Magnetic Nanomaterials-based Biosensors For The Detection Of Trace Substances

The development history of biosensor could date back to the 1960s when scientists firstly proposed the concept of glucose enzyme electrodes and developed the first generation of enzyme biosensors.For the past 50 years,the research field of biosensor is growing rapidly.Various types of biosensors based on different biometric components,methods of signal conversion and analysis techniques have been developed.In the 21^st century,the flourishing development of micro-nano technology,especially the novel nanotechnology,has opened a new era for biosensor-related research.Magnetic nanomaterials,a new type of nanomaterials,have the characteristics of small size,large specific surface area,and superparamagnetism.At the same time,they are well biocompatible and surface modifiable.Application of magnetic nanomaterials in biosensors improved the sample processing efficiency and detection sensitivity.In addition,the use of external magnetic fields for material recycling reduced its application costs,which opened up broad prospects for the commercial application.This project comprehensively utilizes scientific technologies such as nano-synthesis technology,inorganic chemistry technology,and analytical chemistry technology,explores the synthesis methods of magnetic nano-materials,builds related biosensor systems.By adopting electrochemical impedance spectroscopy detection technology,fluorescence detection technology and colorimetric detection technology,the identification and analysis of related trace substances were realized and the application scenarios of functional nanomaterials in biosensors was expanded.The main contents and results are summarized as follows:(1)An aptamer impedance biosensor based on magnetic nanomaterials was constructed for the detection of trace transgenic protein Cry1Ab.The nucleic acid aptamer with high specificity and affinity was used instead of the traditional antibody as the biorecognition element and was modified on the surface of magnetic nanoparticles.New types of disk microelectrode cell system and microfluidic cell system were designed and processed.The disc microelectrode system and nanomagnetic beads were used for signal conversion and transmission,and a microfluidic analysis system was constructed to realize rapid and highly sensitive detection of trace transgenic proteins in samples.In order to optimize the detection performance for Cry1Ab protein,we found the optimal solvent system was 0.01 M mannitol and the optimal detection frequency was 358.3 Hz.The final linear detection range of Cry1Ab protein was 0?0.2 n M,the coefficient of determination value of linear regression was 0.92,and the detection limit was0.015 n M.Four different transgenic proteins(Cry1Ab,Cry1Aa,Cry1Ac,and Cry1B)were used to evaluate the specificity of this novel microfluidic impedance sensing method.At the same concentration,the impedance response to the target Cry1Ab protein was significantly higher than the other three non-specific homologous proteins.Transgenic maize and rice samples with positive Cry1Ab protein had significantly higher impedance response signals than non-transgenic samples with negative Cry1Ab.The analysis system was used to perform impedance test on the reconstituted supernatant samples of transgenic corn,transgenic rice,non-transgenic corn,and non-transgenic rice.The impedance response at the optimal frequency of 358.3 Hz was recorded.It indicated that the analysis system can effectively distinguish between transgenic crops and non-transgenic crops.The difference of results between ELISA and our microfluidic impedance analysis system was within 8%.In addition,at a frequency of358.3 Hz,the relative standard deviation(RSD)of impedance signal was only 1.38%.The data were generated by the electrode chip after 25 regeneration processes.(2)An aptamer fluorescent biosensor based on magnetic nanomaterials was constructed for the detection of trace mercury ions in water.A nucleic acid aptamer that was rich of T bases and had a fluorescent label was used as a biorecognition element.Due to the high cost of carbon nanotube materials,a low-cost magnetic bamboo-like carbon nanotube synthesis method was proposed.The synthesized nanotube was used as a transducer to realize the conversion of biometric signals.With the help of the magnetic separation method and recyclable characteristics of the transducer,the rapid and low-cost detection of trace mercury in water was finally realized.The structure and element of the bamboo-like carbon nanotubes were characterized by SEM and TEM.The results showed that the length of the carbon nanotubes was in micron level and the average diameter was about 100 nm.Nickel nanoparticles were embedded in the top and the graphite phase carbon layer of the carbon tube.Mercury ions with a concentration range from 0.05 to 20.0?M were added to the ss DNA/BMCNTs detection system,and the detection sensitivity of the system based on the FRET principle was evaluated by the concentration gradient detection of mercury ions.Fluorescence detection results showed that the fluorescence emission characteristics of FAM-labeled ss DNA probes had better sensitivity to mercury ions at nanomolar concentration.With the increase of mercury ion concentration,its fluorescence emission intensity also gradually increased(?ex/?em=480/520 nm).When the mercury ion concentration was higher than 10 n M,the intensity growth rate gradually decreases.This phenomenon could be attributed to the interaction between ss DNA and mercury ion tended to be saturated.When the mercury ion concentration was lower than 1?M,the fluorescence intensity of the ss DNA/BMCNTs detection system had a good linear relationship with the mercury ion concentration,and its detection limit was 0.02?M,which is lower than the World Health Organization(WHO)stipulates that the mercury content in drinking water should be less than 0.03?M.The detection system was then used to test various heavy metal ions at a concentration of 50?M.Under the condition that the concentration of non-target heavy metal ions was 10 times the concentration of mercury ions,the increase in fluorescence intensity caused by non-target heavy metal ions was still significantly lower than the increase caused by mercury ions.This result indicated that among all the heavy metal ions tested,only mercury ions can significantly cause the fluorescence recovery of the system.The reason was the mercury ions induced ss DNA to generate base mismatches and caused the ss DNA to detach from the surface of BMCNTs,which hindered the fluorescence resonance energy transfer between FAM fluorescent label and BMCNTs.At the same time,compared with the atomic fluorescence spectrometry,our detection system had a very good accuracy for the detection of mercury ion in water,which suggested that our system had the potential for practical use.In addition,the cost of synthesized BMCNTs in this study was only 0.95$/g.Compared with reported detection methods based on the FRET principle,the detection system constructed in this research had comparable detection sensitivity,but only our detection system constructed can be recycled and reused due to the magnetic separation of BMCNTs.After ten regeneration cycles,the fluctuating range of fluorescence recovery intensity in a specific concentration of mercury ions sample was95.2 to 106.5%,and the relative standard deviation was within 5%.(3)A colorimetric biosensor based on magnetic nanomaterials was constructed for the detection of trace glucose.Using pre-synthesized Prussian blue nanocubes as template precursors,Fe/N/C magnetic nanocubes were synthesized by two-step high-temperature in-situ transformation under an inert gas atmosphere.Fe/N/C magnetic nanocube consisted of ordered cubic framework of mesoporous graphite carbon and uniformly dispersed core-shell Fe/Fe3C@N C nanoparticles.The activity of Fe/N/C MNCs peroxide mimicking enzyme was evaluated by catalyzing reaction of the chromogenic substrate ABTS in the presence of H₂O₂.When H₂O₂ was present,Fe/N/C MNCs could catalyze the blue-green reaction of ABTS;when there is no Fe/N/C MNCs or H₂O₂,ABTS did not develop a color reaction.Through the SEM and TEM morphology characterization,the synthesis method proved reliable.The supernatant of the Fe/N/C MNCs solution was also unable to catalyze the blue-green reaction of ABTS.It was confirmed that the Fe/N/C MNCs had peroxide mimic enzyme activity.The activity of Fe/N/C MNCs peroxide mimic enzyme depended on conditions such as p H and temperature of the reaction.The catalytic activity of Fe/N/C MNCs was greatly affected by the change of p H value,and the activity was highest at p H value 4.The catalytic activity of Fe/N/C MNCs was higher when the reaction temperature is 50?,and then the catalytic activity decreases with the increase of temperature,but it still remained above 80%of the highest activity.During the optimizing procedure,by adding different concentrations of H₂O₂ and glucose to the reaction system,the UV-visible absorbance of the reaction system at 420 nm was tested after the catalytic reaction.The test results showed that there was a good linear correlation between the absorbance of the reaction system and the concentration of H₂O₂ or glucose.The specificity of the constructed sensing method was verified by using maltose,sucrose,xylose,lactose,galactose,fructose and blank controls.When the concentration of the control samples were ten times that of glucose,the color of the reaction system was still differentiable,which indicated our method had good specificity.In addition,after ten regeneration cycles,the relative activity of the peroxidase mimic enzyme of Fe/N/C MNCs can still be maintained at about 95%.