Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.     

Microfluidic device for label-free measurement of platelet activation

In this work we demonstrate a new microfluidic method for the rapid assessment of platelet size and morphology in whole blood. The device continuously fractionates particles according to size by displacing them perpendicularly to the fluid flow direction in a micro-fabricated post array. Whole blood, labeled with the fluorescent, platelet specific, antibody PE-anti-CD41, was run through the device and the positions of fluorescent objects noted as they exited the array. From this, histograms of platelet size were created which show marked increases in size after exposure to thrombin or a temperature of 4 degrees C. We infer that the well known morphological changes that occur during activation are causing the observed increase in size.     

A low-cost and high-throughput benchtop cell sorter for isolating white blood cells from whole blood

The ability to isolate and purify white blood cells (WBCs) from mixed ensembles such as blood would benefit autologous cell-based therapeutics as well as diagnosis of WBC disorders. Current WBCs isolation methods have the limitations of low purity or requiring complex and expensive equipment. In addition, due to the overlap in size distribution between lymphocytes (i.e., a sub-population of WBCs) and red blood cells (RBCs), it is challenging to achieve isolation of entire WBCs populations. In this work, we developed an inertial microfluidics-based cell sorter, which enables size-based, high-throughput isolation, and enrichment of WBCs from RBC-lysed whole blood. Using the developed inertial microfluidic chip, the sorting resolution is sharpened within 2 μm, which achieved separation between 3 and 5 μm diameter particles. Thus, with the present cell sorter, a full population of WBCs can be isolated from RBC-lysed blood samples with recovery ratio of 92%, and merely 5% difference in the composition percentage of the three subpopulations of granulocytes, monocytes, and lymphocytes compared to the original sample. Furthermore, our cell sorter is designed to enable broad application of size-based inertial cell sorting by supplying a series of microchips with different sorting cutoff size. This strategy allows us to further enrich the lymphocytes population by twofold using another microchip with a cutoff size between 10 and 15 μm. With simplicity and efficiency, our cell sorter provides a powerful platform for isolating and sorting of WBCs and also envisions broad potential sorting applications for other cell types.     

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device

We report a fully integrated, pathogen-specific DNA extraction device utilizing centrifugal microfluidics on a polymer based CD platform. By use of the innovative laser irradiated Ferrowax microvalve (LIFM) together with the rapid cell lysis method using laser irradiation on magnetic particles, we could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood on a CD. As a model study, DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) and E.coli were conducted. The total process of the plasma separation, mixing with magnetic beads conjugated with target specific antibodies, removal of plasma residual, washing and DNA extraction was finished within 12 min with only one manual step, the loading of 100 microL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD using a portable sample preparation device was as good as those by conventional bench top protocol. It demonstrates that our novel centrifugal microfluidics platform enables a full integration of complex biological reactions that require multi-step fluidic control.     

A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes with high efficiency directly from 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R(2) = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings.     

Integrated microelectronic device for label-free nucleic acid amplification and detection

We present an integrated microelectronic device for amplification and label-free detection of nucleic acids. Amplification by polymerase chain reaction (PCR) is achieved with on-chip metal resistive heaters, temperature sensors, and microfluidic valves. We demonstrate a rapid thermocycling with rates of up to 50 degrees C s(-1) and a PCR product yield equivalent to that of a bench-top system. Amplicons within the PCR product are detected by their intrinsic charge with a silicon field-effect sensor. Similar to existing optical approaches with intercalators such as SYBR Green, our sensing approach can directly detect standard double-stranded PCR product, while in contrast, our sensor does not require labeling reagents. By combining amplification and detection on the same device, we show that the presence or absence of a particular DNA sequence can be determined by converting the analog surface potential output of the field-effect sensor to a simple digital true/false readout.     

Optofluidic integrated cell sorter fabricated by femtosecond lasers

The main trend in optofluidics is currently towards full integration of the devices, thus improving automation, compactness and portability. In this respect femtosecond laser microfabrication is a very powerful technology given its capability of producing both optical waveguides and microfluidic channels. The current challenge in biology is the possibility to perform bioassays at the single cell level to unravel the hidden complexity in nominally homogeneous populations. Here we report on a new device implementing a fully integrated fluorescence-activated cell sorter. This non-invasive device is specifically designed to operate with a limited amount of cells but with a very high selectivity in the sorting process. Characterization of the device with beads and validation with human cells are presented.     

Optofluidic detection for cellular phenotyping

Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidics, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection.     

Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.     

Low-cost polymer-film spiral inertial microfluidic device for label-free separation of malignant tumor cells

We developed a low-cost polymer-film spiral inertial microfluidic device for the effective size-dependent separation of malignant tumor cells. The device was fabricated in polymer films by rapid laser cutting and chemical bonding. After fabricating the prototype device, the separation performance of our device was evaluated using particles and cells. The effects of operational flow rate, cell diameter, and cell concentration on the separation performance were explored. Our device successfully separated tumor cells from polydisperse white blood cells according to their different migration modes and lateral positions. Then, the separation of rare cells was carried out using the high-concentration lysed blood spiked with 200 tumor cells. Experimental results showed that 83.90% of the tumor cells could be recovered, while 99.87% of white blood cells could be removed. We successfully employed our device for processing clinical pleural effusion samples from patients with advanced metastatic breast cancer. Malignant tumor cells with an average purity of 2.37% could be effectively enriched, improving downstream diagnostic accuracy. Our device offers the advantages of label-free operation, low cost, and fast fabrication, thus being a potential tool for effective cell separation.     

Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery

This report describes the design, fabrication, and testing of a cross-flow filtration microdevice, for the continuous extraction of blood plasma from a circulating whole blood sample in a clinically relevant environment to assist in continuous monitoring of a patient's inflammatory response during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures (about 400,000 adult and 20,000 pediatric patients in the United States per year). The microfiltration system consists of a two-compartment mass exchanger with two aligned sets of PDMS microchannels, separated by a porous polycarbonate (PCTE) membrane. Using this microdevice, blood plasma has been continuously separated from blood cells in a real-time manner with no evidence of bio-fouling or cell lysis. The technology is designed to continuously extract plasma containing diagnostic plasma proteins such as complements and cytokines using a significantly smaller blood volume as compared to traditional blood collection techniques. The microfiltration device has been tested using a simulated CPB circulation loop primed with donor human blood, in a manner identical to a clinical surgical setup, to collect plasma fractions in order to study the effects of CPB system components and circulation on immune activation during extracorporeal circulatory support. The microdevice, with 200 nm membrane pore size, was connected to a simulated CPB circuit, and was able to continuously extract ~15% pure plasma volume (100% cell-free) with high sampling frequencies which could be analyzed directly following collection with no need to further centrifuge or modify the fraction. Less than 2.5 ml total plasma volume was collected over a 4 h sampling period (less than one Vacutainer blood collection tube volume). The results tracked cytokine concentrations collected from both the reservoir and filtrate samples which were comparable to those from direct blood draws, indicating very high protein recovery of the microdevice. Additionally, the cytokine concentration increased significantly compared to baseline values over the circulation time for all cytokines analyzed. The high plasma protein recovery (over 80%), no indication of hemolysis and low level of biofouling on the membrane surface during the experimental period (over 4 h) were all indications of effective and reliable device performance for future clinical applications. The simple and robust design and operation of these devices allow operation over a wide range of experimental flow conditions and blood hematocrit levels to allow surgeons and clinicians autonomous usage in a clinical environment to better understand the mechanisms of injury resulting from cardiac surgery, and allow early interventions in patients with excessive postoperative complications to improve surgical outcomes. Ultimately, monolithic integration of this microfiltration device with a continuous microimmunoassay would create an integrated microanalysis system for tracking inflammation biomarkers concentrations in patients for point-of-care diagnostics, reducing blood analysis times, costs and volume of blood samples required for repeated assays.     

Optofluidic variable-focus lenses for light manipulation

This paper presents a planar optofluidic lens for light manipulation utilizing a combination of optofluidic biconvex lens with micromixer. Three light manipulation techniques including tunable optical diverging, collimating and focusing are realized by altering the refractive index of the optofluidic variable-focus lenses formed by solid polydimethylsiloxane (PDMS) walls and tunable liquid lens body. The optical power from the laser input can be increased or decreased with the tuning of the variable-focus lenses' refractive indexes. The optical power adjustment capabilities are demonstrated and characterized. The combinations of benefits of all lens' optical manipulation capabilities, greater mechanical stability, significant increase of optofluidic device's life time and seamless integration with other lab-on-a-chip functionalities provide a promising and versatile optofluidic compartment to integrate with lab-on-a-chip excitation and sensing applications. Optofluidic lens-including system for tunable fluorescence sensing is demonstrated showing 186% increase in detected fluorescence intensity.     

Double spiral microchannel for label-free tumor cell separation and enrichment

This work reports on a passive double spiral microfluidic device allowing rapid and label-free tumor cell separation and enrichment from diluted peripheral whole blood, by exploiting the size-dependent hydrodynamic forces. A numerical model is developed to simulate the Dean flow inside the curved geometry and to track the particle/cell trajectories, which is validated against the experimental observations and serves as a theoretical foundation for optimizing the operating conditions. Results from separating tumor cells (MCF-7 and Hela) spiked into whole blood indicate that 92.28% of blood cells and 96.77% of tumor cells are collected at the inner and the middle outlet, respectively, with 88.5% tumor recovery rate at a throughput of 3.33 × 10(7) cells min(-1). We expect that this label-free microfluidic platform, driven by purely hydrodynamic forces, would have an impact on fundamental and clinical studies of circulating tumor cells.     

Isolation of plasma from whole blood using planar microfilters for lab-on-a-chip applications

Researchers are actively developing devices for the microanalysis of complex fluids, such as blood. These devices have the potential to revolutionize biological analysis in a manner parallel to the computer chip by providing very high throughput screening of complex samples and massively parallel bioanalytical capabilities. A necessary step performed in clinical chemistry is the isolation of plasma from whole blood, and effective sample preparation techniques are needed for the development of miniaturized clinical diagnostic devices. This study demonstrates the use of passive, operating entirely on capillary action, transverse-flow microfilter devices for the microfluidic isolation of plasma from whole blood. Using these planar microfilters, blood can be controllably fractionated with minimal cell lysis. A characterization of the device performance reveals that plasma filter flux is dependent upon the wall shear rate of blood in the filtration channel, and this result is consistent with macroscale blood filtration using microporous membranes. Also, an innovative microfluidic layout is demonstrated that extends device operation time via capillary action from seconds to minutes. Efficiency of these microfilters is approximately three times higher than the separation efficiencies predicted for microporous membranes under similar conditions. As such, the application of the microscale blood filtration designs used in this study may have broad implications in the design of lab-on-a-chip devices, as well as the field of separation science.     

Microchip-based plasma separation from whole blood via axial migration of blood cells

Highly efficient cell-free plasma separation from 200 μL of human whole blood was realized via axial migration of blood cells and cross-flow filtration in a microchip. Although various analyses of small volumes of blood have been reported, a large volume of blood is necessary for obtaining blood cells and plasma for the conventional plasma separation technique of centrifugation. A highly efficient plasma separation method using small volumes of blood without hemolysis is an important issue. We developed a plasma separation method based on a microchip with a filter, which utilizes the axial migration of blood cells observed in blood vessels. Clogging and hemolysis on the filter can be prevented by the axial migration of the blood cells. Using this method, 65% of the plasma from 200 μL of whole blood was successfully separated without hemolysis. When the plasma separation microchip interfaced with a micro-ELISA system was applied to C-reactive protein (CRP) analysis, the CRP concentration obtained by the microchip showed good correlation with that obtained by conventional centrifugation. Total analysis time, including plasma separation, was achieved in only 25 min.     

Optofluidic micro-sensors for the determination of liquid concentrations

We present a novel optofluidic device for non-invasive and label-free determination of liquid concentrations. A microfluidic channel filled with the sample solution is hit by laser light in an angle close to the critical angle for total internal reflection. Due to the intentionally defined divergence of the incident beam, parts of the rays will experience total internal reflection while another part will be transmitted. Both reflected and transmitted light signals are recorded and the ratio of these signals is used for sample characterization. The stability compared to single signal analyses is significantly improved, resulting in a resolution of approximately 40 mmol L(-1). The typical working range of the device under investigation is between a few tens of mmol L(-1) and 5 mol L(-1) making it useful for applications in the food industry, for example to determine the amount of phosphates in liquid products.     

Fully integrated lab-on-a-disc for simultaneous analysis of biochemistry and immunoassay from whole blood

We report a fully integrated device that can perform both multiple biochemical analysis and sandwich type immunoassay simultaneously on a disc. The whole blood is applied directly to the disposable "lab-on-a-disc" containing different kinds of freeze-dried reagents for the blood chemistry analysis as well as reagents required for the immunoassay. The concentrations of different kinds of analytes are reported within 22 min by simply inserting a disc to a portable device. Using the innovative laser irradiated ferrowax microvalves together with the centrifugal microfluidics, the total process of plasma separation, metering, mixing, incubation, washing, and detection is fully automated. The analyzer is equipped with an optical detection module to measure absorbances at 10 different wavelengths to accommodate the various kinds of reaction protocols. Compared to the conventional blood analysis done in clinical laboratories, it is advantageous for point-of-care applications because it requires a smaller amount of blood (350 μL vs. 3 mL), takes less time (22 min vs. several days), does not require specially trained operators or expensive instruments to run biochemical analysis and immunoassay separately.     

Enzyme electrode for glucose determination in whole blood

The development of a glucose sensor suitable for use with whole blood is described. It is based on anodic oxidation at +700 mV of hydrogen peroxide with a platinum electrode covered with a gas permeable membrane. Glucose reacts with glucose oxidase immobilised on the external side of the membrane, and forms hydrogen peroxide which is able to cross the gas permeable membrane due to its high vapour tension, while other electroactive substances that are important interferents are completely blocked. This principle was discovered several years ago but no practical application was presented up to now. Therefore in this work a number of different commercial membranes were tested, in order to obtain a resistant, rapidly responding and interference free sensor to be used in conjunction with a blood gas measurement apparatus. Coimmobilisation of glucose oxidase and catalase was found to be useful for fast response and recovery of the electrode. Using some of the tested membranes, the linearity range is 1-15 mM, CV 5%, response time 90 s, recovery time for the next sample 120 s. The membrane's working life is 2-3 weeks.     

A urea-sensing membrane electrode for whole blood measurements

The construction and properties of a new urea-sensing membrane electrode capable of making direct urea measurements in whole blood are described. The electrode has a layered structure in which a small quantity of EDTA-stabilized urease enzyme solution is held between an external dialysis membrane and the gas-permeable membrane of a conventional ammonia selective electrode. It is shown that the electrode functions reliably in whole blood samples, used with minimal pretreatment, as well as in serum or aqueous solutions. The range, dynamic response, lifetime, precision, and accuracy of the electrode system are appropriate for clinical measurements in whole blood or serum, and promise to simplify such analyses with an attendant reduction in costs.     

Polymers as matrices for whole cell immobilization

The immobilization of whole microbial cells has become an important tool in the development of biocatalytic processes in the pharmaceutical and food industry. Not only dead, i.e. non growing cells, but recently with higher priority living and growing cells are the biological species, for which simple and efficient polymeric carriers had to be found. In comparison to other methods, like adsorption or encapsulation, entrapment into a polymer network is the most widely used technique. The network can be formed on the basis of a)ionic interactions (ionotropic gelation of polyelectrolytes), b) of polycondensation reactions (epoxides, polyurethanes, silicones) or c) of polymerization reactions (crosslinking polymerization of vinylic monomers, oligomers or polymers). The characteristic features and the efficiency-controlling parameters of some immobilized cells systems are discussed as illustrative examples.