The Development And Application Of Biosensor Based On Moving Neutralization Boundary

Melamine was sometimes adulterated to dairy product for false protein content increase in developing countries.Long-term consumption of melamine-containing dairy products seriously jeopardizes the health of the body.However,portable sensor has not been developed for on-spot determination of melamine in dairy product yet.Herein,a distance-based sensor,which based on chip electrophoretic titration(ET)of moving neutralization boundary(NB)and EDTA photo-catalysis was developed and studied its application.The specific research content of this article is divided into the following sections:1.Design of Microfluidic chip and the development of biosensorPDMS was used to design and fabricate multi-channel microfluidic readout chip for rapid and visual detection of melamine in dairy products.The chip has three channels connecting the anode chamber and the cathode chamber,which could be used for simultaneous measurement of multiple channels to speed the analysis of sample.The ET-NB biosensor was composed of chip and a portable power.It could be integrated into a handheld device and was convenient for resource shortages region to ensure food safety as well as avoid economic losses.2.EDTA photocatalytic ET-NB biosensor modelThe EDTA photocatalytic ET-NB sensor model was theoretically proposed to provide a theoretical basis for our next experiment.The working principle of the ET-NB sensor was as follows:In the chip sensor,EDTA,H₂O₂and leucomalachite green(LMG)were added in the anode well.Under UV light,EDTA was used as the peroxidase-like catalyst,photo-catalyzing the substrate of colorless leuco-malachite green(LMG)and H₂O₂ as H₂O and color malachite green(MG)with one positive charge.When applying electric field,the MG in the anode well migrated into the channel and was neutralized with the base in the channel,resulting in colorless MG-OH and NB.If melamine-content dairy sample was added into the EDTA-H₂O₂-LMG system,H₂O₂ reacts with melamine,leading to the decrease of MG.Thus,the higher the melamine content in dairy product,the shorter the distance of NB migration under the given time,implying a distance-based sensor of melamine.3.Verification of the ET-NB sensors for the detection of melamineIn order to verify the feasibility of the developed ET-NB sensor,firstly,the neutralization boundary formed between the MG produced by the oxidation of the LMG-H₂O₂-EDTA system and the alkaline buffer in the anode chamber of the microfluidic chip is stable,clear and there was an excellent linear relationship between distance and running time.Further,when the standard melamine in the aqueous solution was detected,the relationship between boundary distance and the logarithm of the melamine concentration had a good linear fitting curve,and R²was greater than 0.99.Finally,since the high concentration of protein in dairy products could interact with ascorbic acid,the developed ET-NB sensor had good anti-interference properties for ascorbic acid.All of those results not only proved the correctness of our research strategy that combined the LMG-H₂O₂-EDTA redox system and neutralization boundary-electrophoresis titration to detect melamine concentration,but also manifested the validity that the ET-NB sensor was used for detection of melamine.4.Analysis performance of ET-NB sensorsIn this paper,we developed a simple distance-based analysis method to visual detect melamine content in dairy products through the boundary distance of ET-NB sensors.In the analysis of actual samples,the ET-NB sensor achieved excellent performance:The LOD of ET-NB sensor were0.1?M for infant formula sample and 0.2?M for other milk samples,which were 80 and 100 times less than the FDA safety limits of 8?M for infant formula sample and 20?M for milk sample,respectively.The quantitative spiked recovery were 87%-108%for milk,90%-107%for infant formula.In addition,the"+"or"-"judgments can provide an intuitive and rapid evaluate the quality of dairy products.The distance-based readout chip,which combined ET-NB and EDTA photocatalysis,provided a user-friendly,portable sensor.Due to its rapid(within 15 min),low cost as well as good selectivity and easy operation,it has great prospects for quantitative analysis of melamine and other additives in real samples.