The specific aims for this project period are to develop space technologies for (a) 5-part WBC (white blood cell) differential, (b) analysis of WBC subtypes (e.g., CD4+ T helper and natural killer cells). Our approach to achieve the goal is to develop the capability of minimally diluted micro flow cytometer to enable a comprehensive WBC differential, and allow detection of fluorescent labels attached to ligands used for cell surface marker for WBC subtype analysis. Embedded in the two specific aims is a research component on the data analysis software. This software has been developed in Matlab to facilitate both quantitative assessment of fluorescence detection and cell and analyte recognition and quantitation.

For the last funding years, we worked extensively on searching for a new staining method and optimizing the previously proposed Acridine Orange staining. We successfully developed a series of assays including a 4-part differential assay (i.e., Lymphocyte, Monocyte, Neutrophil, and Eosinophil) with a cocktail staining of fluorescent dyes fluorescein isothiocyanate (FITC) and propidium iodide (PI), a 5-part differential assay (i.e., Lymphocyte, Monocyte, Neutrophil, Eosinophil, and Basophil) with a cocktail staining of fluorescent dyes fluorescein isothiocyanate (FITC), propidium iodide (PI), and Basic Orange 21, and a specific assay for the rare cell type basophil differential using fluorescent dye Basic Orange 21. The differential assays were investigated in a correlation study with the commercial hematology analyzer, and further verified with the purified WBC types. For the Acridine Orange assay, the differential capability is also extended from 2-part (Lymphocyte and Neutrophil) into 4-part (Lymphocyte, Monocyte, Neutrophil, and Eosinophil). The time and temperature dependence of the Acridine Orange staining are also investigated.

Within the project period, we have also explored the possibility of improving the (box) platform in terms of spectroscopic detection. Two different approaches have been implemented; one uses a commercial mini-spectrometer and the other approach uses a 8-channel PMT (photomultiplier) module. Measurement of fluorescent emission spectrum from blood cells stained with the dye assay has been successfully demonstrated on the spectroscopic approach. Single cell fluorescence emission spectrum has been measured on the mini-spectrometer prototype. Distinct spectrums were measured from lymphocyte, neutrophil, and eosinophil cells. Besides, multicolor fluorescent beads have been successfully measured on the 8-color reader. Those two approaches enable detection of multiple fluorophore simultaneously from WBC subtype immune-staining. The additional spectral information should provide better discrimination between multiple fluorophores used simultaneously. It may also provide additional information about the intracellular environment in which Acridine Orange fluorescence occurs, leading to efficient WBC subtype discrimination. For WBC subtypes analysis, we have also successfully demonstrated assays that identified and counted CD4 and CD8 WBCs. In addition, we also developed synthesized peptides specifically targeted for leukocyte cells. The binding peptides were custom-synthesized with a fluorescein fluorophore attached to their n-terminus for binding quantification. A library of 72 potential peptide candidates has been tested using a modified protocol for leukocytes utilized in our studies. Among the results, 4 peptides from our initial library exhibited a 2-3x higher binding strength to the B-cells compared to the other peptides, which confirmed the capability of this approach for WBC subtype analysis.

2. Verification of the differential assays with purified WBC types. A procedure of preparing purified WBC individual types (Lymphocyte, Monocyte, Neutrophil, Eosinophil, or Basophil) has been developed. The differential capability of the 5-part assay (PI, FITC, BO21) and the Basophil specific assay (BO21) was verified with the purified WBC types. The staining pattern observed from the purified WBC types also provided a useful tool to study new assays.

3. Spectrum analysis capability. One unit of the prototype has been upgraded from two-color detection to spectrum analysis with a commercial mini-spectrometer. Fluorescence spectrum measurment of dye (Acridine Orange) stained white blood cells were successfully demonstrated on the microfluidic chip. Distinct spectrums were measured from the Lymphocyte, Neutrophil, and Eosinophil cells. In addition, the detection of lymphocyte subtype cells were also demonstrated with the spectrum measurement system, which paved the way for simultaneous measurement of multiple subtype cells.

4. Planning for the new generation cartridge. Components of the next generation cartridge were successfully demonstrated. In the current cartridge, manual handling was involved to process the blood sample before test, and an external pump and waste collection tube were need for the fluidic operation. In the next generation cartridge, the whole test will be integrated into a 1cm x 1cm x 3mm chip without external fluidic connection. We successfully demonstrated the on-chip staining of blood sample with fluorescent dyes on the microchip. Besides, basic components of on-chip pump, on-chip valve, and long term reagent storage capability were also demonstrated.

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 WBC count and 2-part differential with a portable prototype micro flowcytometer.

For the current project, a major effort is proposed to extend the 2-part WBC differential to a 5-part WBC differential, add cell surface marker detection and analysis capability to the platform repertoire, and add plasma protein detection and analysis capability to the platform repertoire. Our approach to achieve the objectives is to extend the capability of the micro flowcytometer to enable a more comprehensive WBC differential, and allow detection of fluorescent labels attached to ligands used for cell surface marker and plasma protein detection. The second component necessary for extending the platform capability is the offline data analysis software. This software is being developed in Matlab to facilitate both quantitative assessment of fluorescence detection and cell and analyte recognition and quantitation.

In the last funded year, we worked on searching for new staining method and optimizing the previously proposed Acridine Orange staining. We successfully developed a 4-part differential assay (i.e. Lymphocyte, Monocyte, Neutrophil and Eosinophil) with a cocktail staining of fluorescent dyes fluorescein isothiocyanate (FITC) and propidium iodide (PI). The differential assay is investigated in a correlation study with the commercial hematology analyzer, and further verified with the purified WBC types. For the Acridine Orange assay, the differential capability is also extended from 3-part (Lymphocyte, Monocyte and Neutrophil) into 4-part (Lymphocyte, Monocyte, Neutrophil and Eosinophil). The time and temperature dependence of the Acridine Orange staining are also investigated.

We also worked on improving the proposed platform on its detection end. Two different approaches including a commercial mini-spectrometer and a 8-color fluorescence reader with an 8-channel PMT module have been implemented. Single cell fluorescence emission spectrum has been measured on the mini-spectrometer prototype. The information about the subtle differences of the spectrum can be used to optimize the wavelength range of the multi-color measurement for capturing these differences. Multicolor fluorescent beads have been successfully measured on the 8-color reader. Those two approaches are investigated in order to extract as much useful information as possible from the emitted signal. The additional spectral information should provide better discrimination between multiple fluorophores used simultaneously. It may also provide additional information about the intracellular environment in which Acridine Orange fluorescence occurs, leading to efficient WBC subtype discrimination.

To analyze WBC subtypes, we initiated the development of synthesized peptides, which can be utilized to selectively target and bind to leukocytes. The binding peptides were custom synthesized with a fluorescein fluorophore attached to their n-terminus for binding quantification. A library of 72 potential peptide candidates has been tested using a modified protocol for leukocytes utilized in our studies. Several peptide binding assays has been performed to screen for potential peptides that will preferentially bind to leukocytes. Among the results, 4 peptides from our initial library exhibited a 2-3x higher binding strength to the B-cells compared to the other peptides, but showed minimal preference compared with the control group. We are in the process of running additional peptide binding assays using a new peptide library, against cultured cell lines (B- and T-cells).

For the coming year, we plan to work on the prototype with the spectrum analysis capability and the one with the 8-color reader for developing the staining assay. Ability to discriminate among multiple color emissions will provide the capability to detect multiple ligands simultaneously, and may help in performing a 5-part WBC differential with a single stain such as Acridine Orange. Further, we will continue to work on developing the staining assay for the test platform, expanding the differential capability of the staining assays to 5-part (i.e. Lymphocyte, Monocyte, Neutrophil, Eosinophil and Basophil). A staining assay with a dye cocktail of PI, FITC and BO21 is currently being investigated. For the WBC subtype differential with synthetic peptides, we are in the process of running additional peptide binding assays using a new peptide library. These assays will be run using cultured cell lines (B- and T-cells) and additional leukocyte subtypes will also be tested when promising peptide candidates are identified.

1. Blood staining and testing procedure optimization:

A 4-part WBC differential (Lymphocyte, Monocyte, Neutrophil and Eosinophil) assay using a staining cocktail of FTIC and PI has been developed. The differential capability has been investigated with a correlation study with a commercial hematology analyzer and further verified with purified individual WBC types. The differential capability of the previously proposed assay with metachromatic dye, Acridine Orange, was also expanded from 3-part differential (Lymphocyte, Monocyte and Neutrophil) to 4-part differential (Lymphocyte, Monocyte, Neutrophil and Eosinophil).

2. Purified WBC types preparation procedure for developing differential assays: A procedure of preparing purified WBC individual types (Lymphocyte, Monocyte, Neutrophil, Eosinophil or Basophil) has been developed. The purified WBC samples can be used to verify the differential capability of the proposed staining assays, and also provide new capability to identify new clusters in developing the 5-part differential assay.

3. Upgraded detection capability - spectrum analysis:

One unit of the prototype has been upgraded from two-color detection to spectrum analysis with a commercial mini-spectrometer. The spectrum analysis capability can be used to identify the subtle differences of the fluorescence spectrum of different WBC types, and then the wavelength range of the multi-color measurement can be optimized to capture those differences.

4. Upgraded detection capability - 8-color fluorescence emission reader:

Two units have been upgraded for 8-color fluorescence measurement reader using an 8-channel PMT module. One has been delivered to Caltech, and one remains in IRIS for additional tests and further development. Ability to discriminate among multiple color emissions will provide the capability to detect multiple ligands simultaneously, and may help in performing a 5-part WBC differential with a single stain such as Acridine Orange.

5. Synthetic peptides development for WBC type differential:

A library of 72 potential peptide candidates has been tested using a modified protocol for leukocytes utilized in our studies. Several modifications were made to this protocol to minimize cellular damage and optimize the effectiveness of the binding assays. Purified human B-cells and peripheral blood mononuclear cells (PBMCs) were purchased for peptide binding assay studies. Our results demonstrate that peptides have potential to be used as targeting molecules to capture leukocytes. We are in the process of running additional peptide binding assays using a new peptide library, which would be run using cultured cell lines (B- and T-cells).

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 WBC count and 2-part differential with a portable prototype micro flowcytometer.

For the current project, a major effort is proposed to extend the 2-part WBC differential to a 5-part WBC differential, add cell surface marker detection and analysis capability to the platform repertoire, and add plasma protein detection and analysis capability to the platform repertoire. Our approach to achieve the objectives is to extend the capability of the micro flowcytometer to enable a more comprehensive WBC differential, and allow detection of fluorescent labels attached to ligands used for cell surface marker and plasma protein detection. The second component necessary for extending the platform capability is the offline data analysis software. This software is being developed in Matlab to facilitate both quantitative assessment of fluorescence detection and cell and analyte recognition and quantitation.

In the last funded year, we successfully tested the proposed micro flowcytometer in a zero-G parabolic flight test in collaboration with research scientists from Wyle cooperation. The test demonstrated the facility of doing WBC differential count in zero/micro gravity environment with the proposed prototype. Results similar to on-ground test are 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 new staining method and optimizing the previously proposed Acridine Orange staining. We successfully demonstrated 4-part WBC differential count (i.e. Lymphocyte, Monocyte, Neutrophil and Eosinophil) with a two color laser-induced fluorescence (LIF) detection scheme. The dye combination FITC and PI stains WBC in blood with selective affinity and shows different fluorescence signatures for each of the 4 types. The differential capability of the platform is largely improved from 2-part differential (i.e. Lymphocyte, Non-Lymphocyte) to 4-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 2-part to 3-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 capable to detect fluorescence signals from the fluorophore after the conjugated antibody adhering to the cell surface, which provided a useful method of approaching out specific aim for WBC subtype analysis.

To analyze WBC subtypes, we proposed to use continuous flow separation at upstream and dielectric properties characterization at 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-dependant groups. Electrical Impedance Spectroscopy (EIS) is explored for dielectric properties characterization which employed a microelectrode array in combination of a novel cell patterning method for cell impedance measurements on the single-cell basis. Utilizing photolithographically patterned SAMs and stepwise protein immobilization enable 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 platform on its detection part. To expand the detecting spectrum range, we plan to explore of using mini spectrometer on the micro flowcytometer platform instead of the PMT detectors. Spectrum analysis could provide the potential of 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, integration of micromixer and deionier as well as DEP focusing devices and Coulter counters will be investigated. The fluorescent particle immunoassay (FPIA) will be investigated for on-chip plasma protein detection.

1. Blood staining and testing procedure optimization: We demonstrated 4-part WBC differential count including Lymphocyte, Monocyte, Neutrophil and Eosinophil, with a combination staining of FTIC and PI. We also demonstrated 3-part WBC differential count by an optimized Acridine Orange staining.

2. Fluorophore conjugated antibody staining of whole blood: As an alternative to chemical staining, we demonstrated WBC differential and WBC subtype counting with fluorophore conjugated antibody. By using a laser module for excitation, the sensitivity of the prototype is largely improved to detect signals from fluorophore conjugated antibody. Monocyte count and WBC subtype count such as CD4+ have been demonstrated on the proposed micro flowcytometer.

3. Zero-G parabolic flight test: We tested the portable micro flowcytometer prototype on a zero-G parabolic flight test, which is important to validate using the prototype in a zero/micro gravity environment. WBC differential similar to on ground test has been achieved. Experiences from the flight test are being used for improvement of the prototype, such as reinforcing the mechanical structure and increasing the pumping efficiency under low environmental pressure.

4. Hardware improvement: The excitation source of the prototype has been upgraded to a blue laser from a LED. The laser excitation provides stronger fluorescence intensity for detection. Now we are working on improving the detection component. Analysis of fluorescence spectrum would be explored with a mini-spectrometer detector.

5. Optimization of hydrodynamic separator for WBC subtype separation: The geometry of the sorting region has been further optimized to improve sorting efficiency and enhance continuous operation. Computational simulations were utilized to improve the design of various components including the hydrodynamic focusing region, the sorting region and the collection bins. Experiments have been performed to successfully separate particles and embryoid bodies into size-dependant groups.

6. WBC subtype analysis with EIS: Characterization of single-cell dielectric properties using EIS requires immobilization of the target cells onto detection electrodes with accurate position control at the single-cell level. We have refined our previously developed cell patterning technique through optimizing the fabrication process to achieve high selectivity protein patterns enabling for the precise formation of single-cell arrays. We have also improved the detection sensitivity by increasing the effective electrode surface area through a polypyrrole (PPy)-electrode coating and by using a low conductivity cell suspension buffer.

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 WBC count and 2-part differential with a portable prototype micro flowcytometer. For the current project, a major effort is proposed to extend the 2-part WBC differential to a 5-part WBC differential, add cell surface marker detection and analysis capability to the platform repertoire, and add plasma protein detection and analysis capability to the platform repertoire. Our approach to achieve the objectives is to extend the capability of the micro flowcytometer to enable a more comprehensive WBC differential, and allow detection of fluorescent labels attached to ligands used for cell surface marker and plasma protein detection. The second component necessary for extending the platform capability is the offline data analysis software. This software is being developed in Matlab to facilitate both quantitative assessment of fluorescence detection and cell and analyte recognition and quantitation. A total of four demonstration units will be available at the end of the first funding year. Two units are complete and operational. The remaining two units are awaiting power supply components that are scheduled to arrive shortly. Of the completed units one is in use at Caltech, and the second is in use at IRIS International. It will serve as a baseline comparison for the modifications planned to the current 2-color design. Both the excitation and detection capability will be enhanced for the modified units. At the excitation end we will be investigating more powerful sources to provide increased emission intensity. At the detection end we will be investigating methods of increasing the number of colors that can be detected simultaneously. Ability to discriminate among multiple color emissions, even those that do not differ significantly in wavelength will provide the capability to detect multiple ligands simultaneously, and may help in performing a 5-part WBC differential with a single stain such as acridine orange.

For the demonstration unit at Caltech, we are working with scientists from Wyle and NASA Johnson Space Center to test this prototype on one of their zero-G flights. We have been investigating the operation of the unit to suit the zero-G flight environment on ground, including mechanical robustness of the system, electrical safety of the system, long-term reliability of the testing, the actual testing protocol, and the data interpretation. We have also been providing instrument documentation, safety documentation, and experimental design to enable the proposed zero-G flight testing. We believe successful completion of the zero-G flight testing will be a major milestone for this project.

To analyze WBC subtypes, we propose to use continuous flow separation at upstream and dielectrophoretic (DEP) enabled Coulter counting at downstream. For the flow separation part, we improved from previous design and achieved a very compact design capable of continuous cell separation. Pillars are placed within the microchannel to alter the fluid flow pathway, allowing particles of a certain size to be diverted toward a specified route. In order to accurately calibrate the dielectric parameters (i.e. membrane capacitance, cytoplasm resistance) of different types of white blood cells, a microelectrode-array (MA) was designed and fabricated to simultaneously carry out the impedance measurement for a large amount of cells under a wide range of frequency. The challenge of this task is to immobilize single cells onto this MA with precise position controllability. During the last year, we developed a novel cell immobilization method to accurately control the white-blood cells adhesive and repellent molecules functionality with high spatial resolution.

Photolithographically patterned hexamethyldisilazane (HMDS) micron-sized patterns present hydrophobic terminal that were used to physically adsorb the cell capturing antibodies. The non-specific antibody binding was prevented by passivating the other surface without HMDS micropatterns by poly(ethylene glycol) (PEG). Specific biotin-streptavidin complexation was explored to immobilized cell-specific antibodies. High patterning selectivity was achieved and the immobilized antibodies retained their bioactivities to a great extent. By controlling the size of the antibody micropatches, single-cell patterning resolution was achieved using cultured DG75 B lymphocytes as model cells. We believe that using microfluidic networks to accurately control the shear stress imparted on the immobilized cells can further improve the patterning qualities.

For the coming year, with the improvement of excitation with laser diode and detection with PMT array on the micro flowcytometer platform, we will explore WBC counting and differential with fluorophore conjugated antibodies. This can greatly expand the capability of the platform. Also a cocktail of fluorescent dyes including acridine orange will be investigated to stain blood for five part WBC differential. For WBC subtype separation and counting, with the successful optimization of the continuous flow separation device, integration of micromixer and deionier as well as DEP focusing devices and Coulter counters will be investigated. The fluorescent particle immunoassay (FPIA) will be investigated for on-chip plasma protein detection.

1. Acridine orange staining and testing procedure optimization: We are in the process of improving the previous two color micro flowcytometer. Excitation with laser diode and detection with diffraction grating and multi-channel PMT will improve the system sensitivity and capability. We also made progress in system software to enable more comprehensive and accurate data processing.

2. Fluorophore conjugated antibody staining of whole blood: As an alternative to chemical staining, we are investigating using fluorophore conjugated antibody to staining whole blood. Antibody staining has been proposed recently as a potential new standard way for five part differential. Antibody staining followed by micro flow cytometer detection might provide a more accurate and specific way to count some subtypes of WBCs.

3. Preparation for zero-G flight test: As a crucial step toward in space use, we are preparing the portable micro flowcytometer prototype for zero-G flight test. We are in the process of reinforcing the mechanical structure, performing the reliability test, and customizing the system to fit the zero-G flight test scenario. We plan to run the testing continuously for the whole duration of the flight and record data for both low-G and high-G periods. The data processing will be performed after the flight. Intervention and troubleshooting by crew members are not necessary.

4. Optimization of hydrodynamic separator for WBC subtype separation: Based on our previous devices for continuous size based particle separation in microfluidic devices, a new design offers two orders of magnitude reduction in the separation region, while still achieving the same functional purposes for particle separation.

5. Plasma preparation for plasma biomarker detection: The same continuous size based particle separation device can be used to separate plasma from whole blood as demonstrate in the previous funding period.

Blood analysis, if possible, should be the first important step of health monitoring for sick and healthy astronauts. Blood analysis can also be a powerful technique to monitor bone loss and radiation effects. Therefore, NASA should have an in-space, real-time blood analysis capability. However, NASA still does not have blood analysis capability other than blood gas and electrolyte analysis. This project is specifically intended to develop an in-box blood analysis technology for NASA. As a whole, we believe that the lab-on-a-chip technology is the best choice for multiple blood analyses in space. Therefore, our long-term objective is to develop blood analysis in-a-box using lab-on-a-chip technology specifically for space applications, emphasizing small form factor, lightweight and autonomous operation to accommodate the Crew Exploration Vehicle (CEV) and International Space Station (ISS) size requirements for medical kits.

More specifically, this project is to extend our current platform for both blood cell and molecular biomarker analysis using <100nL blood. The specific aims include:

1. 5-part WBC differential;

2. Analysis of WBC subtypes (e.g., CD4+ T helper and killer cells); and

3. Serum/plasma protein biomarker analysis.

All three aims will use the same on-chip blood sample preparation, which allows the cell/plasma separation and RBC/WBC separation. The 5-part WBC differential will use fluorescence labeling to differentiate cell types. The WBC subtype differential will utilize cell specific surface antigen to antibody binding reaction for separation. The protein biomarker will adopt fluorescent particle immunoassay. This projects success can benefit many other areas such as smart medical systems because blood analysis gives the most basic information of an astronauts response to countermeasures. The proposed lab-on-chip technology is independent of gravity and radiation.