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Protein macroarray is a new,simple and multiple detection system,whose spots are more than 1mm diameter and results are visible to naked eyes without any instrument.It,especially applied in the detection of food-borne pathogens,only needs to achieve qualitative or semiquantitative detection of targets.This useful technology,however,possesses recognized spot quality and uniformity issues that can limit its application.Some methods were developed for spot quality control,however,they were complicated or required professional instruments.We therefore generated,for the first time,spotting buffer for protein macroarray directly incorporated with Ponceau S as a simple means of visualizing spot morphology on nitrocellulose membrane prior to hybridization.In this report,1%(w/v)Ponceau S and 10%(v/v)glycerol in spotting buffer were found to be optimal for spot quality control and targets detection.Furthermore,the results of sensitivity and specificity for protein macroarray indicated that there was no difference between the protein macroarrays created by spotting buffer with and without Ponceau S.Under optimal conditions,we make the important observation that macroarray imaging prior to hybridization is predictive of hybridization performance.