Study On The Detection Of Food-borne Pathogenic Salmonella By The Paper-based Microfluidic Device

Salmonella is a common kind of food-borne pathogenin the nature.Poisoning accidents caused by salmonella accounted for the first in the food poisoningofworldwide.The main reason is that the salmonella can produce endotoxin which can cause fever, diarrhea, vomiting, blood coagulation, shock and other poisoning symptoms. In view of the great harm to human health and public hygiene, the detection of salmonellas is extremely important.The traditional detection methods,which have many limitations including time-consuming, operation-complicating, high-cost, poor selectivity and so on, have limitedtheir applications, especially in field application. Therefore, to develop a rapid, simple and highly sensitive method for the detection of salmonellas is helpful in preventing and controlling the salmonellas caused poisoning. In recent years, with the rapid development of microfluidic chip technology, the microfluidicpaper-basedanalytical device(μPAD), with porous filter paper as the substrate material, is increasingly used in medical diagnosis, drugs development, water quality monitoring and other fields due to its distinct advantages such as portable, no pollution and so on. However, at present most of the preparations of the μPAD pathway require expensive equipments or professional operationand cannot be carried out in ordinary laboratory, which limit the practical application of μPADs. Based on the above issues, this study developed a rapid, simple, low-cost and no equipment requirment preparation method for paper-based microfluidic chips.Then the μPAD was applied to the rapid detection of salmonellas to provide a new technical means for the food safety monitoring of salmonellas.In this study, the polyclonal antibody of anti-salmonella enteritidis was prepared, purified and identified. Then, a new preparation method of paper based microfluidic chip was developed, the process parameters were optimized and the mechanism was explored. Finally, a rapid method for the detection of salmonella enteritidis based on the paper-based microfluidic was established, the sensitivity and specificity of the method were evaluated. The main results are as follows:1. The female Japanese rabbits were immunized with the whole bacterial antigen of salmonella obtained by ultrasonication and inactivation with formaldehyde, then the antisera were collected.The titer of the anti-salmonella serum was 4.8 ×10~5 and increased to 6.4 × 105 after purification.The concentration of the polyclonal antibody is 7.03mg/mL determined by the bicinchoninic acid method(BCA).2. Compared with the traditional methods, the preparation method of this μPADhad the advantages of simple operation, safe and low cost. The preparation period is short which could be completed in only 7 min. The optimized process parameters were as follows: the concentration of trimethoxy silane(TOS) was 4%(v/v); the corrosion time and concentration of Tween-20 were 3.5min and 1% respectively(m/m);the corrosion time and concentration of sodium dodecyl sulfate(SDS) were 4.5min and 1%(m/m) respectively;the corrosion time and concentration of cetyl trimethyl ammonium bromide(CTAB) were 5min and 1%(m/m) respectively. The three kinds of surface activity had similar effect on the preparation of μPAD, while the corrosion time of Tween-20 was the shortest, therefore, Tween-20 was chosen in the following study for the preparation of hydrophilic channels. In addition, the formation mechanism of μPAD’s hydrophilic and hydrophobic channels was explored by using scanning electron microscopy(SEM) and X ray photoelectron spectroscopy(XPS).Results showed that the formation reason of the hydrophobic pathway was that as a silane coupling agent, the trimethoxysilane reacted with the hydroxyl groups of the papercelluloseand then covered on the surface of the paper making the whole piece of paper changed from hydrophilic into hydrophobic. Subsequently, the hydrophilic pathwayswere formed on the surface of the hydrophobic paper because the hydrophilic substrate model soaked by surfactant solution could selectively etch the hydrophobic filter paper.3. Asandwich immunoassay pattern was established on the paper based microfluidic chip by using a commercial monoclonal antibody as the capture antibodyandthe lab-made polyclonal antibody as the detecting antibody. Determined by the sandwich ELISA, the working concentrationof the monoclonal antibody and polyclonal antibody were1: 8000 and1: 4000 respectively. The lowestdetectable limit of the method for the pure culture liquid of salmonellaswas 106 cfu/mL. Besides, the reaction of the method to other bacteria of the same species was very weak and had no reaction to the heterologous bacteria.In summary, this paper developed a new type of paper-based microfluidic chip, the preparation of which needed no expensive instruments, professional personnel and so on. And a new method for rapid detection of salmonella enteritidis was established using the developed μPAD.The results provide reference information for the further development of other simple microfluidic chips and broaden applications of μPADs in the field of food safety monitoring.