that will be used to accomplish the project objectives and to further improve upon the use and manufacturing of LEC.Luminescence Measurements - Light emissions from all reaction mixtures will be recorded with either a bench-top luminometer (Promega Glomax, 20/20) with an internal heat block (which is adjustable from 22 to 70 degrees C), or a Novalum luminometer (Charm Sciences) with external dry block heater, where appropriate. Emission wavelengths will be determined using 25 mm optical filters (Omega Optical, Brattleboro, VT), of varying wavelength cutoffs, placed in the luminometer to measure luminescence at specific wavelength ranges. Results will typically be reported in 50 nm wavelength ranges (e.g., 500 to 550 nm, 550 to 600 nm, etc) with the ends of the photomultiplier tubes detection ranged measured with from the 450 short path (450sp) and 650 long path (650lp) filters, respectively. Accuracy of wavelength cutoffs for all optical filters were confirmed using an Ocean Optics HR4000 UV/Vis spectrometer (Dunedin, FL) equipped with a USB-DT light source (producing wavelengths from 200 to 900 nm), QP200-2-UV-Vis optical cables, CUV-FL-DA cuvette holder and SpectraView software.Iodometric Peroxide Value (PV) Determination will be carried out precisely according to the American Oil Chemists' Society (AOCS) official method Cd 8b-90 with results expressed as meq/kg oil.Cyclic Voltometry will be conducted on a PalmSens EmStat Blue using screen-printed carbon electrodes (CH Instruments, TE100) or standard electrodes. Light-emitting materials will be attached with a binder such as polyvinylidene fluoride (PVDF) matrix. In addition to CV other potentiometric assays will be examined such as linear sweep voltometery.Electron paramagnetic resonance (EPR) spectroscopy will be performed on a Bruker EMX EPR spectrometer (Billerica, MA). The EPR parameters for paramagnetic materials will be optimized for microwave power; microwave frequency (typically 9.86 GHz); receiver gain; modulation amplitude; modulation frequency; time constant; conversion time; resolution; center field at in Gauss; and field sweep width in Gauss. Detector response, where appropriate will be calibrated using the same EPR parameters with a standard curve prepared with Fremy's salt solutions in quartz capillary tubes. Analyses of EPR peaks and calibration of g-value relative to DPPH standard (2.0036) were performed using the Bruker WinEPR System softwareAtomic Absorption Spectrometry - Select metal contents will be analyzed with a PerkinElmer AAnalyst 200 atomic absorption spectrometer (PerkinElmer, Waltham, MA).. Powdered samples were ashed according to AOAC Official Method 985.35.Peroxidase Reaction Mechanisms and Kinetics will be monitored using gas chromatography/mass spectrometry, high performance liquid chromatography and UV-Vis spectrometry. In aqueous reaction mediums peroxidase activity will be monitored using ortho-Phenylenediamine dihydrochloride (OPD). Composition and structural analysis will be conducted at the University of Kentucky's Electron microscopy Center using a Zeiss EVO MA 10 scanning electron microscope (SEM), a FEI Talos F200X transmission electron microscope (TEM) and a Thermo K-Alpha XPS x-ray photoelectron spectroscopy (XPS).Statistical analysis will be conducted in triplicate unless specified otherwise. Statistical analysis was performed using Statistix software 9.0 (Analytical Software, Tallahassee, FL, USA). A linear model was used for the overall analysis of variance. When a significant treatment effect was found (P < 0.05), multiple comparisons were performed using Tukey HSD all-pairwise comparison test (P < 0.05).