Synergistic Applications of Graphene-Based Materials and Deep Eutectic Solvents in Sustainable Sensing: A Comprehensive Review.

The recently explored synergistic combination of graphene-based materials and deep eutectic solvents (DESs) is opening novel and effective avenues for developing sensing devices with optimized features. In more detail, remarkable potential in terms of simplicity, sustainability, and cost-effectiveness of this combination have been demonstrated for sensors, resulting in the creation of hybrid devices with enhanced signal-to-noise ratios, linearities, and selectivity. Therefore, this review aims to provide a comprehensive overview of the currently available scientific literature discussing investigations and applications of sensors that integrate graphene-based materials and deep eutectic solvents, with an outlook for the most promising developments of this approach.

A smartphone aptasensor for fipronil detection in honey samples.

In this study, an electrochemical smartphone-based aptasensor for the determination of fipronil was developed by modifying a screen-printed carbon electrode (SPCE). Fipronil is a broad-spectrum insecticide that has been widely used in various applications such as agriculture, veterinary, and household pest control. Recently, its use has raised concerns over the potential impact on the environment and human health. The absence of effective methods for this purpose poses a significant obstacle. To tackle this problem, we have developed a cutting-edge aptamer-based portable sensor capable of rapidly and conveniently detecting fipronil in situ. Considering that the detection of small molecules, such as fipronil, can be a challenging task, a competitive replacement assay was set up based on the aptamer's preference for the free form of fipronil over the immobilized one on the electrode. The analytical performance provided by the sensor on standard solutions of a known fipronil content made it possible to estimate a limit of detection (LOD) equal to 1.07 µg kg-1 and a limit of quantification (LOQ) of 3.21 µg kg-1. Selectivity tests were conducted using atrazine as a possible interferent. The use and performance of the developed portable aptasensor was assessed on honey samples, which were simultaneously analyzed using an HPLC-MS method. This aptasensor could be an affordable and effective tool for accurately quantifying fipronil not only in honey samples but also in other food products.

Electrochemical Evaluation of Tyrosinase Enzymatic Activity in Deep Eutectic Solvent and Aqueous Deep Eutectic Solvent.

The use of green, inexpensive, and biodegradable deep eutectic solvents as nonaqueous solvents and electrolytes could be a useful way to potentially improve the enzyme biosensor performance as well as a profitable strategy to extend their use in the gas phase. However, enzyme activity in these media, although fundamental for their implementation in electrochemical analysis, is still almost unexplored. In this study, an electrochemical approach was employed to monitor tyrosinase enzyme activity in a deep eutectic solvent. This study was performed in a DES consisting of choline chloride (ChCl) as a hydrogen bond acceptor (HBA) and glycerol as a hydrogen bond donor (HBD), while phenol was chosen as the prototype analyte. The tyrosinase enzyme was immobilized on a gold-nanoparticle-modified screen-printed carbon electrode, and its activity was monitored following the reduction current of orthoquinone produced by the tyrosinase biocatalysis of phenol. This work represents a first step toward the realization of green electrochemical biosensors capable of operating in both nonaqueous and gaseous media for the chemical analysis of phenols.

A portable electrochemiluminescence aptasensor for ß-lactoglobulin detection.

Cow's milk allergy is one of the most common food allergies in children with a prevalence of around 2.5%. Milk contains several allergens; the main ones are caseins and ß-lactoglobulin (ß-LG). At regulatory level, ß-LG is not explicitly named, but milk is included in the list of substances or products causing allergies or intolerances. Hence, the presence of ß-LG can be a useful marker for determining the presence of milk in food. In this work, we present an aptasensor based on electrochemiluminescence (ECL) for the quantification of ß-LG in real food matrices displaying integrated advantages consisting of high specificity, good sensitivity, portability, and cost effectiveness. The performance and applicability of this sensor were tested by analyzing a sample of skimmed milk and an oat-based drink proposed as a vegetable substitute for milk of animal origin. We obtained a linear correlation between the intensity of the signal and the concentration of ß-LG standard solutions (y = x * 0.00653 + 1.038, R2 = 0.99). The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 1.36 and 4.55 µg L-1, respectively.

An Effective Label-Free Electrochemical Aptasensor Based on Gold Nanoparticles for Gluten Detection.

Nanomaterials can be used to modify electrodes and improve the conductivity and the performance of electrochemical sensors. Among various nanomaterials, gold-based nanostructures have been used as an anchoring platform for the functionalization of biosensor surfaces. One of the main advantages of using gold for the modification of electrodes is its great affinity for thiol-containing molecules, such as proteins, forming a strong Au-S bond. In this work, we present an impedimetric biosensor based on gold nanoparticles and a truncated aptamer for the quantification of gluten in hydrolyzed matrices such as beer and soy sauce. A good relationship between the Rct values and PWG-Gliadin concentration was found in the range between 0.1-1 mg L-1 of gliadin (corresponding to 0.2-2 mg L-1 of gluten) with a limit of detection of 0.05 mg L-1 of gliadin (corresponding to 0.1 mg L-1 of gluten). The label-free assay was also successfully applied for the determination of real food samples.

Paper-based aptamer-antibody biosensor for gluten detection in a deep eutectic solvent (DES).

Paper has been widely employed as cheap material for the development of a great number of sensors such as pregnancy tests, strips to measure blood sugar, and COVID-19 rapid tests. The need for new low-cost analytical devices is growing, and consequently the use of these platforms will be extended to different assays, both for the final consumer and within laboratories. This work describes a paper-based electrochemical sensing platform that uses a paper disc conveniently modified with recognition molecules and a screen-printed carbon electrode (SPCE) to achieve the detection of gluten in a deep eutectic solvent (DES). This is the first method coupling a paper biosensor based on aptamers and antibodies with the DES ethaline. Ethaline proved to be an excellent extraction medium allowing the determination of very low gluten concentrations. The biosensor is appropriate for the determination of gluten with a limit of detection (LOD) of 0.2 mg L-1 of sample; it can detect gluten extracted in DES with a dynamic range between 0.2 and 20 mg L-1 and an intra-assay coefficient of 10.69%. This approach can be of great interest for highly gluten-sensitive people, who suffer from ingestion of gluten quantities well below the legal limit, which is 20 parts per million in foods labeled gluten-free and for which highly sensitive devices are essential.

Kinetics of electron transfer reactions by humic substances: Implications for their biogeochemical roles and determination of their electron donating capacity.

Humic substances (HS) possess redox active groups covering a wide range of potentials and are used by facultative anaerobic microorganisms as electron acceptors. To serve as suitable electron shuttles for anaerobic respiration, HS should be able to re-oxidize relatively quickly to prevent polarization of the surrounding medium. Mediated electrochemical oxidation and decolorization assays, based on the reduction of the radical ion of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS•-) allow to determine the electron donating capacity (EDC) of HS, but uncertainties remain about the reaction time that should be allowed to obtain environmentally meaningful EDC values. In this work, we performed a kinetic analysis of the time trend of the reduction of ABTS•- by HS by Vis and Electron Paramagnetic Resonance (EPR) spectroscopies and by cyclic voltammetry. We found evidences of two concomitant separate mechanisms of electron exchange: a fast and a slow transfer processes which may have different environmental roles. These results can set a base to identify the appropriate conditions for the spectrophotometric determination of the fast and slow components of the EDC of HS.

Deep Eutectic Solvents (DESs) and Their Application in Biosensor Development.

Deep Eutectic Solvents (DESs) are a new class of solvents characterized by a remarkable decrease in melting point compared to those of the starting components. The eutectic mixtures can be simply prepared by mixing a Hydrogen Bond Acceptor (HBA) with a Hydrogen Bond Donor (HBD) at a temperature of about 80 °C. They have found applications in different research fields; for instance, they have been employed in organic synthesis, electrochemistry, and bio-catalysis, showing improved biodegradability and lower toxicity compared to other solvents. Herein, we review the use of DESs in biosensor development. We consider the emerging interest in different fields of this green class of solvents and the possibility of their use for the improvement of biosensor performance. We point out some promising examples of approaches for the assembly of biosensors exploiting their compelling characteristics. Furthermore, the extensive ability of DESs to solubilize a wide range of molecules provides the possibility to set up new devices, even for analytes that are usually insoluble and difficult to quantify.

Characterization of Inherently Chiral Electrosynthesized Oligomeric Films by Voltammetry and Scanning Electrochemical Microscopy (SECM).

A voltammetric and scanning electrochemical microscopy (SECM) investigation was performed on an inherently chiral oligomer-coated gold electrode to establish its general properties (i.e., conductivity and topography), as well as its ability to discriminate chiral electroactive probe molecules. The electroactive monomer (S)-2,2'-bis(2,2'-bithiophene-5-yl)-3,3'-bibenzothiophene ((S)-BT2T4) was employed as reagent to electrodeposit, by cyclic voltammetry, the inherently chiral oligomer film of (S)-BT2T4 (oligo-(S)-BT2T4) onto the Au electrode surface (resulting in oligo-(S)-BT2T4-Au). SECM measurements, performed in either feedback or competition mode, using the redox mediators [Fe(CN)6]4- and [Fe(CN)6]3- in aqueous solutions, and ferrocene (Fc), (S)-FcEA, (R)-FcEA and rac-FcEA (FcEA is N,N-dimethyl-1-ferrocenylethylamine) in CH3CN solutions, indicated that the oligomer film, as produced, was uncharged. The use of [Fe(CN)6]3- allowed establishing that the oligomer film behaved as a porous insulating membrane, presenting a rather rough surface. This was inferred from both the approach curves and linear and bidimensional SECM scans, which displayed negative feedback effects. The oligomer film acquired semiconducting or fully conducting properties when the Au electrode was biased at potential more positive than 0.6 V vs. Ag|AgCl|KCl. Under the latter conditions, the approach curves displayed positive feedback effects. SECM measurements, performed in competition mode, allowed verifying the discriminating ability of the oligo-(S)-BT2T4 film towards the (S)-FcEA and (R)-FcEA redox mediators, which confirmed the results obtained by cyclic voltammetry. SECM linear scans indicated that the enantiomeric discriminating ability of the oligo-(S)-BT2T4 was even across its entire surface.

Truncated aptamers as selective receptors in a gluten sensor supporting direct measurement in a deep eutectic solvent.

Enzyme-linked immunosorbent assays are currently the most popular methods to quantify gluten in foods. Unfortunately, the antibodies used as specific receptors in such methods are not compatible with the usual solvents for the extraction of gluten proteins. In consequence, commercial tests require a high dilution of the sample after the extraction, increasing the limit of quantification and decreasing convenience. In this work, we have rationally truncated an aptamer capable of recognizing gliadin in a deep eutectic solvent (DES). The truncated aptamer is a 19-nucleotides-long DNA that minimizes self-hybridization, allowing the development of an electrochemical sandwich-based sensor for the quantification of gluten in the DES ethaline. The sensor incorporates two identical biotin-labeled truncated aptamers, one of which is immobilized on a carbon screen-printed electrode and the other reports the binding of gliadin after incubation in streptavidin-peroxidase. This sensor can detect gliadin in DES, with a dynamic range between 1 and 100 µg/L and an intra-assay coefficient of variation of 11%. This analytical performance allows the quantification of 20 µg of gluten/kg of food when 1 g of food is extracted with 10 mL of ethaline. We demonstrate the ability of this method to achieve the measurement of gluten in food samples, after the extraction with pure ethaline. The assay is useful for the analysis of residual gluten levels in foods, thus facilitating the evaluation of any potential health risk associated with the consumption of such food by people with celiac disease or other gluten-related disorders.

Modified Screen Printed Electrode Suitable for Electrochemical Measurements in Gas Phase.

We describe a convenient assembly for screen printed carbon electrodes (SPCE) suitable for analyses in gaseous samples which are of course lacking in supporting electrolytes. It consists of a circular crown of filter paper, soaked in a RTIL or a DES, placed upon a disposable screen printed carbon cell, so as to contact the outer edge of the carbon disk working electrode, as well as peripheral counter and reference electrodes. The electrical contact between the paper crown soaked in RTIL or DES and SPCE electrodes is assured by a gasket, and all components are installed in a polylactic acid holder. As a result of this configuration, a sensitive, fast-responding, membrane-free gas sensor is achieved where the real working electrode surface is the boundary zone of the carbon working disk contacted by the paper crown soaked in the polyelectrolyte. This assembly provides a portable and disposable electrochemical platform, assembled by the easy immobilization onto a porous and inexpensive supporting material such as paper of RTILs or DESs which are characterized by profitable electrical conductivity and negligible vapor pressure. The electroanalytical performance of this device was evaluated by voltammetric and flow injection analyses of oxygen which was chosen as prototype of electroactive gaseous analytes. The results obtained pointed out that this assembly is very profitable for the analysis of gaseous atmospheres, especially when used as detector for FIA in gaseous streams.

A Portable Setup for the Voltammetric Determination of Total Mercury in Fish with Solid and Nanostructured Gold Electrodes.

A simple procedure for field fish sample pretreatment was developed. This treatment in combination with square wave anodic stripping voltammetry (SW-ASV) with solid gold electrodes (SGE) and gold nanoparticle-modified glassy carbon electrodes (AuNPs-GCE) was applied for the determination of total mercury content. A certified reference material (CRM, Tuna Fish BCR 463), ten freeze-dried samples of canned tuna and two fresh fish samples were analysed both with a bench-top voltammetric analyser after microwave digestion and with a portable potentiostat after mild eating using a small commercial food warmer. The results obtained by the two SW-ASV approaches and by a Direct Mercury Analyser (DMA), the official method for mercury determination, were in very good agreement. In particular, (i) the results obtained with in field procedure are consistent with those obtained with the conventional microwave digestion; (ii) the presence of gold nanoparticles on the active electrode surface permits an improvement of the analytical performance in comparison to the SGE: the Limit of Quantification (LOQ) for mercury in fish-matrix was 0.1 µg L-1 (Hg cell concentration), corresponding to 0.06 mg kg-1 wet fish, which is a performance comparable to that of DMA. The pretreatment proposed in this study is very easy and applicable to fresh fish; in combination with a portable potentiostat, it proved to be an interesting procedure for on-site mercury determination.

Electroanalytical cells pencil drawn on PVC supports and their use for the detection in flexible microfluidic devices.

A simple, effective and low-cost technique is here presented for assembling flexible and robust electrochemical devices on transparent PVC supports, using ordinary tools, all installed on a commercial desktop digitally controlled plotter/cutter. Small diamond burs were first set up to rough precise and well defined patterns on the surface of smooth and flexible PVC transparent films. Subsequently, reference, counter and working carbon electrodes were drawn onto abraded patterns by using micropencils (4B graphite leads, 0.5 mm in diameter), in their turn installed on the plotter/cutter. The effective active working surface of electrochemical cells was then defined by a thin adhesive strip or by covering the patterned support with a suitably cut adhesive layer, depending upon whether they were intended for use in batch or drop mode. After optimization of fabrication parameters, such as pressure and speed adopted during bur abrasion and pencil drawing, the electrochemical characterization of these cells was performed by using potassium hexacyanoferrate(II) as redox probe. Voltammetric responses displayed a good inter-device reproducibility (5.6%), thus confirming the effectiveness of this easy and fast assembling strategy. These PVC-based pencil-drawn electrochemical cells were then integrated as thin-layer detectors in adhesive-tape based microfluidic channels, cut and prepared in their turn using the digitally controlled plotter/cutter. These detectors offer the advantage given by the impermeability of PVC supports, thus avoiding absorption of the flowing carrier and consequent analyte broadening, instead occurring when electrochemical cells are pencil drawn on hydrophilic materials as paper. After optimization of the complete fabrication process, the effectiveness of these devices was tested by a proof-of-concept direct quantification of ascorbic acid in commonly used drugs.

Volatile aldehydes sensing in headspace using a room temperature ionic liquid-modified electrochemical microprobe.

The cyclic voltammetric behaviour of propionaldehyde (PA) and hexanaldehyde (HA), in 1-butyl-3-methylimidazolium bis(trifluoromethyl-sulfonyl) imide ([BMIM][NTF2]), 1-butyl-3-methylimidazolium hydrogen sulphate ([BMIM][HSO4]) and 1-butyl-3-methylimidazolium hydroxide ([BMIM][OH]) was investigated at a platinum microelectrode. A clear oxidation process for both aldehydes was recorded only in [BMIM][OH]. On the basis of these evidences, an electrochemical microprobe (EMP), incorporating [BMIM][OH] as electrolyte, was assembled for sensing these aldehydes in gaseous phases. The EMP exposed in the headspace of the liquid aldehydes displayed voltammetric and amperometric responses, which depended on the aldehyde vapour pressures and, consequently, on the temperature employed. The usefulness of the [BMIM][OH] coated EMP for practical applications was assessed in the detection of HA vapour released from squalene (i.e., a lipid simulant matrix) samples spiked with known amounts of the aldehyde. Calibration plots were constructed at 40 °C, 50 °C and 60 °C, using both voltammetry and chronoamperometry. In both cases, good linearity between current and HA concentration in squalene was obtained over the range 3-300 ppm, with correlation coefficients higher than 0.991. Reproducibility, evaluated from at least three replicates, was within 5%. Detection limits, evaluated for a signal-to-noise ratio of 3, were in any case lower than 1.7 ppm. These analytical performances are suitable for monitoring VAs coming from lipid oxidation processes in food. An application concerning the determination of VAs in headspace of sunflower oil during an induced oxidative test to establish its thermal stability was also performed.

A cotton thread fluidic device with a wall-jet pencil-drawn paper based dual electrode detector.

A simple and sensitive device is presented based on the use of pencil-drawn paper based electrochemical detector placed at the end of a cotton thread fluidic channel in wall-jet configuration. This innovative and fast responding electroanalytical system can be adopted for both single and dual electrode electrochemical detection, this last achieved by applying two different potentials at two independent working electrodes drawn on the opposite faces of the paper based detector. Its performance was preliminarily optimized by adopting hexacyanoferrate(II) as probe species undergoing reversible electrochemical processes. These devices were then used for the single electrode detection of ascorbic acid in aqueous samples and the dual electrode detection of orthodiphenols in extra virgin olive oils (EVOOs). In fact, these devices enable hydrophilic orthodiphenols, typically present in EVOOs (extracted by a 80:20% v/v acetonitrile/water mixture), to be discriminated from hydrophilic monophenols instead present in almost all vegetable oils. Flow-injections runs were conducted by using a 0.01 M H2SO4 + 0.5 KCl running electrolyte allowing the rapid and selective detection of hydrophilic orthodiphenols with satisfactory sensitivity and a low enough detection limit (2 µM). Different real samples of EVOOs and sunflower oils were analyzed. Abundant enough contents of orthidiphenols were found in EVOO samples, while no trace of these antioxidants was found in sunflower oils.

Selection of Anti-gluten DNA Aptamers in a Deep Eutectic Solvent.

Herein, we show the feasibility of using deep eutectic solvents as a faster way of selecting aptamers targeting poorly water-soluble species. This unexplored concept is illustrated for gluten proteins. In this way, aptamer-based gluten detection can be performed directly in the extraction media with improved detectability. We envision deep implications for applications not only in food safety control but also in biomedicine.

Digitally Controlled Procedure for Assembling Fully Drawn Paper-Based Electroanalytical Platforms.

A simple, reliable, and low-cost fabrication method is proposed here for assembling paper-based electrochemical devices (PEDs) using a commercial desktop digitally controlled plotter/cutter, together with ordinary writing tools. Permanent markers (tips of 1 mm) were used to create effective hydrophobic barriers on paper, while micromechanical pencils (mounting 4B graphite leads, 0.5 mm in diameter) were adopted for automatically drawn precise reference, counter, and working carbon electrodes. Fabrication parameters, such as writing pressure and speed, were first optimized, and the electrochemical performance of these devices was then evaluated by using potassium hexacyanoferrate(II) as redox probe. The good interdevice reproducibility (4.8%) displayed by the relevant voltammetric responses confirmed that this strategy can be profitably adopted to easily assemble paper-based electrochemical devices in a highly flexible manner. The simplicity of the instrumentation used and the low cost of each single device (about $0.04), together with the speed of fabrication (about 2 min), are other important features of the proposed strategy. Finally, to confirm the effectiveness of this prototyping method for the analysis of real samples and rapid controls, PEDs assembled by this simple approach were successfully exploited for the analysis of vitamin B6 in food supplements.

Oxidative behavior of (+)-catechin in the presence of inactive dry yeasts: a comparison with sulfur dioxide, ascorbic acid and glutathione.

BACKGROUND: The antioxidant capacity of an inactive dry yeast preparation (YD) was investigated by conventional analytical methods (spectrophotometry, high-performance liquid chromatography) as well as by cyclic voltammetry in a (+)-catechin model wine and compared with that of some of the most common antioxidants found in wine: sulfur dioxide, ascorbic acid and glutathione. RESULTS: Sulfur dioxide was the highest-performing substance in protecting (+)-catechin against browning, followed by ascorbic acid and the YD preparation. Sulfites were the only antioxidant whose activity was clearly detectable in the model wines after 29 days of storage. Voltammetric studies demonstrated that the antioxidant capacity of the products tested was connected to their intrinsic characteristics and their molar concentrations (catechin/antioxidant molar ratio). CONCLUSION: The YD preparation displayed a certain ability to protect polyphenols against browning. The antioxidant activity of YDs towards (+)-catechin appeared to be based on different mechanisms with respect to that of the other products tested: the insoluble portion of these preparations (cell wall residues) might have a non-negligible role, even if the ability of YDs to release compounds able to suppress oxidation cannot be rejected. Direct comparison of the different antioxidants led to interesting indications concerning their mechanism of action in wine-like solution, depending on their concentration and intrinsic characteristics. © 2017 Society of Chemical Industry.

Anodic stripping voltammetry with gold electrodes as an alternative method for the routine determination of mercury in fish. Comparison with spectroscopic approaches.

The applicability to the determination of mercury in tuna of square wave anodic stripping voltammetry (SW-ASV) conducted at both solid gold electrode (SGE) and a gold nanoparticle-modified glassy carbon electrode (AuNPs-GCE) was demonstrated. Mercury content in two certified materials and in ten samples of canned tuna was measured. The performances of the electrodes were compared with one another as well as with two spectroscopic techniques, namely cold vapour atomic absorption spectroscopy (CV-AAS) and a direct mercury analyser (DMA). The results found pointed out that both SW-ASV approaches were suitable and easy-to-use method to monitor mercury concentration in tunas, since they allowed accurate quantification at concentration values lower than the maximum admissible level in this matrix ([Hg]=1mg/kgwet weight,ww). In particular, mercury detection at the AuNPs-GCE showed a LOQ in fish-matrix of 0.1µg/l, corresponding to 0.06mg/kgww, with performance comparable to that of DMA.

A paper-based platform with a pencil-drawn dual amperometric detector for the rapid quantification of ortho-diphenols in extravirgin olive oil.

A simple, sensitive and fast responding device is described for the discrimination of hydrophilic ortho-diphenols, whose presence in abundant enough amounts is typical for extra virgin olive oils (EVOOs), from hydrophilic mono-phenols instead present in almost all vegetable oils. It consists of a dual electrode detector pencil-drawn at the end of a paper microfluidic channel, defined by hydrophobic barriers, where samples of these antioxidants, extracted from vegetable oils by a 80:20% v/v acetonitrile/water mixture, were applied. Thin-layer chromatographic runs conducted by using a 0.01 M H2SO4 + 1 M KCl running buffer allowed the selective detection of hydrophilic ortho-diphenols by profiting from the fact that they undergo reversible oxidation at less positive potentials than those required by monophenols for displaying their irreversible anodic process. On this basis, a potential for the oxidation of hydrophilic ortho-diphenols was applied to the upstream pencil-drawn electrode (W1) (at which a minor fraction of mono-phenols was also oxidized), while a potential for the reverse process involving the sole product (ortho-quinones) of the reversible oxidation of ortho-diphenols was imposed at the downstream pencil-drawn working electrode (W2). Thus, cathodic peak currents linearly dependent on analyte concentrations could be recorded at W2 which led to a satisfactory detection limit (8 µM, equivalent to 1.23 mg/L) even when working electrodes W1 and W2 with same dimensions were employed. Improved sensitivities and lower detection limits were achieved by increasing the dimensions of W2 with respect to W1, thanks to the improvement of the collection efficiency. Throughout this investigation, hydroxytyrosol (HTy) and tyrosol (Ty) were adopted as models of ortho-diphenols and mono-phenols, respectively, in view of their abundant presence in EVOOs. Real samples of EVOO from different production companies, of a simple olive oil and of a sunflower oil were analyzed. Different hydrophilic ortho-diphenol contents were found in EVOO samples (up to 40.8 mg/kg), while only a negligible amount turned out to be present in simple olive oil. No trace of these antioxidants were instead found in sunflower oil, as expected. All concentrations found were in good agreement with those detected by a more frequently employed spectrophotometric method used for the sake of comparison.