| The issue of odor in drinking water is a huge challenge for the global water industry.Conventional detection techniques require complex pre-treatment and costly detection,which makes an urgent need to develop methods or instruments for quick on-site odor detection.Olfactory sensors based on odor-binding proteins（OBPs）have been widely used to detect contaminants in food and air samples,as researchers have gained a better understanding of olfactory mechanisms.However,the application of such techniques to detect the typical odor-causing compounds in drinking water were rare due to a lack of understanding of the binding properties of odorants.In this work,14 typical odorants in drinking water are investigated in terms of ligand binding mechanisms.The 14 odorants include 7 aldehydes,2 terpenes,2thioethers,bis（2-chloro-1-methylethyl）ether（DCIP）,2-ethyl-4-methyl-1,3-dioxolane（2E4MDL）,and 2-isobutyl-3-methoxypyrazine（IBMP）.The binding characteristics of the human odor binding protein OBP2a to 14 odorants were investigated and odorants with good binding ability to OBP2a were screened.An OBP2a-based electrochemical sensor was constructed for the detection of two typical aldehydes.The repeatability,specificity and retention time of the biosensors were assessed in relation to different water quality contexts.The main contents are as follows:（1）This work investigated the binding characteristics and mechanism of OBP2a with typical odorants in drinking water using fluorescence competition binding experiments and molecular docking techniques.OBP2a has been shown to bind to nine odorants,including terpenes,pyrazines,thioethers and aldehydes,and its broad binding spectrum is due to the large hydrophobic cavity.Lys112 and Phe97 within the hydrophobic pocket of the protein have been shown to be important amino acid residues involved in binding.All aldehydes and IBMP can form hydrogen bonds with Lys112 and all aromatic ring-containing substances can formπ-πstacking with Phe97.In the experimental aldehydes,it was found that OBP2a relies on the formation of hydrogen bonds with Lys112 to enhance the binding of the protein to the aldehydes,thus showing a good binding affinity for the aldehydes.Correlation analysis demonstrates a moderate negative correlation between the hydrophobicity of the aldehyde molecule and the dissociation constant,indicating that the more hydrophobic the odorant,the stronger the binding ability between the two.（2）Immobilization of OBP2a on the surface of the electrode was achieved by the physical adsorption of the nitrocellulose membranes.A screen-printed gold electrode was used to determine benzaldehyde in the concentration range of 100 nmol/L-1 mmol/L andβ-cyclocitral in the concentration range of 100 ng/L-100μg/L,based on cyclic voltammetry detection using the transient peak current changeΔI caused by different concentrations of odorants.The data fitting revealed that the binding of the odorant molecule to OBP2a is a reversible reaction.The concentration dependence curve of the screen printed gold electrode for the detection of benzaldehyde conforms to the Hill’s formula:ΔI=2.702×（X/（X+3.024））0.090.A Hill coefficient of less than 1 indicates a negative synergistic relationship between the two,i.e.when OBP2a binds to the benzaldehyde molecule,it inhibits the binding of other odorants to OBP2a.（3）The sensor was evaluated for specificity,repeatability,interference resistance and storing conditions.Comparison of the response of the OBP2a sensor to 14 odorants showed that the current response to aldehydes is generally higher than that of other odorants,indicating that the OBP2 has good specificity for the detection of aldehydes;At low and medium concentrations,the sensor showed good repeatability（error less than 10%）;The results of the detection in different water quality contexts showed a certain degree of immunity to interference and a more sensitive redox peak;The sensor could be stored at low temperatures and still retain good biological activity when stored at-20℃ for a week. |
