Xuanhong Cheng

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.

Enhancing the performance of a point-of-care CD4+ T-cell counting microchip through monocyte depletion for HIV/AIDS diagnostics

CD4+ T cell counts are important tests used to stage HIV-positive patients, enabling clinicians to make informed antiretroviral treatment decisions and to monitor the therapeutic outcomes. However, state-of-the-art CD4 counting methods based on flow cytometry are not applicable in resource-limited settings, due to their high cost and technical requirements. In previous work, we reported the development of a cell isolation microchip that can be used at the point of care for CD4 counts. In that microfluidic chip, CD4+ T cells were separated from 10 microL of whole blood, and enumerated via either light microscopy or impedance sensing. The microchip counts matched flow cytometry results in the intermediate CD4 count range, between 200-800 cells/microL, but displayed a positive bias at absolute CD4 counts below 200 cells/microL, due largely to monocyte contamination. To enhance the performance in the low CD4 count range, we report here an improved design of a two-stage microfluidic device to deplete monocytes from whole blood, followed by CD4+ T cell capture. Using the double-stage device combined with a high viscosity rinsing solution, we obtained microchip CD4 counts comparable to flow cytometry results in the full clinically relevant range. In addition to CD4 counting, the strategy of contaminant depletion prior to target cell isolation can be easily adapted to immunoaffinity capture of other cell types that lack a unique surface marker from a complex biological fluid.

Emerging technologies for point-of-care CD4 T-lymphocyte counting

A CD4 T-lymphocyte count determines eligibility for antiretroviral therapy (ART) in patients recently diagnosed with HIV and also monitors the efficacy of ART treatment thereafter. ART slows the progression of HIV to AIDS. In the developing world, CD4 tests are often performed in centralized laboratories, typically in urban areas. The expansion of ART programs into rural areas has created a need for rapid CD4 counting because logistical barriers can delay the timely dissemination of test results and affect patient care through delay in intervention or loss of follow-up care. CD4 measurement at the point-of-care (POC) in rural areas could help the facilitation of ART and monitoring of treatment. This review highlights recent technology developments with applications towards determining CD4 counts at the POC.

Plasmonic Mach-Zehnder interferometer for ultrasensitive on-chip biosensing.

We experimentally demonstrate a plasmonic Mach-Zehnder interferometer (MZI) integrated with a microfluidic chip for ultrasensitive optical biosensing. The MZI is formed by patterning two parallel nanoslits in a thin metal film, and the sensor monitors the phase difference, induced by surface biomolecular adsorptions, between surface plasmon waves propagating on top and bottom surfaces of the metal film. The combination of a nanoplasmonic architecture and sensitive interferometric techniques in this compact sensing platform yields enhanced refractive index sensitivities greater than 3500 nm/RIU and record high sensing figures of merit exceeding 200 in the visible region, greatly surpassing those of previous plasmonic sensors and still hold potential for further improvement through optimization of the device structure. We demonstrate real-time, label-free, quantitative monitoring of streptavidin-biotin specific binding with high signal-to-noise ratio in this simple, ultrasensitive, and miniaturized plasmonic biosensor.

A microchip approach for practical label-free CD4+ T-cell counting of HIV-infected subjects in resource-poor settings.

Simple affordable CD4 cell counting is urgently needed to stage and monitor HIV-infected patients in resource-limited settings. To address the limitations of current approaches, we designed a simple, label-free, and cost-effective CD4 cell counting device using microfluidic technology. We previously described the fabrication of a microfluidic system for high-efficiency isolation of pure populations of CD4+ T cells based on cell affinity chromatography operated under controlled flow. Here, we compare the performance of a microfluidic CD4 cell counting device against standard flow cytometry in 49 HIV-positive subjects over a wide range of absolute CD4 cell counts. We observed a close correlation between CD4 cell counts from the microchip device and measurements by flow cytometry, using unprocessed whole blood from HIV-positive adult subjects. Sensitivities for distinguishing clinically relevant thresholds of 200, 350, and 500 cells/μL are 0.86, 0.90, and 0.97, respectively. Specificity is 0.94 or higher at all thresholds. This device can serve as a functional cartridge for fast, accurate, affordable, and simple CD4 cell counting in resource-limited settings.

Temperature dependent activity and structure of adsorbed proteins on plasma polymerized N-isopropyl acrylamide.

Thorough studies of protein interactions with stimulus responsive polymers are necessary to provide a better understanding of their applications in biosensors and biomaterials. In this study, protein behavior on a thermoresponsive polymer surface, plasma polymerized N-isopropyl acrylamide (ppNIPAM), is investigated using multiple characterization techniques above and below its lower critical solution temperature (LCST). Protein adsorption and binding affinity are probed using radiolabeled proteins. Protein activity is estimated by measuring the immunological activity of an antibody adsorbed onto ppNIPAM using surface plasmon resonance. Conformation/orientation of the proteins is probed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and principal component analysis (PCA) of the TOF-SIMS data. In this work, we find that at low protein solution concentrations, ppNIPAM-treated surfaces are low fouling below the LCST, but protein retentive above it. The protein adsorption isotherms demonstrate that apparent affinity between soluble protein molecules and the ppNIPAM surface are an order of magnitude lower at room temperature than at 37 °C. Although direct protein desorption is not observed in our study when the surface temperature drops below the LCST, the binding affinity of surface adsorbed protein with ppNIPAM is reduced, as judged by a detergent elution test. Furthermore, we demonstrated that proteins adsorbed onto ppNIPAM are functionally active, but the activity is better preserved at room temperature than 37 °C. The temperature dependent difference in protein activity as well as TOF-SIMS and PCA study suggest that proteins take different conformations/orientations after adsorption on ppNIPAM above and below the LCST.

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.

Micro- and nanotechnology for viral detection

Since the identification of viruses at the start of the 20th century, detecting their presence has presented great challenges. In the past two decades, there has been significant progress in viral detection methods for clinical diagnosis and environmental monitoring. The earliest advances were in molecular biology and imaging techniques. Advances in microfabrication and nanotechnology have now begun to play an important role in viral detection, and improving the detection limit, operational simplicity, and cost-effectiveness of viral diagnostics. Here we provide an overview of recent advances, focusing especially on advances in simple, device-based approaches for viral detection.

Broadband Electrical Detection of Individual Biological Cells

To resolve the dilemma of cell clogging or solution parasitics encountered by Coulter counters and to evolve a general-purpose electrical detection technique, we used broadband microwave measurements to overcome electrode polarization, ac dielectrophoresis to precisely place cells between narrowly spaced electrodes, and relatively wide microfluidic channels to prevent cell clogging. This unique combination of approaches resulted in reproducible sensing of single Jurkat and HEK cells, both live and dead, of different cultures at different times.

Plasmonic interferometers for label-free multiplexed sensing

We report a plasmonic interferometric biosensor based on a simple slit-groove metallic nanostructure that monitors the phase changes of surface plasmon polaritons resulting from biomolecular adsorptions. The proposed sensing scheme integrates the strengths of miniaturized plasmonic architectures with sensitive optical interferometry techniques. Sensing peak linewidths as narrow as 7 nm and refractive index resolutions of 1 × 10(-5) RIU were experimentally measured from a miniaturized sensing area of 10 × 30 µm(2) using a collinear transmission setup and a low-cost compact spectrometer. A high-density array of such interferometric sensors was also fabricated to demonstrate its potential for real-time multiplexed sensing using a CCD camera for intensity interrogation. A self-referencing method was introduced to increase the sensitivity and reduce sensor noise for multiplexing measurements. The enhanced sensing performance, small sensor footprint, and simple instrumentation and optical alignment suggest promise to integrate this platform into low-cost label-free biosensing devices with high multiplexing capabilities.