Electrochemical Sensors for Flow-Enhanced Immunoassays and Flow Monitoring of Porous Materials

Department of Chemical EngineeringMicrofluidics enables a miniaturization of laboratory-scale biochemical analysis into a single chip. It has been actively developed and utilized in various biomedical diagnostic devices. Especially, lab-on-a-disc (LOD) and paper microfluidic system can serve as excellent candidates for point-of-care testing (POCT) because of several advantages including relatively simple fluid transfer mechanism, portable size of the device, fast analysis time, reduced cost, automated analysis steps and low consumption of test samples and reagents. In this thesis, electrochemical detection method has been implemented in two kinds of diagnostic devices. First, lab-on-a-disc integrated with screen-printed carbon electrodes (SPCEs) was developed for electrochemical detection of protein biomarkers, C-reactive protein. Compared to conventional optical detection, the electrochemical detection could provide enhanced sensitivity as well as significant cost reduction because it is not necessary to use optical grade plastic materials for the fabrication of the disc. In addition, we have developed electrochemical sensors for the measurement of the local fluid velocity based upon the fact that the electrochemical signal is proportional to the flow rate. As a proof of the concept experiment, we have used the electrochemical sensors for real time monitoring of the flow through porous materials, which can provide a practical tool to quantify the fluid velocity on the paper-based microfluidics. Based on chronoamperometric signals measurement using SPCEs, fluid transfer phenomena through various kinds of porous materials, geometries, absorption pads, and flow modifiers were evaluated. In conclusion, we have developed a cost-effective SPCEs-based electrochemical detection method and utilized not only for the immunoassays fully integrated on a disc but also to characterize fluid transfer behavior through the porous materials. The SPCEs can be utilized as a cost-effective sensor not only for the highly sensitive electrochemical detection for bioassays but also for the flow measurements in porous materials.ope

Size-Selective Isolation of Urinary Exosome on Lab-on-a-Disc

A size selective isolation of exosome is demonstrated on a centrifugal microfluidic system. Starting with 1 mL of raw urine sample, we suggest a rapid and easy method for isolation of exosomes by using nanosized filters integrated on a lab-on-a-disc. Our method is a cost effective, quick, and painless method for identification of the bacterium by analyzing the RNA in the exosomes secreted in the patient's urine. It may be used routinely for early diagnostics of hospitalized patients susceptible to sepsis, dramatically lowering its mortality rate

An electrochemical-sensor system for real-time flow measurements in porous materials

Flow monitoring in porous materials is critical for the engineering of paper-based microfluidic bioassays. Here, we present an electrochemical-sensor system that monitors the liquid flow in porous materials without affecting the real flow in paper-strip samples. The developed microfluidic sensor records an amperometric signal created by the solution movement mediated by paper wicking. This approach allows the in situ monitoring of the different hydrodynamic conditions of a specific paper geometry or composition. In addition, the method proposed in this work was employed to characterise the fluid flow of different nitrocellulose paper strips after oxygen-plasma treatment or dextran coating. The dextran fluid-flow modifiers were further used on the paper strip-based assays as means of signal enhancement. The proposed electrochemical-sensing method offers a valuable alternative to existing optical-based monitoring techniques for flow measurement in paper-based microfluidic systems.close1

Fully automated centrifugal microfluidic device for ultrasensitive protein detection from whole blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-carediagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.clos

Characterization of Tauopathy in a Rat Model of Post-Stroke Dementia Combining Acute Infarct and Chronic Cerebral Hypoperfusion

Post-stroke dementia (PSD) is a major neurodegenerative consequence of stroke. Tauopathy has been reported in diverse neurodegenerative diseases. We investigated the cognitive impairment and pathomechanism associated with tauopathy in a rat model of PSD by modeling acute ischemic stroke and underlying chronic cerebral hypoperfusion (CCH). We performed middle cerebral artery occlusion (MCAO) surgery in rats to mimic acute ischemic stroke, followed by bilateral common carotid artery occlusion (BCCAo) surgery to mimic CCH. We performed behavioral tests and focused on the characterization of tauopathy through histology. Parenchymal infiltration of cerebrospinal fluid (CSF) tracers after intracisternal injection was examined to evaluate glymphatic function. In an animal model of PSD, cognitive impairment was aggravated when BCCAo was combined with MCAO. Tauopathy, manifested by tau hyperphosphorylation, was prominent in the peri-infarct area when CCH was combined. Synergistic accentuation of tauopathy was evident in the white matter. Microtubules in the neuronal axon and myelin sheath showed partial colocalization with the hyperphosphorylated tau, whereas oligodendrocytes showed near-complete colocalization. Parenchymal infiltration of CSF tracers was attenuated in the PSD model. Our experimental results suggest a hypothesis that CCH may aggravate cognitive impairment and tau hyperphosphorylation in a rat model of PSD by interfering with tau clearance through the glymphatic system. Therapeutic strategies to improve the clearance of brain metabolic wastes, including tau, may be a promising approach to prevent PSD after stroke

Exodisc for rapid, size-selective, and efficient isolation and analysis of nanoscale extracellular vesicles from biological samples

Extracellular vesicles (EVs) are cell-derived, nanoscale vesicles that carry nucleic acids and proteins from their cells of origin and show great potential as biomarkers for many diseases, including cancer. Efficient isolation and detection methods are prerequisites for exploiting their use in clinical settings and understanding their physiological functions. Here, we presented a rapid, label-free, and highly sensitive method for EV isolation and quantification using a lab-on-a-disc integrated with two nanofilters (Exodisc). Starting from raw biological samples, such as cell-culture supernatant (CCS) or cancer-patient urine, fully automated enrichment of EVs in the size range of 20-600 nm was achieved within 30 min using a tabletop-sized centrifugal microfluidic system. Quantitative tests using nanoparticle-tracking analysis confirmed that the Exodisc enabled >95% recovery of EVs from CCS. Additionally, analysis of mRNA retrieved from EVs revealed that the Exodisc provided >100-fold higher concentration of mRNA as compared with the gold-standard ultracentrifugation method. Furthermore, on-disc enzyme-linked immunosorbent assay using urinary EVs isolated from bladder cancer patients showed high levels of CD9 and CD81 expression, suggesting that this method may be potentially useful in clinical settings to test urinary EV-based biomarkers for cancer diagnostics.clos

Malnutrition, inflammation, progression of vascular calcification and survival: Inter-relationships in hemodialysis patients.

Background and aimsMalnutrition and inflammation are closely linked to vascular calcification (VC), the severity of which correlate with adverse outcome. However, there were few studies on the interplay between malnutrition, inflammation and VC progression, rather than VC presence per se. We aimed to determine the relationship of malnutrition, inflammation, abdominal aortic calcification (AAC) progression with survival in hemodialysis (HD) patients.MethodsMalnutrition and inflammation were defined as low serum albumin (ResultsAAC progressed in 54.6% of 97 patients (mean age 58.2±11.7 years, 41.2% men) at 1-year follow-up. Hypoalbuminemia (Odds ratio 3.296; 95% confidence interval 1.178-9.222), hs-CRP (1.561; 1.038-2.348), low LDL-cholesterol (0.976; 0.955-0.996), and the presence of baseline AAC (10.136; 3.173-32.386) were significant risk factors for AAC progression. During the mean follow-up period of 5.9 years, 38(39.2%) patients died and 27(71.0%) of them died of cardiovascular disease. Multivariate Cox regression analysis adjusted for old age, diabetes, cardiovascular history, and hypoalbuminemia determined that AAC progression was an independent predictor of all-cause mortality (2.294; 1.054-4.994).ConclusionsMalnutrition and inflammation were significantly associated with AAC progression. AAC progression is more informative than AAC presence at a given time-point as a predictor of all-cause mortality in patients on maintenance HD