Target peptide enrichment microfluidic chip for rapid detection of oral squamous cell carcinoma using stable isotope standards and capture by anti-peptide antibodies

The use of antibodies against surrogate peptides for target protein enrichment combined with stable isotope standards is one of the important steps in the quantitative analysis of protein samples via mass spectrometry. Currently, commercially available automated instruments such as KingFisher™ are used to perform immunoprecipitation, wash, and elution of protein samples using magnetic beads and immune reactions. However, the use of these instruments for protein or peptide purification takes over 2 h. Moreover, numerous samples (e.g., 96) need to be processed at once to meet the operating cost, and the consumables used in conjunction with them are expensive. Thus, this study developed an automated microfluidic chip that could rapidly purify target peptides in saliva samples within 50 min. The chip is made of five connected pneumatic micromixers that automate the immunoprecipitation of target peptide from the samples. Under optimum operating conditions of 5 Hz and −75 kPa, the recovery rate of peptides achieved by the micromixer in an operating time of 30 min was similar to that achieved by KingFisher™ in the standard operating time of 2 h. The chip-processed samples could be used for the quantitative detection of the oral cancer biomarker protein MMP1 via mass spectrometry with a detection limit of 6.75 ng/mL. Six saliva samples were used to verify the feasibility of the chip for clinical sample use. The MMP1 concentration measured using our sample treatment method was comparable to that treated using a commercially available instrument.