| Nowadays,food safety is a serious threat to global public health.While various synthetic or natural compounds have promoted the rapid development of food industry,the food safety situation is deteriorating.As the continued increase of food contaminants,market competition and trade challenges,people need to establish and improve the detection technology,food and drug regulatory system and virtuous cycle of food safety to maintain social harmony and stability.Diazepam(DZP)is a drug that belongs to the most widely prescribed 1,4-benzodiazepines for the therapeutic treatment of anxiety,insomnia,epilepsy,acute alcohol withdrawal,and muscular spasms.The family act on the brain and nerves to produce a calming effect by a neurotransmitter chemical(gamma-aminobutyric acid),which nerves use to communicate with one another.Since its metabolites can be enriched in animal bodies and still have biological activity,most countries have classified DZP as a prohibited compound in livestock and poultry products.Moreover,it has been used in drug-facilitated sexual assault and robberies.In other cases,DZP has been revealed to be used in the illegal adulteration of herbal medicine and functional foods.Such events have aroused widespread concern from the government and the community.A range of analytical approaches have been published to determine DZP in beverages and related samples.At present,such as high-performance liquid chromatography(HPLC),LC/mass spectrometry(MS),gas chromatography/MS and direct electrospray probe/MS have been employed in medical and pharmaceutical analysis.Although high sensitivity and reliability have been achieved,the previously mentioned methods present a common disadvantage,namely,requirement of intricate sample pretreatment,which involves extraction,cleanup,and pre-concentration.Falling prices and increased availability of the drug have also been linked to the frequent use in clinical and forensic cases.Therefore,a rapid,convenient,and interference-free approach is necessary for the “on-the-spot” detection in practical samples.The enzyme thermistor(ET),a thermometric biosensor device,has been developed and studied in numerous application areas because of advantages like superior versatility and general operational stability.It could transform the most common enthalpy changes in biological,physical and chemical reactions into detection signals.The conventional thermometric device consists of thermostat(containing reaction column),syringe pump,injection valve,Wheatstone bridge,signal acquisition station and connecting pipeline.As a new analytical instrument,previous demonstrations have focused on food analysis,environmental monitoring,bioprocess monitoring and clinical laboratory.Thermometric enzyme-linked immunosorbent assay(TELISA)is based on the conventional enzyme-linked immunosorbent assay(ELISA)but utilizes heat production from the enzyme label,which is measured by an ET unit.TELISA offered a variety of advantages in comparison with conventional immunoassays,such as adaptation in complex matrices and diverse labeled enzyme.In addition,the immunosorbent could be used repeatedly when appropriate reagents are employed for regeneration,and continuous detection is achieved.In this work,we had established a TELISA method for rapid detection of DZP based on an ET unit and immunoassay technology.Our thermometric biosensor was well versatile and pretreatment did not require elaborate extraction procedures to correct the matrix effect of the beverages but simple dilution and filtration.Besides,we had carried out the spike recovery test in various of actual samples to verify the reliability of the method.Finally,in order to improve the detection sensitivity,we constructed an indirect competitive immune detection method through the integration of biotin-avidin system.It had become a more economical and effective detection strategy of DZP,and provided technical support for the application of TELISA in analysis field such as food safety.1 Direct competition TELISA method for the detection of diazepamThe top priority of this study was the development of a FIA-TELISA biosensor that is capable of rapidly detecting and quantifying DZP.In this protocol,Protein G Sepharose? 4 Fast Flow(PGSFF)was used as the solid phase carrier.The TELISA strategy was based on the competitive inhibition of the antigen-antibody reaction,which was reflected by thermal signals generated when β-lactamase-labeled analytes were catalytically degraded by the ampicillin trihydrate.After a series optimization,our scheme was capable of detecting DZP at the lowest limit of 33.71 ng m L-1 with a linear range from 45.37 to 726.71 ng m L-1.Sensitivity was not outstanding but was adequate for the determination of the minimum effective concentration of the sufferer.In the validation of specificity and repeatability,it had no cross reaction with five diazepam structural or functional analogues except temazepam,and no observable deterioration of response was detected in the first 50 samples with the same column which the CV of signals was 6.17% after three months of study.Moreover,we had carried out the sample analysis in beverages,including tap water,juice,rum and functional foods.The recoveries of the spiked samples in TELISA determination ranged from 100.15% to 116.26%,and showed excellent correlation with the HPLC measurement.The method evaluation indicated that this assay was highly reliable for the detection of DZP.Owing to requiring minimal sample pretreatment,such as filtering with 0.22 μm filter and some necessary dilution,the approach provided us with an efficient tool that could obtain detection results in various real samples within 20 minutes.Future efforts will involve further optimization of the loading method to improve the overall efficiency,and transformation of the mobile phase which can extend the life of the adsorbent.Meanwhile,it will be developed for application to diverse targets in biological samples,as the ability to correct the matrix effect and the versatility of the protein G are fully utilized.2 Indirect competition TELISA of diazepam based on the biotin-avidin systemIn order to improve the detection sensitivity,we had constructed an indirect competitive immune detection method through the integration of biotin-avidin system.In this study,diazepam hapten was coated on the solid support,the controlled porous glass(CPG),by the active ester method.It competed with the target in the sample when the monoclonal antibody was present.At the same time,biotinylated goat anti-mouse IgG,streptavidin and biotinylated β-lactamase formed conjugates by amplification effect.Thus,the DZP concentration was reflected by the enzyme-catalyzed heat signal of ampicillin.We had optimized the critical conditions in the reaction system,including the order,concentration and proportion of the reagents,and established the standard curve of detection.The linear range was 0.1 ng m L-1 to 1000 ng m L-1 with the lowest detection limit of 0.12 ng m L-1.A specific study was performed on the diazepam analogues such as temazepam,lorazepam,nitrazepam,flurazepam and zopiclone.No response was observed at the selected concentration within the linear detection range.In the subsequent repeatability experiments,the recoveries of 99.94-106.25% indicated the reliability of the assay.Moreover,this strategy was able to calculate the concentration of DZP after 30 minutes of sample injection.In addition to the advantages of direct competition TELISA method,the indirect competition detection technology greatly improved the detection sensitivity and enhanced the life and stability of the core components in the ET unit.Future research will focus on the installation of the device according to the characteristics of loading,and the exploration of the influence of the mobile phase system in the indirect competition TELISA method.It is possible to establish a more effective and economical TELISA detection strategy,which provides a promising technology platform for high sensitivity detection of illegal additives and pesticide residues. |
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