Candidate: Hesam Abouali
Title: Design and Development of a Real-time Monitoring Microfluidic Platform for Multiplexed Biomarker Detection
Date: December 18, 2024
Time: 11:00 AM
Place: REMOTE ATTENDANCE
Supervisor(s): Poudineh, Mahla
Abstract:
Continuous, multiplexed, real-time measurements of biomarkers can unravel useful information about different health-related problems in patients. In the case of diabetes and obesity, glucose can be rapidly, repeatedly, and now continuously measured. The time course of changes in blood glucose after a glucose intake is well-established, and there has also been progress in defining glucose responses to different foods and how individual glucose responses predict and change in prediabetes and type 2 Diabetes (T2D). However, compared to blood glucose, very little is known about dynamic changes in blood insulin responses, especially highly time-resolved postprandial changes in insulin and related hormones/peptides such as C-peptide and glucagon.
In addition to blood glucose, there is a need to measure highly time-resolved insulin, glucagon, and C-peptide responses using continuous monitoring. This is an important knowledge gap because current methods to assess these hormones/peptides only capture a few time points after ingesting meal or glucose intake, which is a serious limitation in both preclinical and human assessments.
In this work, a multi-module microfluidic-based platform is developed and utilized to go beyond solely glucose measurements. This system, called quantum dot integrated real-time ELISA (QIRT-ELISA), measures glucose, insulin, glucagon, and C-peptide level continuously and simultaneously.
First, a sensitive bead-based quantum dot immunoassay (BQI) has been validated for insulin and glucagon detection. The integration of the BQI facilitated multiplexed and continuous monitoring. Moreover, the QDot technology used in BQI assists with the enhancement in the sensitivity of the immunoassays for both insulin and glucagon. Next, the QIRT-ELISA system was validated for in vitro continuous measurements of insulin and glucagon in whole blood sample without a need for pre-processing the sample. Finally, the results from discrete glucose tolerance tests (GTT) conducted by the developed microfluidic platform from in vivo rat models showed successful cross-validation of the device with the gold-standard ELISA.
In the next step, the QIRT-ELISA was expanded for more complex system measurements. The expanded QIRT-ELISA can monitor four biomarkers continuously in a multiplexed setting. To bridge the gap in diabetes studies, insulin, glucagon, C-peptide, and glucose were selected as the targets of the study. The BQI was employed for insulin, glucagon, and C-peptide, and a new aptamer bead-based assay was developed for glucose measuring. All assays were tested for their specificity against their target since the number of biomarkers was increased in this step of the work. Next, QIRT-ELISA was tested for its ability for continuous and multiplexed in vivo monitoring of insulin, glucagon, C-peptide, and glucose on conscious rat models in a GTT experiment.
With the continuous measurements of all four biomarkers, the QIRT-ELISA provides more data compared to conventional ELISA, which enables us to explore new interplays between the biomarkers under various scenarios.
The results obtained by this system will assist with shedding light on fundamental knowledge gaps of how factors beyond blood glucose are involved in the progression of prediabetes and T2D and add vital information to continuous glucose monitoring for precision nutrition, which finally leads to better diabetes management.
The clinical field requires a reliable approach that ensures continuous, cost-effective, and high-throughput separation of blood plasma, achieving sufficient purity for the accurate detection of biomarkers. Current methods, such as gold-standard centrifugation and microfluidic technologies, do not adequately fulfill these requirements. In the current work, we updated a passive hydrodynamic device to maintain high yield while achieving admissible purity, and high-quality plasma samples.
Through both a computational model and experimental trials, we optimized the employed side channels' lengths, which contributed to improving the plasma extraction rate significantly. These optimized side channels utilize the established cell-free layer areas within the contraction expansion regions to facilitate the reliable and effective extraction of plasma. Named Hydrodynamic Continuous, High-Throughput Plasma Separator (HCHPS) microfluidic device, the optimized device works at high throughput and achieves a purity of 47% with whole and 62% with 1:1 diluted blood, while increasing the yield by three times compared to the previous studies.
Finally, the optimized device was used to extract lactate-containing plasma from whole blood samples and was compared to plasma separated by centrifugation by conducting a bead-based fluorescence biosensing and an electrochemical aptamer-based biosensing.
With comparable results from this experiment, we showed that this device can potentially be integrated with other microfluidic platforms for more sensitive downstream analysis of whole blood samples.