Study And Application Of A New Rapid Detection Method Of Lactoferrin In Infant Formula Milk Powder

Lactoferrin,as an important nutritional ingredient in breast milk,especially as a nutritional fortifier added to infant formula,has become the preferred source of nutrition for infants who can not be breastfed.Prior to this,with the gradual improvement of consumer demand for the quality of infant formula,the market consumption trend tends to be more high-end products,and the core of middle-and high-end infant formula is the use of high-quality milk sources and functional ingredients such as lactoferrin,probiotics,DHA,ARA and so on.Therefore,many international first-tier dairy brands have reached a consensus on adding lactoferrin to improve the quality of milk powder.Therefore,three new methods for rapid detection of lactoferrin were established based on lactoferrin specific aptamers and different signal transduction methods.1.The establishment of colloidal gold colorimetric sensor based on nucleic acid aptamer.Using the unlabeled aptamer as the recognition element and colloidal gold as the signal molecule,the qualitative and quantitative detection of lactoferrin was realized by the color change of colloidal gold in salt solution and the change of UV-visible absorbance ratio of colloidal gold at 620 nm and 520 nm.The specificity of this method is good,the linear range is 5-75 n M,the detection limit is 3.6 n M,and the recovery rate is good in infant formula,but the detection range is narrow.2.Establishment of unlabeled fluorescence sensor based on nucleic acid aptamer.In order to avoid the defects of colorimetry,we choose a simple and sensitive fluorescence detection method,and use DNA structure to selectively embed dyes to design a simple and universal unlabeled fluorescence sensor.Using the unlabeled aptamer as the recognition element and the aptamer binding to the DNA structure selectively embedded dye as the fluorescence probe,the quantitative analysis of lactoferrin was realized by using the fluorescence signal change caused by the change of the number of dyes embedded in the aptamer groove region.Firstly,the traditional fluorescence sensing strategy of Pico Green dye embedded in double-stranded DNA is studied.Under the optimal conditions,the detection range is 300-1500 n M,and the detection limit is 205 n M.By comparison,this strategy is not enough to meet the detection requirements.In view of the fact that the dye can interact with both double-stranded DNA and single-stranded DNA,the fluorescence sensing strategy of Pico Green dye embedded in single-stranded DNA was studied.Under the best conditions,the detection range is 20-500 n M,the detection limit is 3 n M,and the specificity is good.And the fluorescence sensing strategy based on Pico Green dye embedded in single-stranded DNA has the advantages of easier operation,shorter detection time,higher accuracy and sensitivity,and a better recovery rate in the actual sample.Finally,by replacing other targets and their aptamers and another fluorescent dye,it is verified that the fluorescence sensing strategy based on DNA structure selective intercalation dye and single-stranded DNA nucleic acid aptamer is a general,simple,efficient and low-cost sensing strategy.3.The establishment of fluorescence sensor based on nucleic acid aptamer and CRISPR/Cas12 a.In order to further improve the detection performance,we introduce the CRISPR/Cas12 a signal amplification system with trans-cutting activity to design the fluorescence sensor.In this strategy,the aptamer of lactoferrin nucleic acid is not only the target recognition element,but also the activator of CRISPR/Cas12 a trans-cleavage activity.The activated Cas12 a infinite cleavage of any non-specific fluorescence reporter molecule(labeled with fluorophore and quenchant at both ends)leads to the change of fluorescence signal,so as to realize the quantitative detection of lactoferrin.The linear range of the strategy is 100-750 n M,and the detection limit is 53.4 n M.Although the detection range is expanded,the sensitivity is low and the detection cost is high."Food Safety National Standard Food Nutrition fortifier use Standard" GB14880-2012 stipulates that the maximum use of lactoferrin in infant food is 100 mg/100 g.According to the investigation and statistics,the lactoferrin content in domestic brands of infant formula is generally 30-100 mg/100 g,which is in line with the national limit standard.It is calculated that the detection range of the three nucleic acid aptamer biosensors is 150-500 n M.By comparing the detection range and other detection performance of the above three methods,the unlabeled fluorescence sensor based on Pico Green dye embedded in single-stranded DNA is the best,and it is a general,simple,efficient and low-cost sensing strategy.