| Modern industrialization and agricultural practices have resulted in increasing pollution of water.Heavy metal pollution is one of the major problems facing the world.Resulting from the long-term mining,smelting and electroplating,etc,heavy metals generated in the process of rain and natural settlement have entered water and soil,or have been discharged into water with sewage.Its accumulation in nature has an irreversible impact on the ecological environment and poses a huge threat to human health.In addition,the enrichment of shellfish biotoxins into food raw materials will also endanger food safety and human health.In China,the main polluted marine environment are those local sea areas,and the red tide is still in a high incidence period The resulting toxins in the red tide algae accumulate in the shellfish and then form shellfish toxins,leading to serious food safety issues.Therefore,rapid,accurate,easy-to-operate and on-site environmental toxin detection methods are of great practical significance for ensuring food safety and human health.In this study,the heavy metal mercury ions(Hg2+)and the paralytic shellfish toxin saxitoxin(STX)are the main analytes.With nucleic acid aptamers and antibodies as sensitive elements,three kinds of novel biosensors for rapid on-site detection were developed,including electrochemical DNA biosensors,nucleic acid based lateral flow strip sensors and enzyme-linked immunoassay(ELISA)colorimetric sensors.Integrated with portable analysis systems,they were applied to achieve trace detection and rapid analysis of the above toxins in water.The constructed biosensors exhibit outstanding characteristics of being portable,fast,super sensitive and high selective,and shows great potential in practical use.Moreover,the developed ELISA colorimetric sensor was also used to rapid screening of cancer biomarkers,achieving the specificity and rapidity of the the oral squamous cell carcinoma biomarker Cyfra21-1.The main content and innovation of this paper are summarized as follows:1.A label-free electrochemical DNA biosensor based on gold nanoband electrode and single strand DNA was constructed to achieve trace detection of Hg2+An electrochemical DNA biosensor based on gold nanoband electrode and the aptamer for Hg2+was constructed.The specific recognition of Hg2+was realized by modifying the gold working electrode surface with thymine(T)-riched DNA(the Hg2+aptamer).The formation of T-Hg2+-T stimulates charge transfer near the gold electrode surface,which can be used to establish the quantitative relationship between charge transfer impeda1ce and Hg2+ concentration.Experiments were performed to optimize a series of conditions,such as the DNA modification mode,time and ratio with blocking agent.The significant nonlinear diffusion and extremely fast mass transfer rate of gold nanoribbon electrode were used to improve the electrochemical performance,like sensitivity and stability.The detection limit of the constructed biosensor is 8 ng/L with a wide linear range from 0.02 to 200 ?g/L.Compared with the results by using the general disk electrode,the superiority of the nanoribbon electrode detection performance was verified.Though conventional techniques for Hg2+ detection,such as cold atomic absorption spectroscopy and inductively coupled plasma mass spectrometry,exhibit excellent sensitivity.Considering the bulky instruments,complicated procedures,long period and high operating costs,they are not suitable for on-site rapid detection.While the developed sensor simplifies the cumbersome modification process required by the conventional three-electrode system.It can detect Hg2+ ultrasensitively and selectively without electrochemically active labels,which is more suitable for automated detection and on-site applications.2.A novel DNA-based lateral flow strip integrated with a portable colorimetric analyzer was developed for on-site detection of Hg2+A novel lateral flow strip(LFS)based on DNA functionalized AuNPs and a competitive pairing strategy was developed to achieve ultra-sensitive and rapid detection of Hg2+.At present,most Hg2+ LFSs are semi-quantitative and rarely achieve ultra-sensitivity levels.The commonly used methods for improving sensitivity involve more than three DNA strands,which not only increases complexity,reduces stability and repeatability,but also greatly improves cost.It is apparently not suitable for large-scale rapid detection of Hg2+ in the field.The T-rich single-stranded DNA was used as a detection probe to specifically recognize Hg2+,and also served as pretreatment reagent to ensure its full reaction with Hg2+ and avoid consumption on the conjugate pad in those traditional methods.The base complementary strand was designed to compete with Hg2+and modified on AuNPs to develop signal with inverse sensitivity,which was beneficial to the analysis of low concentration samples.Besides,a biotin-avidin binding system was utilized to achieve signal amplification.Optimizations of the preparation of AuNPs-DNA,the pretreatment of strips,the coating concentration of reagents on conjugate pad and test line,and the incubation time were performed to improve detection performance.Integrated with a portable colorimetric analyzer,the developed lateral flow strip achieved rapid and ultrasensitive detection of Hg2+ in water.The whole detection process could be done in one step within 15 min with the detection limit as low as 8 ng/L.It shows the outstanding advantages of low cost,high integration,simple operation and fast detection speed.The operator only needs to drop sample to the sample pad and then gain the result from smartphone.No specialized knowledge is required.Therefore,it is quite suitable for rapid large-scale on-site detection.3.A signal-enhanced enzyme-linked immunosorbent assay(ELISA)based on AuNPs and biotin-avidin binding system was proposed to achieve rapid and sensitive detection of STX by cooperating with a portable smartphone-based microplate detection system,which could also be used for rapid screening of oral squamous cell carcinoma(OSCC)biomarker Cyfra21-1A signal enhancement assay based on AuNPs and biotin-avidin binding system was proposed,which could be applied to both indirect competitive ELISA and sandwich ELISA.Integrated with a portable smartphone-based microplate detection system,it was used to realize fast and ultra-sensitive detection of STX in the field and rapid screening of Cyfra21-1.Biotinylated secondary antibody(IgG-Biotin)was modified on the AuNPs surface to amplify signal due to the large specific surface area of AuNPs.When the complex of streptavidin and horseradish peroxidase(SA-HRP)was added,it could bind with IgG-Biotin and lead to the immobilization of HRP on AuNPs surface.Owing to multiple binding sites for biotin on IgG,the ELISA signal could be further amplified.Compared with the result from traditional ELISA,the improved sensitivity of this signal-enhanced ELISA was ensured,with detection limit as low as 0.4 ng/mL,the detection range from 1 to 200 ng/mL,the recovery rate from 89.1%to 112.2%and the coefficient of variation(CV)in the range of 4.6%-23.2%.Moreover,the detection limit for cyfra21-1 was as low as 0.09 ng/mL and linear range was from 0.25 to 8 ng/mL.The constructed biosensing system is expected to have great potential in large-scale on-site and ultrasensitive detection of seafood and rapid screening and early diagnosis,exhibiting outstanding advantages of convenience,fast speed and being easy to operate. |
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