Nicholas Watkins

A robust electrical microcytometer with 3-dimensional hydrofocusing

In this paper, we present a device to electrically count blood cell populations using an AC impedance interrogation technique in a microfabricated cytometer (microcytometer). Specifically, we direct our attention to obtaining the concentration of human CD4+ T lymphocytes (helper T cells), which is a necessary method to diagnose patients for HIV/AIDS and to give an accurate prognosis on the effectiveness of ARV (anti-retroviral) drug treatments. We study the effectiveness of a simple-to-fabricate 3-dimensional (3D) hydrodynamic focusing mechanism through fluidic simulations and corresponding experiments to increase the signal-to-noise ratio of impedance pulses caused by particle translocation and ensure lower variance in particle translocation height through the electrical sensing region. We found that the optimal 3D sheath flow settings result in a 44.4% increase in impedance pulse signal-to-noise ratio in addition to giving a more accurate representation of particle size distribution. Our microcytometer T cell counts closely with those found using an industry-standard flow cytometer for the concentration range of over three orders of magnitude and using a sample volume approximately the size of a drop of blood (approximately 20 microL). In addition, our device displayed the capability to differentiate between live and dead/dying lymphocyte populations. This microcytometer can be the basis of a portable, rapid, inexpensive solution to obtaining live/dead blood cell counts even in the most resource-poor regions of the world.

Microfluidic CD4+ and CD8+ T Lymphocyte Counters for Point-of-Care HIV Diagnostics Using Whole Blood

A microfluidic biochip enumerates CD4+ or CD8+ T lymphocytes in whole blood of healthy and HIV-infected donors. Tiny T Cell Counter for HIV The amount of CD4 and CD8 T cells in a blood sample can tell a doctor the status of the patient’s immune system and HIV infection. Current methods of counting aren’t always available in resource-poor settings, such as Sub-Saharan Africa, so Watkins et al. created a microfluidic chip for these point-of-care (POC) settings, which incorporates all steps of sample preparation and accurate T cell counting. The microfluidic differential T cell counter is based on the Coulter counter principle: In the device, cells are flowed through a tiny pore that has a current passing through it; the cell, which doesn’t conduct electricity, then blocks the current and causes a “spike” in signal. The number of spikes told Watkins et al. how many cells traveled through the pore. The device also integrated sample preparation and cell selection. Red blood cells were lysed and removed from the sample, leaving primarily white blood cells (including T cells). Antibodies decorated the microfluidic channels, to capture the population of choice—either CD4 or CD8 T cells. Thus, by obtaining a total cell count at the beginning and a final count at the end, the authors were able to quantify the number of T cells in the sample. Blood samples from HIV-infected donors and healthy volunteers were tested and compared to the gold standard, flow cytometry. Watkins and colleagues found that their differential T cell counter worked as well as flow cytometry in counting CD4+ and CD8+ T cells, thus suggesting that it is a viable platform for tracking HIV infection. Unique to this device is the ability to monitor not only CD4 cells but also CD8 T cells, which can give a more complete picture of infection. By integrating all steps of POC detection—sample preparation, purification, and analysis—and costing less per test than certain flow cytometers, it is possible that this device will be useful in resource-limited settings. Nevertheless, more testing on patients over time will be necessary to determine the device’s clinical utility. Roughly 33 million people worldwide are infected with HIV; disease burden is highest in resource-limited settings. One important diagnostic in HIV disease management is the absolute count of lymphocytes expressing the CD4+ and CD8+ receptors. The current diagnostic instruments and procedures require expensive equipment and trained technicians. In response, we have developed microfluidic biochips that count CD4+ and CD8+ lymphocytes in whole blood samples, without the need for off-chip sample preparation. The device is based on differential electrical counting and relies on five on-chip modules that, in sequence, chemically lyses erythrocytes, quenches lysis to preserve leukocytes, enumerates cells electrically, depletes the target cells (CD4 or CD8) with antibodies, and enumerates the remaining cells electrically. We demonstrate application of this chip using blood from healthy and HIV-infected subjects. Erythrocyte lysis and quenching durations were optimized to create pure leukocyte populations in less than 1 min. Target cell depletion was accomplished through shear stress–based immunocapture, using antibody-coated microposts to increase the contact surface area and enhance depletion efficiency. With the differential electrical counting method, device-based CD4+ and CD8+ T cell counts closely matched control counts obtained from flow cytometry, over a dynamic range of 40 to 1000 cells/μl. By providing accurate cell counts in less than 20 min, from samples obtained from one drop of whole blood, this approach has the potential to be realized as a handheld, battery-powered instrument that would deliver simple HIV diagnostics to patients anywhere in the world, regardless of geography or socioeconomic status.

A microfabricated electrical differential counter for the selective enumeration of CD4+ T lymphocytes†

We have developed a microfabricated biochip that enumerates CD4+ T lymphocytes from leukocyte populations obtained from human blood samples using electrical impedance sensing and immunoaffinity chromatography. T cell counts are found by obtaining the difference between the number of leukocytes before and after depleting CD4+ T cells with immobilized antibodies in a capture chamber. This differential counting technique has been validated to analyze physiological concentrations of leukocytes with an accuracy of ∼9 cells per µL by passivating the capture chamber with bovine serum albumin. In addition, the counter provided T cell counts which correlated closely with an optical control (R(2) = 0.997) for CD4 cell concentrations ranging from approximately 100 to 700 cells per µL. We believe that this approach can be a promising method for bringing quantitative HIV/AIDS diagnostics to resource-poor regions in the world.

On a Chip

Biological or biomedical microelectromechanical systems (BioMEMS) are poised to have a significant impact on clinical and biomedical applications. These devices-also termed lab-on-chip or point-of-care (POC) sensors-rep- resent a significant opportunity in various patient-centric settings, including at home, at the doctors office, in ambulances on the way to the hospital, in emergency rooms (ERs), at the hospital bedside, in rural and global health settings, and in clinical or commercial diagnostic laboratories. The potential impact of these technologies on the early diagnosis and management of disease can be very high for sensing and reporting on parameters ranging from physiological to biomolecular. As health-care delivery and management be- come increasingly personalized and individualized and as genomic, proteomic, and metabolic technologies unravel the human genetic and epigenetic dispositions to disease, detection of multiple markers (at any of the Omics scale) at an individualized level to assess the state of health and disease will become even more important.

Flow metering characterization within an electrical cell counting microfluidic device

Microfluidic devices based on the Coulter principle require a small aperture for cell counting. For applications using such cell counting devices, the volume of the sample also needs to be metered to determine the absolute cell count in a specific volume. Hence, integrated methods to characterize and meter the volume of a fluid are required in these microfluidic devices. Here, we present fluid flow characterization and electrically-based sample metering results of blood through a measurement channel with a cross-section of 15 μm × 15 μm (i.e. the Coulter aperture). Red blood cells in whole blood are lysed and the remaining fluid, consisting of leukocytes, erythrocyte cell lysate and various reagents, is flown at different flow rates through the measurement aperture. The change in impedance across the electrodes embedded in the counting channel shows a linear relationship with the increase in the fluid flow rate. We also show that the fluid volume can be determined by measuring the decrease in pulse width, and increase in number of cells as they pass through the counting channel per unit time.

Micro- and Nanotechnology for HIV/AIDS Diagnostics in Resource-Limited Settings

Thirty-four million people are living with HIV worldwide, a disproportionate number of whom live in resource-limited settings. Proper clinical management of AIDS, the disease caused by HIV, requires regular monitoring of both the status of the hosts immune system and levels of the virus in their blood. Therefore, more accessible technologies capable of performing a CD4+ T cell count and HIV viral load measurement in settings where HIV is most prevalent are desperately needed to enable better treatment strategies and ultimately quell the spread of the virus within populations. This review discusses micro- and nanotechnology solutions to performing these key clinical measurements in resource-limited settings.

Microfluidic differential immunocapture biochip for specific leukocyte counting

Enumerating specific cell types from whole blood can be very useful for research and diagnostic purposes—e.g., for counting of CD4 and CD8 T cells in HIV/AIDS diagnostics. We have developed a biosensor based on a differential immunocapture technology to enumerate specific cells in 30 min using 10 μl of blood. This paper provides a comprehensive stepwise protocol to replicate our biosensor for CD4 and CD8 cell counts. The biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies. Capture of other specific cells requires immobilization of their corresponding antibodies within the capture chamber. Therefore, this protocol is useful for research into areas surrounding immunocapture-based biosensor development. The biosensor production requires 24 h, a one-time cell capture optimization takes 6–9 h, and the final cell counting experiment in a laboratory environment requires 30 min to complete.

Separating beads and cells in multi-channel microfluidic devices using dielectrophoresis and laminar flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Electrical flow metering of blood for point-of-care diagnostics

We have developed a microfabricated chip that creates a purified white blood cell (WBC) population from whole blood samples and then electrically analyzes the WBCs at the same time as measuring sample volume flown. The flow metering is based on the measurement of the electrical admittance between microelectrodes inside a microfluidic channel. We found that the admittance related to the flow rate linearly. WBC counts which correlated with the flow rate shows how this technique is a viable method in metering and analyzing blood and other biological samples in a point-of-care environment.