This project is a continuation of a related project entitled Handheld Body-Fluid Analysis System for Astronaut Health Monitoring, in which we explored electrical impedance sensing, fluorescence optical sensing and flow separation of blood cells in microfluidic devices and portable platforms. We successfully demonstrated fluorescent sensing and counting for white blood cell (WBC) count and two-part differential with a portable prototype micro flow cytometer.

For the current project, a major effort is to extend the two-part WBC differential to a five-part WBC differential, add cell surface marker detection and analysis capability to the platform repertoire, as well as add plasma protein detection and analysis capability to the platform repertoire.

Specific Aims

1. Five-part WBC differential.
2. Analysis of WBC subtypes (e.g., CD4+ T helper and natural killer cells).
3. Serum/plasma protein biomarker analysis (e.g., for infection, radiation and bone loss monitoring).

In the last year, we successfully tested the proposed micro flow cytometer in a microgravity parabolic flight test in collaboration with research scientists from Wyle. The test demonstrated the facility of doing WBC differential count in a microgravity environment with the proposed prototype. Results similar to on-ground tests were obtained. The prototype was shown to be convenient for operation. One flight crew learned to operate the prototype and carried out the test after a brief training.

For improving the differential capability of the prototype, we worked on searching for a new staining method and optimizing the previously proposed acridine orange staining. We successfully demonstrated four-part WBC differential count (i.e. lymphocyte, monocyte, neutrophil and eosinophil) with a two-color laser-induced fluorescence (LIF) detection scheme. The dye combination fluorescein isothiocyanate and propidium iodide stains WBC in blood with selective affinity and shows different fluorescence signatures for each of the four types. The differential capability of the platform is largely improved from two-part differential (i.e., lymphocyte, non-lymphocyte) to four-part differential (i.e., lymphocyte, monocyte, neutrophil and eosinophil). The previously proposed fast staining with acridine orange was also optimized so that the differential capability was expanded from two-part to three-part (lymphocyte, monocyte and neutrophil).

We also worked on improving the proposed platform on its excitation source. By replacing the LED with a laser excitation, the induced fluorescence intensity is largely enhanced. With the improved prototype, WBC subtype counting, such as CD4+ T cells with fluorophore conjugated antibody staining whole blood was also demonstrated. The sensitivity of the improved prototype was able to detect fluorescence signals from the fluorophore after the conjugated antibody adhering to the cell surface, which provided a useful method of approaching our specific aim for WBC subtype analysis.

To analyze WBC subtypes, we proposed to use continuous flow separation upstream and dielectric properties characterization downstream. Our previous design had achieved a very compact design capable of continuous cell separation. The geometry of the sorting region has been further optimized to improve sorting efficiency and enhance continuous operation. Experiments have been performed to successfully separate particles and embryoid bodies into size-dependent groups. Electrical impedance spectroscopy (EIS) was explored for dielectric properties characterization, which employed a microelectrode array in combination with a novel cell-patterning method for cell impedance measurements on the single-cell basis. Utilizing photolithographically patterned self-assembled monolayers and stepwise protein immobilization enabled the precise formation of single-cell arrays. Target cells are immobilized onto detection electrodes, and their impedance spectra are measured to discriminate different WBC subtypes.

For the coming year, we plan to work on improving the platforms detection capabilities. To expand the detecting spectrum range, we plan to explore using a mini spectrometer on the micro flow cytometer platform instead of the photomultiplier tube detectors. Spectrum analysis could provide the potential for analyzing multi-color fluorescence in a compact size prototype. Further, we will explore WBC counting and differential with fluorophore conjugated antibodies. A cocktail of fluorescent dyes, including acridine orange, will be investigated to stain blood for five-part WBC differential, also for WBC subtype counting. For WBC subtype separation and counting, with the successful optimization of the continuous flow separation device, the integration of a micromixer, deionier as well as dielectrophoretic focusing devices and Coulter counters will be investigated. The fluorescent particle immunoassay (FPIA) will be investigated for on-chip plasma protein detection.

Earth-based Applications of Research Project
The devices under development can be used for Earth-based applications. The proposed hand-held device uses a cartridge. The cartridge will be cheap and disposable. The results will be available almost immediately to patients without going through central lab facilities. The device can be used in emergency rooms, on ambulances as well as at home. As the senior population continuous to grow, this kind of device will become more and more appealing in point-of-care applications.

Project Description
NASA Task Book Entry