Isolation of exosomes from whole blood by a new microfluidic device: proof of concept application in the diagnosis and monitoring of pancreatic cancer

Background: Exosomes are endocytic-extracellular vesicles with a diameter around 100 nm that play an essential role on the communication between cells. In fact, they have been proposed as candidates for the diagnosis and the monitoring of different pathologies (such as Parkinson, Alzheimer, diabetes, cardiac damage, infection diseases or cancer). Results: In this study, magnetic nanoparticles (Fe3O4NPs) were successfully functionalized with an exosome-binding antibody (anti-CD9) to mediate the magnetic capture in a microdevice. This was carried out under flow in a 1.6 mm (outer diameter) microchannel whose wall was in contact with a set of NdFeB permanent magnets, giving a high magnetic field across the channel diameter that allowed exosome separation with a high yield. To show the usefulness of the method, the direct capture of exosomes from whole blood of patients with pancreatic cancer (PC) was performed, as a proof of concept. The captured exosomes were then subjected to analysis of CA19-9, a protein often used to monitor PC patients. Conclusions: Here, we describe a new microfluidic device and the procedure for the isolation of exosomes from whole blood, without any need of previous isolation steps, thereby facilitating translation to the clinic. The results show that, for the cases analyzed, the evaluation of CA19-9 in exosomes was highly sensitive, compared to serum samples

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Label-free multi-step microfluidic device for mechanical characterization of blood cells: Diabetes type II

The increasing interest to establish significant correlations between blood cell mechanical measurements and blood diseases, has led to the promotion of microfluidic devices as attractive clinical tools for potential use in diagnosis. A multi-step microfluidic device able to separate red and white blood cells (RBCs and WBCs) from plasma and simultaneously measure blood cells deformability (5 and 20% of hematocrit) is presented in this paper. The device employs passive separation based on the cross-flow filtration principle, introduced at each daughter channel. At the outlets, hyperbolic geometries allow single-cell deformability analysis. The device was tested with blood from five healthy and fifteen diabetic type II voluntary donors. The results have shown that WBCs have lower deformability than RBCs, and no significant differences were observed in WBCs from healthy and pathological blood samples. In contrast, RBCs have shown significant differences, with pathological cells exhibiting lower deformability. Shear rheology has shown that blood from patients with type II diabetes has higher viscosity than blood from healthy donors. This microfluidic device has demonstrated the ability to reduce cell concentration at the outlets down to 1%, an ideal cell concentration for assessing the blood cells deformability, under healthy and pathological conditions. The results provide new insights and quantitative information about the hemodynamics of in vitro type II diabetes mellitus RBCs. Thus, such device can be a promising complement in clinical diagnosis and biological research as part of an integrated blood-on-a-chip system.This work was supported by Projects NORTE-01-0145-FEDER- 028178, NORTE-01-0145-FEDER-029394, NORTE-01-0145-FEDER- 030171 funded by COMPETE2020, NORTE2020, PORTUGAL2020, and FEDER. This work was also supported by Fundação para a Ciência e a Tecnologia (FCT) under the strategic grants UIDB/04077/2020 and UIDB/00532/2020. D. Pinho and V. Faustino acknowledge the Ph.D. scholarships SFRH/BD/89077/2012 and SFRH/BD/99696/2014, respectively, both provided by FCT. Susana Catarino thanks FCT for her contract funding provided through 2020.00215.CEECIND. F. T. Pinho is thankful to FCT for financial support through projects LA/P/0045/2020 of the Associate Laboratory in Chemical Engineering (ALiCE) and projects UIDB/00532/2020 and UIDP/00532/2020 of Centro de Estudos de Fenómenos de Transporte.info:eu-repo/semantics/publishedVersio

Blood cells separation and sorting techniques of passive microfluidic devices: From fabrication to applications

Since the first microfluidic device was developed more than three decades ago, microfluidics is seen as a technology that exhibits unique features to provide a significant change in the way that modern biology is performed. Blood and blood cells are recognized as important biomarkers of many diseases. Taken advantage of microfluidics assets, changes on blood cell physicochemical properties can be used for fast and accurate clinical diagnosis. In this review, an overview of the microfabrication techniques is given, especially for biomedical applications, as well as a synopsis of some design considerations regarding microfluidic devices. The blood cells separation and sorting techniques were also reviewed, highlighting the main achievements and breakthroughs in the last decades.This work was supported by projects UID/EEA/04436/2019, UID/EMS/04077/2019, UID/EMS/00532/2019 from FCT; and by projects NORTE-01-0145-FEDER-028178, NORTE-01-0145-FEDER-029394, and NORTE01-0145-FEDER-030171 funded by NORTE 2020 Portugal Regional Operational Programme, under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund and by Fundação para a Ciência e Tecnologia (FCT), IP. Conflicts of Interest: Th

REVERSE INSULATOR DIELECTROPHORESIS: UTILIZING DROPLET MICROENVIRONMENTS FOR DISCERNING MOLECULAR EXPRESSIONS ON CELL SURFACES

Lab-on-a-chip (LOC) technologies enable the development of portable analysis devices that use small sample and reagent volumes, allow for multiple unit operations, and couple with detectors to achieve high resolution and sensitivity, while having small footprints, low cost, short analysis times, and portability. Droplet microfluidics is a subset of LOCs with the unique benefit of enabling parallel analysis since each droplet can be utilized as an isolated microenvironment. This work explored adaptation of droplet microfluidics into a unique, previously unexplored application where the water/oil interface was harnessed to bend electric field lines within individual droplets for insulator dielectrophoretic (iDEP) characterizations. iDEP polarizes particles/cells within non-uniform electric fields shaped by insulating geometries. We termed this unique combination of droplet microfluidics and iDEP reverse insulator dielectrophoresis (riDEP). This riDEP approach has the potential to protect cell samples from unwanted sample-electrode interactions and decrease the number of required experiments for dielectrophoretic characterization by ~80% by harnessing the parallelization power of droplet microfluidics. Future research opportunities are discussed that could improve this reduction further to 93%. A microfluidic device was designed where aqueous-in-oil droplets were generated in a microchannel T-junction and packed into a microchamber. Reproducible droplets were achieved at the T-junction and were stable over long time periods in the microchamber using Krytox FSH 157 surfactant in the continuous oil FC-40 phase and isotonic salts and dextrose solutions as the dispersed aqueous phase. Surfactant, salts, and dextrose interact at the droplet interface influencing interfacial tension and droplet stability. Results provide foundational knowledge for engineering stable bio- and electro-compatible droplet microfluidic platforms. Electrodes were added to the microdevice to apply an electric field across the droplet packed chamber and explore riDEP responses. Operating windows for droplet stability were shown to depend on surfactant concentration in the oil phase and aqueous phase conductivity, where different voltage/frequency combinations resulted in either stable droplets or electrocoalescence. Experimental results provided a stability map for strategical applied electric field selection to avoid adverse droplet morphological changes while inducing riDEP. Within the microdevice, both polystyrene beads and red blood cells demonstrated weak dielectrophoretic responses, as evidenced by pearl-chain formation, confirming the preliminary feasibility of riDEP as a potential characterization technique. Two additional side projects included an alternative approach to isolate electrode surface reactions from the cell suspension via a hafnium oxide film over the electrodes. In addition, a commercially prevalent water-based polymer emulsion was found to adequately duplicate microchannel and microchamber features such that it could be used for microdevice replication

Label-free multi-step microfluidic device for mechanical characterization of blood cells: diabetes type II

The increasing interest to establish significant correlations between blood cell mechanical measurements and blood diseases, has led to the promotion of microfluidic devices as attractive clinical tools for potential use in diagnosis. A multi-step microfluidic device able to separate red and white blood cells (RBCs and WBCs) from plasma and simultaneously measure blood cells deformability (5 and 20% of hematocrit) is presented in this paper. The device employs passive separation based on the cross-flow filtration principle, introduced at each daughter channel. At the outlets, hyperbolic geometries allow single-cell deformability analysis. The device was tested with blood from five healthy and fifteen diabetic type II voluntary donors. The results have shown that WBCs have lower deformability than RBCs, and no significant differences were observed in WBCs from healthy and pathological blood samples. In contrast, RBCs have shown significant differences, with pathological cells exhibiting lower deformability. Shear rheology has shown that blood from patients with type II diabetes has higher viscosity than blood from healthy donors. This microfluidic device has demonstrated the ability to reduce cell concentration at the outlets down to 1%, an ideal cell concentration for assessing the blood cells deformability, under healthy and pathological conditions. The results provide new insights and quantitative information about the hemodynamics of in vitro type II diabetes mellitus RBCs. Thus, such device can be a promising complement in clinical diagnosis and biological research as part of an integrated blood-on-a-chip system.This work was supported by Projects NORTE-01-0145-FEDER-028178, NORTE-01-0145-FEDER-029394, NORTE-01-0145-FEDER-030171 funded by COMPETE2020, NORTE2020, PORTUGAL2020, and FEDER. This work was also supported by Fundacao para a Ciencia e a Tecnologia (FCT) under the strategic grants UIDB/04077/2020 and UIDB/00532/2020. D. Pinho and V. Faustino acknowledge the Ph.D. scholarships SFRH/BD/89077/2012 and SFRH/BD/99696/2014, respectively, both provided by FCT. Susana Catarino thanks FCT for her contract funding provided through 2020.00215.CEECIND. F. T. Pinho is thankful to FCT for financial support through projects LA/P/0045/2020 of the Associate Laboratory in Chemical Engineering (ALiCE) and pro-jects UIDB/00532/2020 and UIDP/00532/2020 of Centro de Estudos de Fenomenos de Transporte

NEW MICROFLUIDIC SYSTEM TO INCREASE ROBUSTNESS OF ELECTRODE PERFORMANCE AND DEVELOP POINT-OF-CARE HEMATOCRIT DEVICE

The present dissertation aimed to develop a new microfluidic system for a point-of-care hematocrit device. Stabilization of microfluidic systems via surfactant additives and integration of semipermeable SnakeSkin® membranes was investigated. Both methods stabilized the microfluidic systems by controlling electrolysis bubbles. Surfactant additives, Triton X-100 and SDS stabilized promoted faster bubble detachment at electrode surfaces by lowering surface tension and decreased gas bubble formation by increasing gas solubility. The SnakeSkin® membranes blocked bubbles from entering the microchannel and thus less disturbance to the electric field by bubbles occurred in the microchannel. Platinum electrode performance was improved by carbonizing electrode surface using red blood cells. Irreversibly adsorbed RBCs lysed on platinum electrode surfaces and formed porous carbon layers while current response measurements. The formed carbon layers increase the platinum electrode surface area and thus electrode performance was improved by 140 %. The microfluidic system was simplified by employing DC field to use as a platform for a point-of-care hematocrit device. Feasibility of the microfluidic system for hematocrit determination was shown via current response measurements of red blood cell suspensions in phosphate buffered saline and plasma media. The linear trendline of current responses over red blood cell concentration was obtained in both phosphate buffered saline and plasma media. This research suggested that a new and simple microfluidic system could be a promising solution to develop an inexpensive and reliable point-of-care hematocrit device

3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood

Detection of circulating tumor cells has emerged as a promising minimally invasive diagnostic and prognostic tool for patients with metastatic cancers. We report a novel three dimensional microfilter device that can enrich viable circulating tumor cells fromblood. This device consists of two layers of parylene membrane with pores and gap precisely defined with photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimize the stress concentration on cell membrane and sustain cell viability during filtration. Viable cell capture on device was investigated with scanning electron microscopy, confocal microscopy, and immunofluorescent staining using model systems of cultured tumor cells spiked in blood or saline. The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application. The device provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings