A method for the detection of hCG β in spent embryo culture medium based on multicolor fluorescence detection from microfluidic droplets

The evaluation of embryo quality via human chorionic gonadotropin beta (hCG β) and other proteins secreted by embryos in a spent embryo culture medium (SECM) receives a close review in the field of assisted reproduction. However, accurate and quantitative detection of these trace proteins is still a challenge. In this study, a highly sensitive protein detection method using microfluidic droplets and multicolor fluorescence detection was developed and used to detect hCG β secreted by embryos in SECM. β-Galactosidase (β-Gal) was used to label hCG β and can catalyze the conversion of nonfluorescent substrate fluorescein di-β-d-galactopyranoside to produce fluorescein to amplify the signal strength. Compared with previous studies, the proposed method requires only a simple microfluidic chip and can eliminate false-positive signals generated by free β-Gal through simultaneous detection of fluorescence, which can ensure the accuracy of the results. The lower detection limit of hCG β was 0.1 pg/ml. Using the developed method, hCG β in SECM was successfully detected; the hCG β secreted by top-quality blastocysts was significantly higher than that of non-top-quality blastocysts and embryos that do not develop into blastocysts. The proposed method can be used to detect secretory proteins from embryos in SECM and has application value in the screening of other biomarkers.