Biomarker assay systems for onsite monitoring of fatigue

Abstract

Overtraining is one of the most underestimated physiological states of athletes. Being recognized as a serious complication for high performance, overtraining is diagnosed based only on verbal complaint of a sportsman, making it highly subjective, or on exhibiting underperformance. Besides, numerous diseases and psychological disorders have the same symptoms. No specific biomarkers have been proposed for detection of overtraining, neither in blood or urine, nor in saliva. Various physiological, behavioral and biochemical markers are constantly examined. Recently, metabolic and hormonal biomarkers demonstrated their potential in assessing overtraining. The variations in concentration levels of salivary cortisol, testosterone and α-amylase are currently being used to evaluate the state of a sportsman. However, these changes can be caused by a variety of other disorders that greatly complicates the work of sport doctors. Presumably, constant monitoring of biomarkers' response to the influence of external and internal factors can provide tools for timely detection of early signs of overtraining development. In this work, saliva has been chosen as a biological fluid that can be tested onsite, being non-invasive and rapidly accessible object.Recently, the ratio of two salivary heptapeptides was reported to be indicative of overtraining condition. However, saliva as a complex matrix needs to be cleaned up in order to remove high weight proteins, debris and bacteria that otherwise would interfere with the detection of peptides. Few different extraction protocols were explored with the aim to extract the peptides from saliva. High Performance Liquid Chromatography with UV detection (HPLC/UV) and Matrix-Assisted Laser Desorption Ionization-Time of Flight Spectroscopy (MALDI/ToF) were used to identify peptides in extract. Quartz Crystal Microbalance (QCM) platform was developed with the aim to confirm the presence of peptides in salivary extract. A series of antibodies raised against each of peptide were tested in order to distinguish the one with the highest affinity towards a peptide. The surface chemistry was optimized to achieve the best binding of antibody to an immobilized peptide.