Blood Plasma Separation Research Articles

Development and Characterization of Monoclonal Antibodies Against Glycophorin A Applicable for Blood Sample Processing.

The separation of plasma from blood cells is critical for the accuracy of blood tests because cellular fractions can create discrepancies in analysis. The most common method to separate blood cells from the liquid part of the blood is centrifugation, which is not always applicable in resource-constrained areas and countries. In this study, we describe the generation of monoclonal antibodies (mAbs) against glycophorin A (GPA) of human erythrocytes. BALB/c mice were immunized with human erythrocytes followed by purified GPA. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. Hybridoma clones were screened by hemagglutination assay and enzyme-linked immunosorbent assay (ELISA). Six hybridoma clones were obtained and subcloned. The characterization of the purified mAbs demonstrates that they are able to bind and retain erythrocytes in hemagglutination assay. Furthermore, one of the mAbs 1A9 recognizes purified GPA in ELISA, whereas the other mAb 1G7 is able to immunoprecipitate GPA from human erythrocyte lysates, and a band of 38 kDa is detected. In conclusion, the anti-GPA mAbs are useful tools in developing a quick and easy way to separate blood plasma from whole blood for clinical tests, and in developing bi-specific antibodies for other clinical applications.

Paper-Based Lateral Flow Immunoassay for Detection of Biomarkers in Human Fluids

Traumatic brain injuries (TBI) patients suffer from neurological deficits and increased risk of neurological disease, so there is a need for accurate, high throughput, and rapid diagnosis. Currently, TBI severity is detected and assessed using neurological examination and CT imaging, which require expensive hospital and doctors office visits. Protein biomarkers have emerged as an objective means to diagnose TBI; however, proteins are typically detected using time consuming and expensive methods such as ELISA and Western blot. In this work, a paper-based lateral flow assay (PLFA) has been developed for a rapid, low-cost means for detecting TBI biomarkers in blood plasma. PLFAs have received increasing attention due to their low cost and ease-of-use. However, these devices suffer from poor sensitivity and interference from the blood plasma matrix. In this study, surface-enhanced Raman scattering (SERS) was applied into a PLFA as the signal transducer to improve sensitivity and overcome biological interference. These advantages are attributed to the optical excitation in the near-infrared biological transparency window and SERS nanostructure design. The PLFA is designed using a plasmonic nanoarray chip and a blood plasma separation unit for highly sensitive TBI biomarker detection.

Elastocapillary flow driven lab-on-a-membrane device based on differential wetting and sedimentation effect for blood plasma separation

We report an elastocapillary flow—driven lab on a membrane device based on differential wetting and sedimentation effect for the separation of plasma from whole blood. Interaction between a thin polydimethylsiloxane (PDMS) membrane (thickness ~35 μm) bonded to the edge of a PDMS substrate and a sample blood drop (of volume ~70 μl) gives rise to deformation of the soft membrane due to the capillary forces providing a conduit and consequent elastocapillary flow of blood. The surface of the PDMS membrane is hydrophilic up to a certain length along the flow direction to support the elastocapillary flow and hydrophobic thereafter to impede the flow. In the hydrophobic region, owing to a much smaller sedimentation time scale (~100 s) as compared to the capillary flow time scale (~1000 s), sedimentation of blood cells occurs thus facilitating separation of plasma from the blood cells in the hydrophobic region. The role of differential wetting and sedimentation effects on the blood plasma separation is studied. The effects of membrane width and thickness, length of the hydrophilic region, erythrocyte sedimentation rate (ESR) on the separation of plasma were investigated. Using a membrane of width 3 mm, thickness 35 μm, total length 25 mm and hydrophilic length of 4 mm and 70 μl of whole blood with ESR varying in the range 4 mm h−1 to 40 mm h−1, the volume of plasma was in the range of 7.5 μl to 20 μl, respectively, which corresponds to a plasma recovery of 22%–49%, respectively. Purity of the plasma from the proposed device was compared with that obtained from centrifugation which showed a good match. The device was integrated with a commercially available detection strip to detect the level of glucose present in the plasma from blood samples of healthy and diabetic patients which are in qualitative agreement with that obtained from conventional tests.

Integrated microfluidic pneumatic circuit for point-of-care molecular diagnostics

Developing simple, portable, rapid, and easy-to-use diagnostic technologies is essential for point-of-care (POC) blood molecular testing. Integrated microfluidic devices that include the functionalities of blood separation, microfluidic pumping, and molecular detection are desirable for POC testing; however, current technologies still rely on off-chip sample processing or require bulky equipment. We report a fully-integrated microfluidic diagnostic device, i.e., an integrated pneumatic microfluidic circuit (iPC), that can autonomously pump whole blood, continuously sort blood plasma, and readily enable blood plasma proteomic analysis. The iPC contains vacuum pillars as a vacuum source and waste reservoir, as well as microchannels connecting the pillars as a plasma separator or a flow stabilizer. We combined the iPC and a miniaturized fluorescence microscope to create a portable diagnostic platform that enables fluorescence-based biomarker detection. First, we performed systematic parametric studies to establish design rules for determining the transport and distribution of fluid streams in the iPC. We then demonstrated the capability of the iPC-based diagnostic platform by successfully separating blood plasma from microliter quantities of whole blood while simultaneously quantifying thrombin in blood samples using an aptamer beacon within 5 min of sample injection. Our platform holds potential as a rapid, field-deployable, essentially universal diagnostic tool in POC settings.

A passive two-way microfluidic device for low volume blood-plasma separation

Blood-plasma separation is one of the main concerns for Point-of-Care devices seeking to retain cells of interest or analyze plasma biomarkers. The present work describes a novel passive device based on size-exclusion method. Starting from a particulate-trap-like filter, numerical simulations in ANSYS Fluent were performed in order to optimize the geometry for blood-plasma separation. The final layout was then embedded inside a microfluidic channel and the related master produced with standard cleanroom processes. A 1 mm thick Cyclic Olefin Copolymer wafer was chosen for hot embossing replication, since Cyclic Olefin Copolymer is a transparent, solvent-resistant and medical-grade material. Moreover, the adopted process is easily scalable to industrial levels. The filtering efficiency was finally assessed with a 4 μm fluorescent beads dispersion, mimicking the erythrocytes average diameter of 8 μm. No fluorescent particles were observed downstream of the filter after filtration, thus all the beads were retained by the optimized filtering structure.

Localized surface plasmon resonance (LSPR) biosensor based on thermally annealed silver nanostructures with on-chip blood-plasma separation for the detection of dengue non-structural protein NS1 antigen

Early diagnosis of dengue biomarkers by employing a technology that is less labor- and time-intensive and offers higher sensitivity and lower limits of detection would find great significance in the developing world. Here, we report the development of a biosensor that exploits the localized surface plasmon resonance (LSPR) effect of silver nanostructures, created via thermal annealing of thin metal film, to detect dengue NS1 antigen, which appears as early as the onset of infection. The biosensor integrates membrane-based blood-plasma separation to develop lab-on-chip device that facilitates rapid diagnosis (within 30 min) of dengue NS1 antigen from a small volume (10 µL) of whole blood. The refractive index (RI) sensitivity of the LSPR biosensor was verified by using aqueous glycerol (0–100 wt%) which showed that it is sufficiently sensitive to detect 10−3 change in RI, which is comparable to that observed with protein-protein interaction. The RI sensitivity was utilized to demonstrate protein binding by using bovine serum albumin and detection of antibody-antigen immune reaction by binding human chorionic gonadotropin antigen to immunoglobulin antibody immobilized in our LSPR biosensor. Next, we demonstrated the detection of NS1 in plasma obtained via centrifugation and in plasma separated on-chip. From 10 µL of whole blood spiked with NS1 antigen, our biosensor reliably detects 0.06 µg/mL of NS1, which lies within the clinical limit observed during the first seven days of infection, with a sensitivity of 9 nm/(µg/mL). These results confirm that the proposed LSPR biosensor can potentially be used in point-of-care dengue diagnostics.

Fidget-spinner centrifuge separates blood from plasma

A fidget spinner, the toy twirled endlessly between the fingers of schoolkids, can act as a makeshift centrifuge and separate blood plasma sufficiently for an HIV test (Anal. Chem. 2018, DOI: 10.1021/acs.analchem.8b04860). For blood tests, red blood cells must be separated from the plasma, usually with a fast-spinning centrifuge, but these are expensive and require electricity to operate, which may not be feasible in resource-limited regions of the world, says Chien-Fu Chen of the Institute of Applied Mechanics at National Taiwan University. Along with Chien-Cheng Chang and colleagues, Chen put human blood spiked with a common HIV-1 diagnostic protein in 50 mm long plastic tubes, sealed them, and taped them to each of the three spinning arms of a commercially available fidget spinner. After spinning the device for 4–7 min, the team separated about 30% of the plasma from the blood sample—with 99% purity. A paper-based HIV test detected the

A micro-dispenser for long-term storage and controlled release of liquids

The success of lab-on-a-chip systems may depend on a low-cost device that incorporates on-chip storage and fluidic operations. To date many different methods have been developed that cope separately with on-chip storage and fluidic operations e.g., hydrophobic and capillary valves pneumatic pumping and blister storage packages. The blister packages seem difficult to miniaturize and none of the existing liquid handling techniques despite their variety are capable of proportional repeatable dispensing. We report here on an inexpensive robust and scalable micro-dispenser that incorporates long-term storage and aliquoting of reagents on different microfluidics platforms. It provides long-term shelf-life for different liquids enables precise dispensing on lab-on-a-disc platforms and less accurate but proportional dispensing when operated by finger pressure. Based on this technology we introduce a method for automation of blood plasma separation and multi-step bioassay procedures. This micro-dispenser intends to facilitate affordable portable diagnostic devices and accelerate the commercialization of lab-on-a-chip devices.

A passive portable microfluidic blood–plasma separator for simultaneous determination of direct and indirect ABO/Rh blood typing

The blood typing test is mandatory in any transfusion, organ transplant, and pregnancy situation. There is a lack of point-of-care (POC) blood typing that could perform both direct and indirect methods using a single droplet of whole blood. This study presents a new methodology combining a passive microfluidic blood-plasma separator (BPS) and a blood typing detector for the very first time, leading to a stand-alone microchip which is capable of determining the blood group from both direct and indirect methods simultaneously. The proposed design separates blood cells from plasma by applying hydrodynamic forces imposed on them, which overcomes the clogging issue and consequently maximizes the volume of the extracted plasma. An axial migration effect across the main channel is responsible for collecting the plasma in plasma collector channels. The BPS novel design approached 12% yield of plasma with 100% purity in approximately 10 minutes. The portable BPS was designed and fabricated to perform ABO/Rh blood tests based on the detection of agglutination in both antigens of RBCs (direct) and antibodies of plasma (indirect). The differences between agglutinated and non-agglutinated samples were distinguishable by the naked eye and also validated by particle analysis of microscopic pictures. The results of this passive BPS in ABO/Rh blood grouping verified the quality and quantity of the extracted plasma in practical applications.

Glucose and Cholesterol Sensing in Blood Plasma Using Zno-Paper Based Microfluidics

This work aims at investigating the reliability of ZnO-paper based microfluidic analytical devices (ZnO-µPAD) for blood plasma separation as well as bio-sensing of glucose and cholesterol. Two-layer paper-based microfluidic analytical devices coated with ZnO nanoflowers/ZnO spherical aggregates (ZnO-µPAD) for a rapid blood plasma separation and glucose sensing were developed. Plasma separation in ZnO-µPAD was evaluated experimentally and numerically using computational fluid dynamics package for a flow over porous networks. Glucose and cholesterol detection was carried out using spectroscopic techniques, both Fourier-transform infrared (FTIR) and Raman measurements. The plasma separation process on ZnO NF-μPAD requires 250 s. The spectroscopic investigation reveals that the IR absorptions and Raman signals at the typical vibrational frequencies of glucose and cholesterol are increasing at higher concentration. After subtraction from absorption background arising from ZnO and the paper, a specific and linearly increasing IR absorption (913 and 1349 cm-1) and Raman signals (1346 and 1461 cm-1) are observable with a relatively good sensitivity.

An anti-clogging method for improving the performance and lifespan of blood plasma separation devices in real-time and continuous microfluidic systems

On-chip blood plasma separators using microfluidic channels are typically developed as disposable devices for short-term use only because blood cells tend to clog the microchannels, limiting their application in real-time and continuous systems. In this study, we propose an anti-clogging method. We applied dielectrophoresis to prevent microchannel clogging in a plasma separator that can be used over long periods for real-time and continuous monitoring. Prior to applying the anti-clogging method, the blood plasma separator stopped working after 4 h. In contrast, by manipulating the separator with the new anti-clogging method at a voltage of 20 V, it continued working in a long-term experiment for 12 h without performance deterioration or an increase in cell loss. Two critical performance parameters of the manipulated separator, the purity efficiency and the plasma yield, were 97.23 ± 5.43% and 38.95 ± 9.34%, respectively, at 20 V after 15 min. Interestingly, the two performance parameters did not decrease during the long-term experiment. Hence, the blood plasma separator with the anti-clogging method is an interesting device for use in real-time and continuous blood plasma separation systems because of its consistent performance and improved lifespan.

Electrokinetics with blood.

Microfluidics based lab-on-a-chip technology holds tremendous promises towards point-of-care diagnosis of diseases as well as for developing engineered devices aimed towards replicating the intrinsic functionalities of human bodies as mediated by blood vessel mimicking circulatory networks. While the analysis of transport of blood including its unique cellular constituents has remained to be the focus of many reported studies, a progressive interest on understanding the interplay between electric field and blood flow dynamics has paved a new way towards further developments from scientific engineering as well as clinical viewpoint. Here, we briefly outline the interconnection between electrokinetics and blood flow through micro-capillaries, in an effort to address several challenging propositions in a wide variety of applications encompassing biophysical transport to medical diagnostics. We first present the fundamentals of interaction of electric field with cellular components. In conjunction with the unique rheological features of blood, we show that this interaction may turn out to be compelling for the use of electric fields for transporting blood samples through microfluidic conduits. We discuss the perspectives of both direct current and alternating current electrokinetics in the context of blood flow. In addition, we provide a brief outline of the concerned theoretical developments. We also bring out the relevant biophysical perspectives and focus on applications such as blood plasma separation and separation of circulatory tumor cells. Finally, we attempt to provide a futuristic outlook and envisage the potential of combining electrokinetics with blood microcirculation towards developing futuristic biomimetic microdevices that can replicate a novel control mechanism over micro-circulatory transport in the entire connective network of human bodies. This may effectively pave the way towards the realization of a next-generation medical simulation device, significantly advanced from what is available under the ambit of the state of art technology in the field.

Paper‐Based Antibody Detection Devices Using Bioluminescent BRET‐Switching Sensor Proteins

AbstractThis work reports on fully integrated “sample‐in‐signal‐out” microfluidic paper‐based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET‐based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume‐independent and fully reagent‐free operation, including on‐device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti‐HIV1, anti‐HA, and anti‐DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user‐friendly point‐of‐care testing in low‐resource environments.

Improved Understanding of Acoustophoresis and Development of an Acoustofluidic Device for Blood Plasma Separation

Separating blood plasma in microchannels is of great relevance to microfluidics-based biodetection. However, the physics behind the separation of plasma from whole blood using acoustophoresis (movement driven by sound waves) is not well understood. This study uses experiments and simulations to provide an improved understanding of plasma separation from whole blood using acoustophoresis. It is seen that acoustophoretic focusing of cells depend on two time scales: that of shear-induced diffusion, and that of actual acoustophoresis. These results will help in designing highly efficient blood-plasma separation devices for lab-on-a-chip applications.

Paper‐Based Antibody Detection Devices Using Bioluminescent BRET‐Switching Sensor Proteins

This work reports on fully integrated “sample‐in‐signal‐out” microfluidic paper‐based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET‐based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume‐independent and fully reagent‐free operation, including on‐device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti‐HIV1, anti‐HA, and anti‐DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user‐friendly point‐of‐care testing in low‐resource environments.

A whole blood sample-to-answer polymer lab-on-a-chip with superhydrophilic surface toward point-of-care technology

An innovative sample-to-answer (S-to-A) polymer lab-on-a-chip (LOC) with a blood plasma separator based on asymmetric capillary force has been proposed, developed, and completely characterized for point-of-care technology (POCT) applications. A spray layer-by-layer (LbL) nanoassembly coating has been applied for the superhydrophilic surface onto the cyclic olefin copolymer (COC). Then, the developed superwetting surfaces were designed and optimized for three device applications such as lateral transportation of whole blood in the device by capillary pumping, on-chip whole blood/plasma separation with an asymmetric capillary force, and detection using a capillary-driven lateral flow colorimetric assay. Integrating three primary components of the devices, the S-to-A polymer LOC platform has been effectively confirmed for the lateral flow colorimetric assay of bovine serum albumin (BSA) from unprocessed human whole blood without an external power resource.

Potential Point-of-Care Microfluidic Devices to Diagnose Iron Deficiency Anemia.

Over the past 20 years, rapid technological advancement in the field of microfluidics has produced a wide array of microfluidic point-of-care (POC) diagnostic devices for the healthcare industry. However, potential microfluidic applications in the field of nutrition, specifically to diagnose iron deficiency anemia (IDA) detection, remain scarce. Iron deficiency anemia is the most common form of anemia, which affects billions of people globally, especially the elderly, women, and children. This review comprehensively analyzes the current diagnosis technologies that address anemia-related IDA-POC microfluidic devices in the future. This review briefly highlights various microfluidics devices that have the potential to detect IDA and discusses some commercially available devices for blood plasma separation mechanisms. Reagent deposition and integration into microfluidic devices are also explored. Finally, we discuss the challenges of insights into potential portable microfluidic systems, especially for remote IDA detection.

Fiber-based optic sensor for detecting human blood clot: present and future revival

Sustaining human’s life-frame away from being impeded by the clot - ghost term, we attempt to approach a mobile fiber-based optical sensor (f-s) for detecting blood clot in a blood vessel (intra-arteries/veins). Blood vessels are the part of the circulatory system that transport blood throughout the human body, thus their significance of being protected arise to the monograph focus. MRI (magnetic resonance imaging), X-rays and other medical instruments are diagnostic immobility techniques with a slackest interval. The corer causation of fiber-based optical sensor is to detect a clump of blood in the bloodstream by providing a prompt mobile diagnostic intervals preserving last-minutes-breath of human’s life. The detector (f-s) has been etched by diluting sulphuric acid ~10% at certain zone to sensate its function. The in-vitro monograph peaks its maximal monitoring when the sensor is attached to Raman Spectroscopy (RS) setup. RS quantifies the relative intensities of fibrinogen bond, which is the first type of blood coagulation elements of blood plasma. Blood coagulation parameters are the major concern of the monograph investigation, such as total haemoglobin (tHb), clotting reaction time (t), clot progression time (t2), maximum clot amplitude (ma) and mean refractive index (r). A blood sample will be drawn from the patient and after centrifugation to separate blood plasma from its constituents, then an immediate sloshing of blood plasma in the (f-s) packet which has its plug-in to RS. Estimating the quantitative analysis of blood sample concentration, RS will determine the presence of coagulation in terms of intensity and medical procedures will dominate the treatment process. Thus, the suggestive monograph provides a definite instrument for investigating blood coagulation intra-arteries/veins promptly.

Motorized smart pipette for handheld operation of a microfluidic blood plasma separator

Simple and institutionalized testing of circulating biomarkers is essential for rapid cancer diagnosis and prognosis. However, blood sample preparation typically requires time-consuming separation steps and bulky, costly equipment. Although microfluidic separators have been developed for rapid and simple blood separation, their fluidic accessories such as syringe pumps still need to be miniaturized at a lower price before translating the separators into clinical practice. Here, we present a motorized smart pipette as a portable, cost-effective, and controllable pumping tool that can generate stable and high-pressure conditions suitable for high-throughput microfluidic blood separators. The pumping technology is based on a miniature air pump and its feedback control using an open-source microcontroller board. We characterized the pumping performance (controllability and stability) of the proposed technology using a pressure divider and a bang-bang feedback control method in a range of flow rate (166.5–529.0 μL/min). Additionally, we demonstrated the clinical utility of the pumping technology by rapidly separating blood plasma from whole blood and successfully recovering cancer cell-derived exosomes spiked in blood. We achieved high-throughput (≈422.3 μL/min) and high-purity (≈99.8%) plasma separation without hemolysis, recovering ≈90.5% of cancer cell-derived exosomes spiked in blood. With the advantages of high usability and versatility, this method can potentially accelerate the widespread adoption and clinical translation of microfluidics-based liquid biopsy technologies.

C-MEMS Device for Plasma Separation from Whole Blood

In this work, we report the fabrication of carbon-microelectromechanical system (C-MEMS) based contactless dielectrophoresis chip for the separation of Red Blood Cell (RBC) and plasma from whole blood. Blood is the most important functional body fluid and it is necessary to separate plasma and RBC for common disease diagnosis. Use of centrifugal force and microfilters are the conventional methods for this separation. To make a portable and cost effective device, researchers found a method of geometry driven separation by means of changing the blood flow channel shapes. This method was not popular because of the pressure driven flow of blood in the channel and the need of the apparatus including syringe pump, tubing, etc. Dielectrophoresis is one of the best methods for this application. But the contact dielectrophoresis (sample is in contact with the electrodes) damages the RBC because of the use of high voltage (more than 100V). It may also cause electrochemical effect, bubble formation and joule heating in the sample. In our development, one Indium Tin Oxide (ITO) coated glass was used as the bottom substrate. ITO coated side was used as the bottom electrode and microchannel was made in SU-8 by UV photolithography on the other side of the substrate. In a separate process, SU-8 derived carbon electrode was fabricated on Si/SiO2 substrate using the conventional C-MEMS process and transferred to 200µm thick PDMS layer. This polydimethylsiloxane (PDMS) layer was used to cover the microchannel in such a way that the carbon electrode was on the upper side of the device, aligned with the microchannel. These two electrodes (ITO and carbon) were capacitively coupled with the microchannel. The eletrostatic force is induced in the blood cells flow in the channel upon applying high frequency (1MHz) electric field to the electrodes. When blood flows in the channel, the electrostatic force combines with the capillary force and produces velocity difference between RBC and plasma resulting in their separation. In the preliminary experiment, we observed the separation of RBC and plasma at less than 1Vp-p with 1MHz frequency. Figure 1