State of the art
Glyphosate
Glyphosate, a widely used herbicide in both agriculture and urban areas, was first synthesized in 1950 by Swiss chemist Henri Martin for potential pharmaceutical applications Its herbicidal properties were identified in 1970 by John Franz at Monsanto, leading to its registration by the EPA in 1974 under the brand name Roundup Over the years, glyphosate has been marketed under various names, including Roundup Pro, Roundup PowerMAX, Roundup WeatherMAX, and AquaMaster, becoming the most commonly applied herbicide globally As of now, global glyphosate usage exceeds 825 million kg, with agricultural use increasing dramatically from 43 million kg in earlier years.
1994 to 747 million kg in 2014 2 Today, glyphosate is applied to more than 130 countries 8
Glyphosate is a phosphonomethyl derivative of the amino acid glycine (Figure 1.1) 9
Glyphosate is an amphoteric compound featuring a secondary amine group in its structure, along with monobasic (carboxylic) and dibasic (phosphonic) acidic sites Its physicochemical properties reveal that glyphosate has functional groups capable of both donating and accepting hydrogen The acid dissociation constants for glyphosate are recorded at 2.0, 2.6, and 5.6 for its phosphoric and carboxylic acid moieties, with an estimated pKa of approximately 10.6 attributed to the amine group Consequently, glyphosate can exhibit cationic and anionic forms depending on the pH, indicating its zwitterionic nature between pH 1 and 10 At room temperature, glyphosate appears as a colorless crystal with a low vapor pressure of 9.3×10 −3 (mm Pa) at 25 °C It is highly polar and soluble in water (12 g/L at 25 °C), yet remains insoluble in organic solvents like acetone, ethanol, and xylene.
Figure 1 1 The chemical structure of N-(phosphonomethyl)glycine 20
Table 1 1 The physicochemical properties of glyphosate 15-19
Over the last 45 years, glyphosate becomes the most commonly used herbicide in the
Glyphosate's effectiveness in managing perennial weeds and overwintering rhizomes and tubers has contributed to its widespread success in the EU and globally Its capacity to bind with soil colloids and undergo degradation by soil microbes, along with the development of transgenic glyphosate-resistant crops, further enhances its utility in agriculture.
Glyphosate operates by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), a crucial component of the shikimate pathway This pathway is vital for the synthesis of aromatic amino acids in plants and microorganisms, making EPSP synthase the sixth step in this essential biochemical process.
The shikimate pathway is a crucial metabolic route for synthesizing aromatic amino acids like phenylalanine, tyrosine, and tryptophan, which are vital for metabolic regulation This pathway involves seven steps, starting with the condensation of phosphoenolpyruvate and erythrose-4-phosphate, leading to the production of chorismic acid Found in bacteria, archaea, yeasts, algae, fungi, and plants, the shikimate pathway is absent in animals and humans, making EPSP synthase a promising target for new antimicrobial agents against various pathogens Glyphosate, a highly effective herbicide, mimics phosphoenolpyruvate and competes with it in the EPSPS–S3P complex, inhibiting EPSPS activity and disrupting the shikimic acid pathway, ultimately causing plant death due to amino acid starvation As a small and simple molecule, glyphosate is readily absorbed by plants through their leaves and transported to growing shoots and roots, where it interacts with EPSPS Following glyphosate uptake, plant death can occur within 4 to 20 days To enhance its water solubility for easy application, glyphosate is often formulated with various salts such as isopropyl ammonium and potassium salts.
Figure 1 2 The shikimate pathway for the biosynthesis of aromatic amino acids, and the mode of action of glyphosate on the reaction catalyzed by EPSPS 36
1.1.3 The fate of glyphosate in the environment
Glyphosate, commonly used in large quantities for agricultural purposes, has the potential to leach into soil and groundwater Consequently, there has been extensive research published on its sorption, degradation, and leaching in the environment The behavior and environmental fate of glyphosate are illustrated in Figure 1.3.
Figure 1 3 Behavior and environmental fate of glyphosate 40
Sorption of glyphosate in soil
Glyphosate is a small, water-soluble molecule characterized by various polar functional groups, including carboxyl, amino, and phosphonate groups, which contribute to its low mobility in soil due to strong sorption to soil particles The sorption of glyphosate is influenced by several factors, such as soil type, surface charge, pH levels, and the presence of metal cations and organic matter Research by Miles and Moye indicates that the primary mechanisms for glyphosate sorption are hydrogen bonding and ion exchange Typically, Freundlich-type sorption isotherms are employed to describe glyphosate sorption in soils, humic substances, and clay minerals.
A 2015 study by Vera Silva and colleagues revealed that glyphosate was the most prevalent pesticide residue in agricultural soils across Europe Out of 317 soil samples tested from 11 European countries, 67 samples (21%) contained glyphosate, with concentrations reaching up to 2.05 mg/kg.
Glyphosate exhibits strong sorption to soils through the chelation of its functional groups with trace metals Its functional groups can create robust coordination bonds with metal ions, forming bidentate and tridentate complexes The stability constants for these metal complexes indicate that glyphosate has the highest affinity for Fe³⁺, followed by Al³⁺, Cu²⁺, and Zn²⁺.
At a 1:1 metal to glyphosate ratio, the sorption capacity follows the order of Fe 2+ > Mn 2+ > Ca ≈ Mg 2+ In contrast, the impact of humic substances on glyphosate sorption has been less frequently investigated, and glyphosate tends to be more challenging to sorb onto humic substances compared to soil minerals in natural soils.
Table 1 2 Reported stability constants of glyphosate with various metals 49
Metal Stability constants (log K ML )
Glyphosate, once accumulated in natural environments, undergoes degradation through various processes including microbial activity, physical and chemical degradation, and photodegradation Extensive research has focused on the breakdown of glyphosate in both soil and water The rate of glyphosate decomposition is influenced by several factors, particularly the type of soil involved.
11 climatic conditions, and glyphosate bioavailability 59 Table 1.3 shows the half-lives of glyphosate in soils, leading to the formation of AMPA
Table 1.3 The half-lives of glyphosate in topsoil and subsoil 53
Glyphosate half-life (DT50) (days)
Glyphosate degradation in bacteria occurs primarily through two pathways The first pathway involves decarboxylation, where glyphosate is cleaved by glyphosate oxidoreductase, resulting in the formation of aminomethylphosphonic acid (AMPA) and glyoxylate Glyoxylate is then converted to glycine via the glyoxylate cycle, while AMPA is further degraded to inorganic phosphate and methylamine through the action of C-P lyase The second pathway is dephosphorylation, where C-P lyase catalyzes the breakdown of glyphosate into phosphate and sarcosine Subsequently, sarcosine oxidase facilitates the transamination of sarcosine into glycine, which is ultimately utilized for microbial biomass.
The degradation of glyphosate in the environment is primarily influenced by abiotic factors rather than physical and chemical pathways Research by Kathleen A Barry and Murray B McBride highlights that the cleavage of the C-P bond at the Mn oxide surface is crucial for glyphosate degradation, which accelerates with rising temperatures Additionally, the presence of sulfate does not impact the reaction rate Ascolani Yael and colleagues found that metallic ions, specifically Cu²⁺, can facilitate the abiotically degradation of glyphosate to AMPA in aqueous solutions Furthermore, photodegradation studies have shown that various factors, including illumination time, photocatalysts, initial pH, anions, electron acceptors, and metal ions, significantly influence the degradation process of glyphosate.
Figure 1 4 Main glyphosate biodegradation pathways in the environment 53
1.1.4 Residues of glyphosate in food and water
Glyphosate, extensively used in agriculture, can accumulate in the environment and leach into soil and aquatic systems Its widespread application has led to the presence of glyphosate in crops, water, and animal feed, raising concerns about potential side effects on human health.
Recently, many scientific research papers have been published on residues of glyphosate in the environment 70-72
Glyphosate, after being absorbed by soils, can be degraded to AMPA through microbial activity, physical and chemical processes, or photodegradation Research indicates that due to its strong sorption and degradation in environmental conditions, glyphosate has a relatively low potential for soil transport and contamination of aquatic systems However, numerous studies have demonstrated the presence of glyphosate in soils and water systems, primarily attributed to water erosion and wind.
Method for glyphosate ex-situ analysis
Sampling is a crucial step in the analytical process, involving the selection of a manageable portion of material for laboratory analysis It significantly contributes to errors in the analytical process, particularly when measuring pesticide residues in water Due to the physical-chemical complexity of water, an appropriate sample-handling procedure is essential to ensure the collection of a representative sample for glyphosate analysis.
1.2.1.1 Direct sampling without pre-concentration step
Direct sampling, a traditional sampling techniques, is the most commonly used method for glyphosate analysis in water The produce of this sampling technique based on a series of steps including 95 :
Sampling Site: it is must be selected with respect to the objectives of a monitoring program or a survey and assured that the location is suitable for taking representative samples
The required sample volume for glyphosate detection varies based on the analysis method used, such as gas chromatography (GC) or high-performance liquid chromatography (HPLC) combined with mass spectrometry or UV–vis detection Typically, the total volume of water samples collected from the site can range from 10 mL to 1-2 liters.
Sampling for water quality analysis can be conducted using bottle collection or water pump systems in rivers or drinking water sources It is essential to sample at a depth of 0.3–1.0 meters in streams For sample storage, plastic bottles made of polypropylene, polyethylene, or high-density polyethylene are recommended Key parameters such as water pH, electrical conductivity, nitrates, ammonia, and dissolved oxygen content should be measured in-situ to ensure accurate results.
The preservation of water samples is critical, as analytes can be lost during transport and storage due to processes like evaporation, adsorption, hydrolysis, photodegradation, and biodegradation Glyphosate, in particular, can form strong complexes with metal ions and colloids at varying pH levels, leading to potential degradation Research by Kylin 97 revealed that glyphosate recovery from spiked natural waters was significantly higher at an acidic pH (2) compared to natural pH after one and three weeks of storage Additionally, maintaining water samples at 4 °C in darkness improved recovery rates Various ISO guidelines for water sampling, including ISO 5667-1:1980/Cor 1996, ISO 5667-3:2003, ISO 5667-6:2005, ISO 5667-9:1992, and ISO 5667-2:1991, have been established to ensure proper sampling practices.
Days after collection pH 2, 4 o C, dark pH 2, 20 o C, dark pH 2, 20 o C, light pH 7, 4 o C, dark pH 7, 20 o C, dark pH 7, 20 o C, light
Figure 1 5 Effects of different treatments on concentrations of glyphosate 97
Direct sampling remains the prevalent method for monitoring glyphosate and other pesticides in water; however, it has significant limitations This approach necessitates large water volumes, only captures the composition at the time of sampling, poses challenges in quality control, and incurs high costs.
1.2.1.2 Passive sampling and pre-concentration
To address the limitations of direct sampling techniques, innovative methods like increased sampling frequency and automatic online monitoring systems have been introduced However, these approaches tend to be costly and labor-intensive Consequently, passive sampling techniques have emerged as a viable option for monitoring pesticides in water Research has demonstrated the effectiveness of passive sampling in detecting glyphosate levels in aquatic environments.
Introduced in 1927 for semi-quantitative CO determination in air, passive sampling has evolved into an effective monitoring tool for time-integrated measurement of pesticide contaminants in water and sediment This technique relies on the in-situ accumulation of analytes from the sampled medium to a receiving phase, driven by differences in chemical potentials The receiving phase can include solvents, chemical reagents, or porous adsorbents that capture the sampled chemicals Typical configurations of passive samplers include Chemcatcher, polar organic chemical integrative samplers (POCIS), and diffusive gradients in thin-film technology (DGT).
Figure 1 6 The typical configurations of passive sampler: Chemcatcher, POCIS, and DGT 104
The exchange kinetics between a passive sampler and water phase can be described by a first-order, one-compartment mathematical model 102, 103 :
𝐶 𝑠 (𝑡) is the concentration of the analyte in the sampler at exposure time t
C W is the analyte concentration in the aqueous environment k 1 and k 2 are the uptake and offload rate constants, respectively
Pollutant adsorption from water to passive sampler is described in Figure 1.7, which is demonstrated in 3 accumulation phases 103 :
Phase 1: which is time-integrative, during which compound accumulation kinetics follow a pseudo-linear curve (when the concentration in the water phase is kept constant)
Phase 2: accumulation kinetics is curvilinear
Phase 3: corresponding to the equilibrium compound distribution between the integrative sampler and aqueous phase being sampled
Passive sampling data can effectively estimate key parameters such as the time-weighted average pollutant concentration in water, mass transfer rates, and contamination levels in the environment This method offers several advantages over traditional sampling, including ease of use, rapid results, reliability, robustness, and cost-effectiveness for pesticide monitoring However, it presents challenges in validation and quality control compared to conventional methods, and the accuracy of the data is significantly influenced by environmental factors like temperature, flow rate, pH, and salinity.
Figure 1 7 Exchange kinetics between the sampler and the water showing 3 phases: linear, curvilinear, and steady-state 103
1.2.2 Analytical methods for the determination of glyphosate
Gas chromatography is a widely used technique for quantifying glyphosate in environmental and food samples Due to glyphosate's low volatility, analytical methods often require derivatization to enhance selectivity and sensitivity Various derivatization approaches have been explored, including trialkysilylation and simultaneous acetylation and esterification, utilizing reagents such as trifluoroacetic anhydride, diazomethane, heptafluorobutyric anhydride, and others.
Since 1977, Monsanto developed a glyphosate derivatization procedure involving trifluoroacetic anhydride for acetylation and alkylation with diazomethane, followed by detection via gas chromatography This method, endorsed by the EPA, was quickly adopted by various laboratories However, it has been criticized for producing irreproducible results and poor analyte recoveries in crop and soil samples Furthermore, the use of diazomethane poses significant toxicity concerns.
21 carcinogenic and explosive reagent Consequently, different methods have been developed to tackle these limitations
Elisabet Borjesson and Lennart Torstensson developed a method for determining glyphosate levels in water and soil, utilizing a mixture of TFAA and TFE for the derivatisation of glyphosate The derivatisation process is depicted in Figure 1.8 For the analysis and separation of the derivatised glyphosate products, gas chromatography–mass spectrometry (GC-MS) was employed in selected ion-monitoring mode The method achieved a limit of quantification of 0.1 μg/L for glyphosate in water and 0.006 μg/g in soil.
Figure 1 8 Derivatisation of glyphosate with trifluoroethanol (TFE) and trifluoroacetic anhydride (TFAA) 110
Kaoqi Lian and colleagues recently explored a novel method for detecting glyphosate in environmental systems, including water and soil, by combining gas chromatography with a flame photometric detector Their research involved the derivatization of glyphosate in samples using heptafluorobutyric anhydride and heptafluorobutanol.
The method, which involves heating samples to 90 °C for 50 minutes and utilizing solid-phase extraction (SPE), has proven effective in detecting glyphosate residues in both water and soil The limit of detection (LOD) was established at 0.10 ng/mL, while the limit of quantification (LOQ) was determined to be 0.37 ng/mL.
Currently, the most effective methods for determining glyphosate involve liquid chromatography combined with various detection techniques, including UV-Vis, fluorescence, and mass spectrometry These liquid chromatography methods offer high adaptability in terms of sensitivity and selectivity for analyzing glyphosate in various matrices such as water, soil, and plant samples Like gas chromatography, a derivatization step is necessary to improve both separation and detection of glyphosate.
Both pre-column and post-column derivatization methods have been established for the analysis of glyphosate, utilizing various derivatization agents based on instrumental design and detector type, as detailed in Table 1.4.
Chemical sensors for quantification of glyphosate
1.3.1 General principle of chemical sensor
To address the limitations of traditional analytical techniques, there is a need for a cost-effective and straightforward method for detecting glyphosate residues in environmental and biological samples The creation of a chemical sensor presents a promising solution for glyphosate sensing, offering significant advantages in terms of efficiency and accessibility.
Chemical sensors offer easy operation, high sensitivity, selectivity, and cost-effectiveness, making them ideal for in-situ measurements and continuous monitoring This capability allows for the provision of analytical information about samples at a high frequency.
According to the International Union of Pure and Applied Chemistry (IUPAC), a chemical sensor is defined as a device that converts chemical information, including specific component concentration and overall composition analysis, into a useful analytical signal A typical chemical sensor consists of two primary components: the recognition element (receptor) and the transducer.
Figure 1 13 Schematic representation of a typical chemical sensor
The recognition element, or receptor, is crucial in a chemical sensor as it provides the active layers that selectively interact with target compounds The interaction between analyte molecules and receptors can result in changes to the receptor's physicochemical properties, such as mass, volume, optical characteristics, or resistance These changes are reversible and can be effectively converted into measurable signals by a suitable transducer.
The chemical sensor can be classified in different ways depending on the operation principle of the transducer (Figure 1.14) 142 According to the measurement types of
Optical Electrochemical Electrical Gravimetric Thermal Magnetic
28 the transducer, the sensor can be grouped into different types such as electrochemical, optical, electrical, mass, magnetic, thermometric, and others 143
Figure 1 14 Classification of chemical sensors 142
Recent studies on glyphosate detection primarily utilize advanced analytical techniques like gas or liquid chromatography, which demand costly equipment, skilled personnel, and intricate procedures These methods are not suitable for on-site rapid screening and monitoring In contrast, electrochemical methods offer a cost-effective, highly sensitive, and selective alternative, with the advantages of easy operation, potential for automation, and rapid analysis capabilities.
Glyphosate, classified as a non-electroactive compound, requires indirect analysis methods under field conditions To address this, researchers have developed electrochemical techniques that involve modifying electrode surfaces or utilizing derivatization methods for effective glyphosate detection.
Moraes et al investigated the electrooxidation of glyphosate using a glassy carbon electrode modified with copper-phthalocyanine and multiwalled carbon nanotubes (GC/MWCNT/CuPc) through differential pulse voltammetry Their findings revealed a significant interaction between glyphosate and CuPc, which altered the electrochemical signal based on glyphosate concentration Under optimal conditions, the GC/MWCNT/CuPc electrode effectively detects glyphosate in drinking water within a concentration range of 140 to 1,673 àg/L, achieving a detection limit of 2.02 àg/L.
Pintado-Benzal et al have investigated a straightforward analytical method for detecting glyphosate in drinking water using electrogenerated copper ions Their approach involves depositing a copper film on a carbon paste electrode through an electrodeposition technique The study reveals a significant coordinated interaction between copper ions and the functional groups of glyphosate molecules—specifically the amino, carboxyl, and phosphate groups—leading to the formation of a complex with glyphosate.
The electroactive complex formed between glyphosate and Cu2+ allows for the indirect determination of glyphosate concentration in solution using voltammetry As illustrated in Figure 1.15, the modified electrode responds to varying glyphosate concentrations This method effectively measures glyphosate levels in drinking water within a concentration range of 5 to 70 µM, achieving recoveries between 96% and 104%.
Figure 1 15 Cyclic voltammograms of 1 mM copper in CPE in the presence of 0, 0.075,
0.15, 0.2, 0.3, 0.37, 0.45, 0.5 and 0.59 mM glyphosate (from a to i) using in 0.1 M phosphate buffer at pH 6.5 144
Guoxiu Wang et al have created a novel platform utilizing metal-organic frameworks (MOFs) for the detection of glyphosate in green vegetable samples Their research employed Copper benzene-1,3,5-tricarboxylate (Cu-BTC) as the MOF material, which was coated on an indium tin oxide (ITO) electrode to enhance glyphosate detection The Cu-BTC MOF provides a large specific surface area, improving both the electrode reaction sites and adsorption capacity, thereby increasing method sensitivity The interaction between Cu-BTC on the electrode and glyphosate allows for the determination of glyphosate concentration through techniques such as electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse stripping voltammetry (DPV) The modified electrode demonstrated an ultralow detection limit of 1.4 × 10 −13 mol/L and a wide detection range from 1.0 × 10 −12 to 1.4 × 10 −5 mol/L.
Recent studies indicate that glyphosate is a non-electroactive compound, necessitating complex procedures for its electrochemical detection However, a breakthrough in 2018 by Noori et al demonstrated that glyphosate can be directly detected using gold (Au) working electrodes in drinking water through electrochemical sensing The cyclic voltammogram of glyphosate in a water background, depicted in Figure 1.16, shows that under optimal conditions, the Au electrode achieves a detection limit of 2.0 μM using amperometric measurements.
Figure 1 16 Cyclic voltammogram of various glyphosate concentration on Au electrode in tap water 147
Optical sensors offer a rapid, simple, and cost-effective method for the sensitive detection of pesticides, particularly glyphosate, which has garnered significant attention in recent research These sensors typically consist of a sensitive layer that selectively interacts with the target analyte and an optical transducer that employs various detection strategies, including colorimetric, surface-enhanced Raman scattering, fluorescence, surface plasmon resonance, time-resolved Förster resonance energy transfer, and chemiluminescence As a result, multiple optical detection modes for glyphosate have been successfully developed.
Wang et al developed a time-resolved Förster resonance energy transfer sensor for direct detection of glyphosate in aqueous solutions, achieving a limit of detection of approximately 0.78 µM using lanthanide-doped nanoparticles Additionally, Dai et al created a prism coupling optical waveguide configuration for trace glyphosate determination in water, capable of detecting glyphosate at a limit of 1.4 nM by exciting highly sensitive ultrahigh-order modes through small incident angle coupling.
Figure 1 17 Schematic diagram of the hollow-core metal-cladded waveguide sensor for glyphosate detection 150
Molecular imprinting in chemical sensor development
In recent years, the application of biosensors has surged across various fields, utilizing biomolecules like antibodies, peptides, and enzymes as recognition elements However, these biomolecules present challenges, including high costs, limited lifespan, and instability, which are heavily influenced by assay conditions such as temperature, pH, and ionic strength Consequently, these limitations have hindered the expected commercial success of biosensors in analytical applications.
To address the limitations of biosensors, researchers have focused on developing materials that can mimic natural binding phenomena A key objective of this research is to create artificial receptors that can serve as substitutes for enzymes, antibodies, cells, or aptamers in biosensor applications The synthetic materials offer significant advantages, including enhanced selectivity and affinity for target molecules.
Molecular imprinting is a highly promising technique for designing materials that mimic molecular recognition, offering significant advantages such as high physical robustness, strength, and resistance to temperature and organic solvents.
Molecular imprinting was first reported by M Polyakov in 1930, demonstrating that silica gels exhibited a preference for binding with solvents used during preparation In 1949, Linus Pauling and colleagues explored the imprinting effect on silica surfaces using dye compounds as templates Subsequent research expanded molecular imprinting to include various organic polymers A significant advancement in this field was made by Guenter Wulff and the teams of Mosbach and Arshady, who utilized both covalent and non-covalent approaches Today, molecular imprinting technology has numerous applications in environmental, food, and pharmaceutical analysis.
Molecular imprinting operates on a principle akin to the "lock and key" theory of enzymes, involving the polymerization of functional monomers, a cross-linker, and an initiator in the presence of a target analyte as a template Following polymerization, the interactions between the template and monomers are disrupted, allowing for the removal of the template molecules, which creates binding sites that precisely match the size, shape, and functional groups of the analyte This results in a polymer with molecular memory capable of rebinding the analyte with exceptional specificity.
Figure 1 18 Schematic representation of the molecular imprinting principle 160
Molecular imprinting techniques can be categorized into three types based on the chemical bonding between the template and functional monomers: covalent, non-covalent, and semi-covalent interactions The non-covalent method, primarily developed by Mosbach et al., relies on weak interactions such as electrostatic forces, hydrogen bonding, π–π interactions, and hydrophobic effects In contrast, the covalent approach, introduced by Wulff’s group, involves the formation of covalent bonds between the template and functional monomers, creating a polymerizable derivative After polymerization, the imprinted template is removed through chemical cleavage, allowing the polymer to rebind the target analyte via covalent bonds Covalent imprinting polymers (MIPs) demonstrate higher imprinting efficiency, making them more sensitive and selective than non-covalent counterparts However, the covalent method often entails complex preparation procedures and intricate extraction methods for template removal.
Non-covalent imprinting has gained popularity in the synthesis of molecularly imprinted polymers (MIPs) due to its straightforward preparation, robust target uptake, and efficient removal However, to address the limitations of traditional methods, a semi-covalent approach that combines both covalent and non-covalent interactions has been proposed for MIP development This semi-covalent method exhibits distinct rebinding kinetics compared to the purely covalent imprinting approach, highlighting the differences in their non-covalent rebinding mechanisms.
In synthetic chemistry, various effective polymerization techniques have been proposed, including bulk polymerization, suspension, two-step swelling, precipitation, and emulsion core-shell polymerization For the development of sensors utilizing molecularly imprinted polymers (MIPs), methods such as surface imprinting, micro-contact imprinting, epitope imprinting, and MIP electropolymerization are also employed.
Figure 1 19 Schematic diagrams of the molecular imprinting process 166
1.4.3 Sensor development for glyphosate analysis using molecular imprinting technology
MIP thin film for electrochemical sensor
Electrochemical sensors have been extensively researched for applications in environmental monitoring and food safety The integration of electrochemistry with molecularly imprinted polymers (MIPs) offers a promising sensing platform, enabling the detection of analytes at very low concentrations with high accuracy Various methods are employed to combine MIPs and electrochemistry, including in-situ polymerization on sensor surfaces through spin coating, surface grafting, electropolymerization, and immobilization techniques.
In 2015, researchers developed an electrochemical sensor utilizing molecular imprinting for the detection of glyphosate in tap water To enhance the specific surface area and increase the number of imprinted sites, gold nanoparticles (AuNPs) were incorporated into the electropolymerization process This innovative sensor, designed for the sensitive and selective detection of glyphosate, was created by electropolymerizing para-aminothiophenol (PATP)-functionalized AuNPs in the presence of glyphosate as the template molecule The fabrication principle of this glyphosate detection sensor is illustrated in Figure 1.20.
Figure 1 20 Steps in the fabrication of the imprinted sensor for glyphosate detection 169
The study demonstrated that the developed method effectively detects glyphosate in water, exhibiting a linear detection range from 5.9 fM (1 pg/L) to 5.9 nM (1 µg/L) and a low detection limit of 5 fM (0.8 pg/L) The sensor displayed excellent selectivity and successfully identified glyphosate in tap water samples, achieving recovery rates of 102.6% and 98.7% Notably, this research presents the first molecularly imprinted metal-organic framework (MOF) electrochemical sensor specifically designed for glyphosate detection.
Based on this method, glyphosate can be measured indirectly using a non-catalytic electrode material
Prasad et al developed a doubly imprinted polymer nanofilm-modified sensor for the ultra-trace simultaneous analysis of glyphosate and glufosinate in aqueous, soil, and human serum samples Utilizing electrochemistry as a transducer, the sensor's fabrication involved complex protocols, incorporating gold nanoparticles and multi-walled carbon nanotubes to enhance electrode conductivity The MIP film was created using N-nitroso derivative of glyphosate and glufosinate as templates, combined with N-methacryloyl-L-cysteine monomers, EGDMA cross-linkers, and chloroform initiators Post-polymerization, template molecules were extracted with a mixture of acetonitrile and trimethylamine The differential pulse anodic stripping voltammetry (DPASV) technique revealed a glyphosate detection range of 3.98 – 176.23 ng/mL, with a detection limit of 0.35 ng/mL The sensor demonstrated recoveries of 97.8 - 101.9% in water, 98.6 – 102.8% in soil, and 98.1 – 110.2% in human serum samples, confirming its sensitive and selective detection capabilities without interferences or matrix effects.
Figure 1 21 Schematic representation of double-template imprinted polymer-modified
GNPs-PGE fabrication for the simultaneous analysis of NGLY and glufosinate 170
Figure 1 22 DPASV runs of NGLY at MIP-modified GNPs-PGE [NGly concentrations
(from a to f): 20.49, 36.38, 63.29, 80.40, 92.21 and 123.61 ng/mL] 170
Zhang et al developed an electrochemical sensor utilizing a molecularly imprinted polymer (MIP) for the selective and sensitive detection of glyphosate in cucumber and tap water samples The MIP film, designed for glyphosate sorption, was created on a gold electrode through the electrodeposition of a pre-polymerization solution that included glyphosate as the template molecule and pyrrole as the functional monomer Under optimal conditions, the sensor effectively measured glyphosate levels using differential pulse voltammetry, demonstrating its potential for accurate monitoring in various water sources.
The MIP electrochemical device effectively detected glyphosate in cucumber and tap water samples, achieving recoveries between 72.70% and 98.96%, with a concentration range of 5 to 800 ng/mL and a detection limit of 0.27 ng/mL Additionally, research by Xu et al utilized pyrrole to create a sensor for voltammetric glyphosate determination in real water samples This sensor, enhanced with various Au nanoparticles and Prussian blue as a signal amplifier, successfully measured glyphosate concentrations from 400 to 1,200 ng/mL, demonstrating a robust detection capability.
Figure 1 23 Schematic illustrations of the fabrication procedure for the electrochemical
MIP sensor for glyphosate detection using pyrrole monomer 171
MIP-based on spectrophotometric sensor
Jensen and his team developed an optical sensor for glyphosate detection using a molecularly imprinted polymer (MIP) and optical fibers as transducers The MIP film was created through the polymerization of a styrene solution containing glyphosate, vinyl benzoate, divinylbenzene, and azobisisobutyronitrile (AIBN) as an initiator During polymerization, lanthanide ions were incorporated to serve as signal transducers Glyphosate molecules were extracted using 1.0 M nitric acid, and detection was achieved by coordinating glyphosate with lanthanide ions, resulting in a change in the lanthanide spectrum and increased luminescence intensity proportional to glyphosate concentration The method demonstrated a wide detection range from 0.009 ng/mL to 100 µg/mL, with a low detection limit of 0.009 ng/mL and a response time of 15 minutes.
Figure 1 24 Spectral response of the optical MIP sensor at different of glyphosate, excited at 465.8 nm 173
Chitosan in sensor technologly
Chitosan (CS), the second most abundant biopolymer next to cellulose, firstly discovered by Henri Braconnot in 1811 177 It consists predominantly of unbranched chains of β-(1→4)-2 acetamido-D-glucose and β-(1→4)-2-amino-D-glucose units 178,
179 CS is composed of two sugars glucosamine and N-acetylglucosamine, the ratio
43 of these sugars in CS depends on the alkaline treatment 178 The chemical structure of
CS at different pH is described in Figure 1.26 180 CS contains two free hydroxyl groups and one primary amine in each monomer 178
Figure 1 26 CS chemical structure At lower pH, CS’s amine groups are protonated At high pH, CS’s amines are deprontonated 180
CS, a polysaccharide, is rarely present in nature 177 The main source of commercial
CS was synthesized by treating the chitin shells of shrimp, prawn, crab, lobster and other crustaceans 178 Deacetylation of chitin to produce CS is shown in figure 1.27 181,
The extraction of calcium carbonate from shells begins with the use of dilute hydrochloric acid, followed by the precipitation of calcium chloride through stirring at room temperature Proteins are then eliminated using dilute aqueous sodium hydroxide The final product, chitosan (CS), is achieved through deacetylation in a 40–45% sodium hydroxide solution at 120 °C for 1–3 hours, resulting in a product that is 70% deacetylated chitosan.
Figure 1 27 Conversion of chitin to CS by deacetylation 183
Chitosan's chemical and physical properties are primarily influenced by its molecular weight and degree of deacetylation, which are affected by the preparation methods and initial sources The average molecular weight of chitosan can range from 50 to 2,000 kDa, with higher molecular weights leading to decreased membrane crystallinity The degree of deacetylation, determined by the acetyl group content, is influenced by the time and temperature during the deacetylation process This degree significantly impacts chitosan's solubility in acidic solutions and its membrane crystallinity; at a low deacetylation degree (55-70%), chitosan is nearly insoluble in water, while at a moderate degree (70-85%), it is partially soluble With a high deacetylation degree (85-100%), chitosan exhibits good water solubility Additionally, chitosan is soluble in various inorganic and organic acids at a pH below its pKa (approximately 6.5) but remains insoluble in most common organic solvents.
1.5.2 CS nanocomposites in sensor technology
In sensor development, CS has been considered as one of the most promising biopolymers due to its excellent properties, such as eco-friendly, substantiality,
Chitosan (CS) is valued for its abundance, nontoxicity, biodegradability, and biocompatibility However, its widespread use is hindered by its high swelling degree in aqueous systems and subpar physical properties Additionally, the amino groups in CS can become protonated at low pH levels To address these challenges, chemical cross-linking is commonly employed to reduce solubility and improve the mechanical properties of CS Various cross-linkers have been investigated for this purpose, including glutaraldehyde, sulfuric acid, glyoxal, and epichlorohydrin The mechanism of cross-linking between CS and these agents is illustrated in Figure 1.28.
Figure 1 28 Scheme of CS cross-linking with glutaraldehyde, epichlorohydrin, sulfuric acid, and glyoxal 189-192
Due to possessing many excellent properties, CS was applied as a platform in the fabrication of different types of chemical sensors including electrochemical, optical, and acoustic wave sensors
Triyana et al developed a chitosan-based quartz crystal microbalance (QCM) sensor for detecting alcohol vapor, as shown in Figure 1.29 The sensor features an active chitosan layer applied to a gold electrode via a self-assembled monolayer of L-cysteine This innovative sensor effectively detects ethanol concentrations within a wide linear range of 1 mg/L to 37 mg/L and boasts a rapid response time of just 15 seconds.
Figure 1 29 Schematic chemical structure and active layer deposition of the CS-based
QCM chip for alcohol sensing 193
Recent advancements in optical sensors have been achieved through the integration of surface plasmon resonance with chitosan (CS) A study by McIlwee et al focused on a surface plasmon resonance sensor designed for detecting ferric ions, utilizing a CS coating on a gold electrode This innovative optical sensor successfully measured ferric ion concentrations ranging from 0 to 2.5 mg/L, with an impressive detection limit of just 0.25 mg/L.
In another study, an electrochemical sensor was developed by Hwang et al for in- situ arsenite detection in mining wastewater and soil leachate 195 In this work, a Fe-
A CS composite was applied to a screen-printed carbon electrode using an electrodeposition technique The resulting sensor demonstrated a strong correlation in its response to arsenite, achieving detection limits of 1.12 ppb for mining wastewater and 1.01 ppb for soil leachate.
Recent research has focused on enhancing the physical-mechanical properties of chitosan (CS) through the development of various CS-based composites These composites have been utilized in sensor technology, including a hybrid nanocomposite of CeO2/ZnO/CS, which was investigated by Sultan Akhtar as a potential sensing platform.
Graphene oxide, known for its excellent electrical and optical properties, has emerged as a promising material for various applications, including the voltammetric detection of paracetamol and its degradation product, p-aminophenol, with a detection limit of 0.98 nM Its incorporation with chitosan (CS) enhances its potential in sensing metal ions.
200 Besides that, different composites based on CS and other nanomaterials such as CuO, TiO2, Ag, and Fe3O4 also used for sensing application 201-203
1.5.3 Use of MIP-CS in sensor technology
Eco-design is increasingly promoted across various sectors to mitigate environmental and health impacts, particularly in the development of Molecularly Imprinted Polymer (MIP) sensors Traditionally, research on MIP sensors has focused on toxic compounds like pyrrole and acrylic acid However, recent advancements have introduced biodegradable polymers derived from natural sources, such as lignin, polysaccharides, gelatin, silk, and polylactic acid, to address the limitations of conventional materials Among these, chitosan (CS) has emerged as a highly suitable option for MIP synthesis.
Current research on chitosan (CS)-based molecularly imprinted polymers (MIPs) focuses on their applications in various fields of analytical chemistry These CS-based MIPs can be integrated with different transducers, including electrochemical, optical, and mass transducers Therefore, the preparation method for CS-based MIPs must be selected based on the specific technique and target analyte Presently, the primary strategies for synthesizing CS MIPs include bulk imprinting and surface imprinting.
Bulk imprinting is a widely utilized technique for preparing chitosan-based molecularly imprinted polymers (MIPs) The general process involves three key steps: first, a solution containing chitosan and a template is prepared in an appropriate solvent Next, crosslinking occurs through noncovalent or covalent interactions, followed by the removal of the template, which creates recognition cavities within the polymer matrix This method allows for the imprinting of the entire template molecule in the polymer MIPs produced via bulk imprinting can be effectively assembled onto electrode surfaces using techniques such as sol-gel systems, electro-polymerization, and both drop and spin coating Among these, cathodic electro-polymerization stands out as the most favored method for developing chitosan-based MIP sensors.
The electrode's surface generates a localized high pH that triggers the sol-gel transition of chitosan (CS), enhancing hydrogel-forming properties While bulk polymerization is a straightforward and widely applicable method, it is often time-consuming and labor-intensive Moreover, this technique presents challenges in sensor fabrication, such as difficulties in template elution and the presence of non-specific binding sites in the resulting molecularly imprinted polymer (MIP) film, which can lead to thick imprints that adversely affect regeneration, cause drift, and result in prolonged response times.
Figure 1 30 Mechanism of CS’s cathodic electrodeposition 211
To address the challenges of bulk and surface imprinting in the creation of CS-based MIP sensors, innovative imprinting techniques have been developed This approach allows for the formation of recognition sites on the substrate's surface, enhancing the effectiveness of these sites, facilitating template removal, and improving access to target analytes Notably, this method utilizes fewer template molecules compared to traditional techniques Various surface imprinting methods, such as soft lithography, template immobilization, micro-contact imprinting, grafting, and emulsion polymerization, are employed in the advancement of MIP sensors.
Numerous molecularly imprinted polymer (MIP) sensors have been created using chitosan (CS) through both bulk and surface imprinting techniques A notable study by Yiyong Wu and colleagues explored a molecularly imprinted electrochemical sensor designed for the detection of tryptophan, highlighting the role of hydrogen bond formation between tryptophan and the chitosan coating.
Multi-walled carbon nanotubes were utilized to enhance electron transfer and conductivity in a glassy carbon electrode modified with sulfuric acid as a cross-linker, resulting in a molecularly imprinted polymer (MIP) sensor with a calibration range of 2 nM to 100 µM and a detection limit of approximately 1 nM Wei et al developed an electrochemical sensor using a CS-graphene oxide composite polymer modified glassy carbon electrode for Cu (II) ion detection, achieving a concentration range of 0.5 to 100 µmol/L and a detection limit of 0.15 µmol/L through chemically crosslinked Cu (II) ion-imprinted polymers In 2019, Wang et al investigated a MIP electrochemical sensor for dopamine, employing CuCo2O4@carbon/3D-KSC to enhance electron and mass transfer, with a detection range of 0.51 μM to 1.95 mM and a detection limit of 0.16 μM Wu et al developed a molecular imprinting sensor for Cr(VI) detection in tap and river water, synthesizing Cr(VI) ion-imprinted polymers via one-step electrodeposition and integrating graphene to improve conductivity This method demonstrated the ability to detect glyphosate with a linear range of 1 nmol/L to 10 µmol/L and a low detection limit of 0.64 nmol/L, while also exhibiting high selectivity for Cr(VI) ions amidst various other ions.
Surface Plasmon Resonance (SPR)
In 1902, Wood discovered narrow dark bands in the spectrum of diffracted light when illuminating a metallic diffraction grating with polarized polychromatic light, identifying an anomalous optical phenomenon While various physicists, including Lord Rayleigh, Thurbadar, and Fano, attempted to interpret this phenomenon, a comprehensive explanation was provided by Kretschmann, Raether, and Otto in 1968 Otto clarified that the decrease in reflectivity observed in the attenuated total reflection method results from the excitation of surface plasmons.
In 1971, Kretschmann and Raether discovered a new configuration for exciting surface plasmons, building on Otto's pioneering research Their work established a practical method for surface plasmon excitation, which has since been widely utilized in various fields, including thin film characterization and the investigation of processes at metal boundaries.
Surface plasmons (SPs) are oscillating electromagnetic waves that occur in the high-density electron liquid of metals at the interface with a dielectric material These evanescent waves decay exponentially into both the metal and dielectric When SPs couple with electromagnetic waves, they are referred to as surface plasmon polaritons (SPPs).
Figure 1 31 Schematic of propagating SPPs on a metal-dielectric interface 219
The theory of Surface Plasmon Resonance (SPR) is mathematically described through the evanescent wave generated by total internal reflection at a reflective surface This evanescent wave can be represented by the electric field E when the exponent is complex.
𝐸 0 is the amplitude of the electric field ω is the angular frequency
𝑟 ≡ (x, y, z) is the position vector, and 𝑖 = √−1
The wave vector contains the propagation direction, which is parallel to the propagation of the electromagnetic wave, thus:
51 where λ is the wavelength, c is the light propagation velocity in vacuum ε is dielectric constant of the medium μ is the magnetic permeability
When an electromagnetic wave encounters the boundary between two media with different refractive indices, n1 and n2, it undergoes refraction, particularly in a two-dimensional system where ky = 0 According to Snell's law, the relationship between the angles of incidence and refraction is expressed as n1 sin θ = n2 sin β Additionally, the perpendicular component of the wavevector, kz2, relative to the interface can be derived using this relationship and the relevant equations.
𝑛 1 2 − 𝑠𝑖𝑛 2 𝜃) (1.4) assuming than n1 sin θ > n2 sin β From Eq 1.4, when sin θ > n2/n1, kz2 is purely imaginary
In medium 2, only longitudinal waves, specifically p-polarized waves, propagate perpendicularly and exhibit exponential decay towards the interface, resulting in the formation of what is known as the evanescent field.
Figure 1 32 Refraction of light at an incident angle θ, at the interface between two media with refractive indexes n1 and n2 218
With the medium 1 is dielectric medium and medium 2 is metallic medium, the evanescent waves traveling in these mediums can be obtained according to the following expressions:
At boundary conditions, the following constraint is necessary:
The equation 1.7 can only be true if ԑ2 < 0 (i.e., a metal) From that, the dispersion relation for surface waves is obtained:
When dielectric medium 1 is air (ε1≈1) and medium 2 is a metal, the dielectric function of free electron gas for metals is given by:
𝜔) 2 (1.10) where 𝜔 𝑝 is the plasma frequency
The dispersion relation for surface plasmons (SPs) in air and in a dielectric medium like glass, illustrated in Figure 1.33, demonstrates that SPs can be excited by electrons or light, with light excitation being more cost-effective Notably, the SP dispersion relation does not intersect with that of light in air, indicating that SPs cannot be excited by a freely propagating light beam striking the metal surface Additionally, SPs cannot be excited when light is directed at the metal/air interface at specific angles, highlighting the need for alternative methods to achieve excitation.
Various experimental techniques have been developed to achieve plasmon excitation, including prism coupling under attenuated total reflectance (ATR) conditions, grating coupling, and waveguides utilizing subwavelength protrusions or holes.
The dispersion relations of surface plasmons (SPs) are illustrated, showing the relationship between wave vector and frequency for light in air, which is represented by the equation kx = ω/c Additionally, the behavior of light in air at a specific incident angle is described with kx = ω/c sinθ, while the dispersion relation for light in a dielectric medium, such as glass, at a certain incident angle is given by kx = n ω/c sinθ.
Photon and plasmon matching can be accomplished using a prism coupling configuration, where light passes through the prism to excite plasmons under attenuated total reflection (ATR) conditions This process allows plasmons to be excited on the opposite side of the interface within the evanescent field of the reflected light, occurring under specific resonance conditions.
𝑐𝑛𝑠𝑖𝑛θ (1.11) and the incident angle corresponding to the excitation of plasmons:
1 +𝜀 2 )𝜀 𝑝 (1.12) where the dielectric constant of the prism is given by 𝜀 𝑝 = 𝑛 2
Prism coupling techniques, introduced by Otto and Kretschmann-Raether in 1968, are essential for exciting surface plasmons (SPs) with incident light The Otto configuration features a thin dielectric layer separating the metal surface from the prism, allowing SP resonance at the metal/dielectric interface when the incident angle exceeds a critical value The effectiveness of this method is influenced by the dielectric medium's width, which should be approximately half the wavelength for optimal coupling In contrast, the Kretschmann-Raether configuration involves a thin metallic layer coated on a glass prism, where light is directed through the prism at a specific angle This setup enables efficient coupling of the evanescent field to SPs, particularly within a film thickness range of 50 ± 10 nm The Kretschmann configuration is favored for sensor development due to its straightforward setup and manageable parameters.
Figure 1 34 Different SPR configurations: Prism coupling ((a) Otto, (b) Kretschmann),
Grating coupling (c), and waveguide coupling (d) 219
Surface plasmons (SPs) can be excited on a metal surface using diffraction effects, a concept first introduced by Wood in 1902 The grating configuration for SPs excitation is illustrated in Figure 1.34 (c) When incident light interacts with a rough surface, it undergoes diffraction, causing changes in the wave vector of the light and leading to SPs excitation, in accordance with the coupling condition.
𝑏 is the reciprocal vector of the grating, being b the lattice constant
In this configuration, surface plasmons (SPs) are excited by light traveling through a waveguide under total internal reflection conditions This process generates an evanescent field at the waveguide/metal interface, which couples to the SPs The excitation occurs when the wave vector of the evanescent field aligns with that of the SPs at the metal/dielectric interface, allowing for a straightforward and flexible method of creating SPs through waveguide coupling.
1.6.3 Interactions analysis and kinetics using SPR sensor
Real-time measurement of analyte interaction with a receptor is visualized through a sensorgram, illustrating the binding response Initially, the response increases as the analyte binds to the receptor in the flow cell, followed by a decrease when the analyte solution is replaced with buffer, leading to dissociation of the binding complex At binding equilibrium, a stable plateau in the Surface Plasmon Resonance (SPR) signal is observed during the association phase before the buffer replacement occurs For more information, visit www.bio-rad.com.
Generally, the interaction between the analyte and the receptor can be described by Langmuir-type binding at a simple 1:1 binding model as the following equation as:
In the context of analyte-receptor interactions, A denotes the analyte while B represents the receptor The association rate constant, denoted as ka, quantifies the formation of AB complexes per second, while the dissociation rate constant, kd, indicates the fraction of these complexes that decay each second.
KD: the affinity value, equilibrium dissociation constant, is used for determining the affinity of the interaction The smaller the KD the higher affinity of molecule A to molecule B
Figure 1 35 Typical SPR sensorgram showing the steps of an analytical cycle (Handbook of MP-SPR – BioNavis)
Under pseudo-first-order conditions free analyte concentration in the flow cell is held constant so the binding described by following equation 220 :
Here, ΔR and ΔRmax are the change in the SPR angle at steady-state and maximum condition, respectively dΔR/dt is the rate of change of the SPR signal
Using a simple Langmuir-type model, the relevant equations for each step can be described the following equation 220 : association step: ∆𝑅 𝑡 = 𝛥𝑅 𝑚𝑎𝑥 [𝐴]
1.6.4 SPR sensor based on molecularly imprinted polymer
Conclusion and motivations of the thesis
This thesis presents a comprehensive overview of the latest techniques for glyphosate analysis, emphasizing the role of chitosan (CS) layers as recognition elements in chemical sensors The focus is specifically on optical and electrochemical sensors, highlighting their significance in advancing glyphosate detection methods.
The diverse range of chitosan (CS)-based receptors discussed in the first chapter, including SPR, SERS, and electrochemistry, highlights the ongoing advancements in CS technology, making them promising candidates for sensing applications Chitosan has demonstrated effectiveness in removing glyphosate due to the electrostatic interactions between glyphosate and its amine groups Additionally, CS thin films are relatively easy to prepare and functionalize, while also being stable and cost-effective Recent developments have explored the combination of chitosan with nanoparticles like ZnO, further enhancing its potential in sensing technologies.
GO has garnered interest for improving the optical properties and electrical conductivity of CS thin films SPR, a fast and portable sensing device, serves as an effective tool for both qualitative and quantitative detection of multiple pollutants in real-time Our primary objective was to create an optical sensor utilizing this technology.
59 incorporation of SPR with CS and its nanocomposites, such ZnO and GO for glyphosate detection, described in chapter 3
Molecular imprinting is emerging as a leading technique for the selective detection of glyphosate This article will discuss an optical sensor utilizing Surface Plasmon Resonance (SPR) technology, featuring a molecularly imprinted polymer (MIP) as the sensitive layer for glyphosate recognition.
The integration of electrochemistry with chitosan-based molecularly imprinted polymers (MIPs) has led to the development of highly accurate glyphosate sensors, capable of detecting this herbicide at extremely low concentrations.