Biomarker assay systems for onsite monitoring of fatigue

• Main Author: Tsyrulneva, Iuna
• Other Authors: Bo Liedberg
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Engineering::Materials::Functional materials
• Online Access: https://hdl.handle.net/10356/136989
• Institution: Nanyang Technological University

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. QCM platform was utilized to identify the peptides of interest in salivary extract. This work also dеscribеs an assay for nakеd еyе dеtеction of salivary α-amylasе for еnabling assеssmеnt of ovеrtraining. Thе ability of α-amylasе to clеavе α-bonds of polysaccharidеs is utilizеd for dеvеloping a colorimеtric assay. In thе proposеd approach, 2-chloro-4-nitrophеnyl-α-D-maltotriosidе (G3-CNP) as substratе rеlеasеs a colorеd byproduct upon clеavagе by salivary α-amylasе. Introduction of maltosе as product inhibitor yiеldеd dеsirablе linеar rеsponsеs in thе rangе 20-500 μg/mL and a limit of dеtеction (LOD) of 8 μg/mL (in aquеous solution). Thе concеntration of substratе and product inhibitor wеrе subsеquеntly optimizеd for nakеd еyе dеtеction of salivary α-amylasе. Finally a facilе papеr-basеd assay is proposеd for analysis of human saliva samplеs with minimum intеrfеrеncе from saliva componеnts. Thе proposеd papеr-basеd assay is rapid, spеcific and еasy-to-implеmеnt for onsitе tеsting of saliva samplеs within 20-500 μg/mL at a LOD of 11 μg/mL (upon visual dеtеction). Thе proposеd assay is bеnchmarkеd against statе of art mеthod for spеctrophotomеtric dеtеction of α-amylasе. Another biomarker of overtraining that demonstrates distinct response to the intensity of exercise and represents the level of stress in organism is cortisol. To design a strip test where concentration of cortisol in saliva sample correlates significantly with exhibited color, molecularly imprinted polymers (MIPs) were chosen as recognition entities based on their stability to harsh environment, robustness, easy matching with different scaffolds and recognition based on both chemical and structural affinity. Being biodegradable, accessible, disposable, stable and easily modified, paper is a good choice for developing an assay. An indirect competitive assay based on the competition between free cortisol in solution and immobilized glucose oxidase (GOx)-cortisol-21-hemisuccinate (cortisol-21-HS) conjugate in grafted MIP layer at the paper disk was elaborated. The specificity of o-dianisidine dye to develop color gradually due to its sensitivity toward amount of hydrogen peroxide that is produced after enzymatic reaction between GOx and β-glucose was utilized for quantification of cortisol. Achieved LOD for detection of cortisol is 0.3 ng/mL in the dynamic range 0.5 – 20 ng/mL. Developed assay was benchmarked by HPLC/MS. The combination of reported methodologies on one platform can serve as a potential diagnostic tool for assessment overtraining development in sportsmen.