University of Twente Student Theses


Development of a microfluidic device for point-of-care testing of free thiol levels in plasma : A lab-on-a-chip for biomarker detection of oxidative stress in IBD patients

Jager, K.E. de (2023) Development of a microfluidic device for point-of-care testing of free thiol levels in plasma : A lab-on-a-chip for biomarker detection of oxidative stress in IBD patients.

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Abstract:Measuring levels of biomarkers, radiology, histopathology and endoscopy are used for the diagnosis, prognosis, and monitoring of disease activity in IBD patients. The current golden standard for measuring disease activity in IBD patients is the fecal biomarker calprotectin. However, all current diagnostic tests are limited in the prediction of long-term outcomes, which emphasizes the need for new biomarkers. In patients with IBD, oxidative stress leads to damage of the mucosal layer and bacterial invasion, which enhances disease progression. The strong correlation between disease activity and reduced antioxidant free thiol levels in plasma is promising for early diagnosis and easy disease monitoring, which has high clinical relevance. The aim of this thesis was to develop a microfluidic device for POCT of free thiol levels in plasma. Also, the underlying goal was to design a LoC for biomarker detection of oxidative stress in IBD patients. The current assay is time-consuming and requires large patient blood samples. It was aimed to create a LoC that has a monolithic approach performing all steps of the current assay: plasma separation through centrifuging, plasma dilution with Tris buffer, a background measurement, diluted plasma mixing with DTNB, and absorbance measurement using the plate reader. For the on-chip blood plasma separation an asymmetric membrane is used. A capillary pump was used to wick the plasma from the membrane, which led to plasma being obtained in 100% of the cases. After the addition of the Tris buffer, the plasma flowed through the microfluidic channels in 66.6% of the devices. Different devices are made allowing the storage of two reagents on-chip, which had a high success rate of meniscus formation and reaching the read-out reservoir (up to 100%). A serpentine structure showed enhancement of the mixing process of these reagents throughout the channel and over time. More research is needed to create a control read-out on-chip for validation of the device. A Quake-valve could be used to prevent backflow and precise volume regulation, which needs 1500 mbar of pressure to be closed. Lyophilization of the DTNB shows a linear relationship between the absorbance and concentration of L-Cysteine until a concentration of 250 µM is reached, after which the calibration curve saturates. No significant differences within the calibration curves obtained in the well plate and with liquid DTNB on-chip were found. Even though, there was a significant difference in the found absorbance between the two methods, no significant difference was found between the measured free thiol concentration in plasma. The intra-CV% of the free thiol concentration determined on-chip is 19.5% compared to the 3.94% of the currently used assay. The accuracy of the according calibration curve was 88.0% with an inter-CV of 9.00%. The LLOQ was 109.6 µM. In conclusion, the first steps toward a sample-in-answer-out microfluidic device for measuring oxidative stress, show promising results. Finally, it was possible to create a calibration curve of L-Cysteine with a linear relationship (R2 = 0.98) utilizing a portable spectrophotometer.
Item Type:Essay (Master)
UMCG, Groningen, The Netherlands
Faculty:TNW: Science and Technology
Subject:35 chemistry, 44 medicine, 50 technical science in general, 51 materials science
Programme:Biomedical Engineering MSc (66226)
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