Isolation, Expansion and Characterization of Circulating Tumor Cells using Microfluidic Technologies.

Circulating tumor cells (CTCs) are the tumor cells shed from primary tumor, which enter the bloodstream and travel to distant anatomic sites to form metastasis. Detecting CTCs, as a means of “liquid biopsy”, allows monitoring cancer progression in real time and predicting therapeutic response. The major challenge that limits the clinical utility of CTCs, especially in early stage cancer patients, is their rarity; specifically, there are only 1-10 CTCs in one milliliter of whole blood. One way to overcome this critical limitation is to ex vivo expand CTCs through culturing. In this thesis, a microfluidic CTC capture device is optimized and tested for the capture and analysis of CTCs. Using a 3D, on chip co-culture model, CTCs were successfully expanded in 70% of the 50 early stage lung and esophageal cancer patients. Cultured CTCs were characterized with immunostaining, RNA profiling, mutation analysis and invasion assays. We found concordant TP53 mutation in CTCs and matched primary tumors. Next-generation sequencing further revealed mutations in additional genes. It was found that, patients whose CTCs exhibited the greatest capacity to expand had earlier recurrence. In one of the patient samples, expanded CTCs implanted in mice resulted in a xenograft tumor, demonstrating the expanded CTCs’ ability to metastasize. Genes related to EMT and tumor microenvironment were enriched in the xenograft. In addition, building upon this co-culture model, CTCs from one of the ALK positive metastatic lung cancer patients were isolated and cultured. The cultured CTCs harbored the concordant EML4-ALK rearrangement as the tumor biopsy specimen and further served as an in vitro model for drug testing. Taken together, this study demonstrated that CTCs from early stage lung cancer are tumorigenic and mirror the phenotypic and genotypic status of primary tumors. Ex vivo culturing of CTCs will make a significant impact in the era of personalized medicine. It will bring about opportunities for individualized drug screening, such as predicting treatment response to targeted therapies and the emergence of acquired drug resistance. Cultured CTCs will also serve as tools for understanding metastatic spread of cancer cells.PhDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120755/1/zzhuo_1.pd

Functional Materials for Electrochemiluminescence Biosensors and Imaging

This doctoral thesis focuses on the investigation of innovative functional materials for the development of sensing applications, which use electrochemiluminescence (ECL) technology as transduction technique. The demand for highly sensitive, selective and reliable sensors able to perform fast, economic and easy detection in clinical and analytical chemistry increases constantly. Among the electrochemical techniques, ECL is the most suitable transduction method for sensing applications. In fact, due to the electrochemical nature of the signal generation, extremely high range of sensitivity and very low detection limits can be reached. Moreover, the generation of the signal is achieved directly in situ, which enables spatial visualization and mapping of the signal distribution. This property of the ECL can be exploited for the imaging of small objects, such as micro-particles or cells, deposited directly on the surface of the electrode. In addition, efforts have been made to understand the complex interplay of the chemical and electrochemical reactions responsible of the signal generation at the electrode surface. The collected information might help towards the improvement of the technology at the basis of the ECL sensors, in order to obtain higher sensitivity and selectivity. The functional materials studied in this thesis present different electrochemical characteristics and are expressly designed for sensor applications. In particular, the proper selection of the material and its optimization assure high performances of the device. For example, the combination of supramolecular chemistry with the ECL technology can be exploited for the development of a sensor for the early diagnosis of prostate cancer, or the excellent electrochemical properties of carbon-based materials can be used for the imaging of biological samples with high spatial resolution

Study of Circulating Tumor Cells using Microfluidic Technology: From Isolation to Analysis

An intimidating aspect of cancer is its ability to spread out to distant organs causing 90% of cancer-associated deaths. This metastatic progression is driven by circulating tumor cells (CTCs) shed from the primary tumor into bloodstream of carcinoma patients. As a result, CTCs hold great promise as a potential biomarker in areas of cancer diagnosis, monitoring, and evaluation of therapeutic efficacy for personalized medicine, which can serve as surrogate for invasive tissue biopsy. However, theses cells are extremely rare with a frequency of only 1-10 cells surrounded by billions of normal blood cells in 1mL of blood. This thesis delineates the shortcomings of existing CTC isolation methods followed by development and implementation of new microfluidic-based platforms to improve the sensitivity, specificity, and throughput for CTC enrichment. First, an affinity-based CTC isolation chip is introduced incorporating functional graphene oxide for high-density tumor specific antibody presentation. The two-dimensional surface-capture approach shows an overall CTC capture efficiency of >82.3% for flow rates up to 3mL/hr, while maintaining high viability (>90%) from low shear stress generated during sample processing. The extremely low blood cell contamination rate in the order of 100 cells/mL enables subsequent downstream analysis of CTCs. The clinical validity of the chip is demonstrated in a cohort of 47 metastatic breast cancer patients. Second, a size based CTC isolation chip is presented utilizing the inertial force effects to isolate CTCs by differentially focusing. Channel design parameters including the height, width, and radius of curvature and flow conditions are investigated to observe their effect on particle/cell focusing and streak migration. Optimal flow regimes to achieve maximum separation of 10/20 μm particles, representing leukocytes and CTCs respectively, in various channel configurations are identified. Based on these results, a cascaded spiral chip is designed for label-free CTC isolation achieving 87.76% recovery rate with 97.91% leukocyte depletion. Finally, a catheter based in-vivo CTC isolation system is implemented for large blood volume CTC screening. The system includes a dual lumen catheter to connect the patient blood veins, a peristaltic pump for continuous blood sampling, heparin injector to prevent blood clogging and clotting, and a CTC capture module.PHDElectrical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138501/1/tztaebo_1.pd

Fabrication of 3D hydrogel-based microscale tissue analog chip with integrated optofluidics

Lab-on-a-chip (LOC) is a device that integrates one or more laboratory functions in a single chip with dimensions ranging from a micrometer to a few millimeters. On-chip optofluidics, which combines microfluidics and tunable micro-optical components, is crucial for bio-sensing applications. However, recently reported optofluidic devices have only two-dimensional (2D) dielectric or metallic regions for sensing cellular activity, which fail to mimic the three-dimensional (3D) in vivo microenvironment of cells. In this research, a 3D hydrogel-based micro-scale-tissue-analog-chip (µTAC) is fabricated with an integrated optofluidic design for biomedical applications. These 3D hydrogels act as a scaffold for the cellular studies and as a waveguide for increasing the signal efficiency in sensing applications. These 3D waveguides, embedded in a Poly(dimethylsiloxane) elastomer-based optofluidic channel, are composed of Poly(ethylene glycol)-diacrylates (PEGDA). The refractive index of the PEGDA waveguides is higher compared to the water-based cladding that surrounds the waveguide. Because of this refractive index difference, waveguides confine the light waves due to the total-internal-reflection phenomenon (TIR). Initially, the characterizations and the sensing efficiency of the µTAC device are successfully demonstrated with a fluorescein detection study. This study demonstrates that the proposed device is in accordance with Beer-Lambert’s law with a limit of detection of 2.54 µM of fluorescein. Further, the sensing efficiency of the µTAC devices is tested in cellular studies by encapsulating cells inside the waveguides. Cellular studies with µTAC devices prove that the device is capable of efficiently sensing the cell density and the cell viability changes inside the waveguides with a limit of detection of ~27 cells/waveguide. In addition, this study also proves that the proposed µTAC device has a potential for long-term cell monitoring applications without compromising cell-viability. Therefore, with integrated 3D hydrogel waveguides, this µTAC-optofluidic device could be a potential platform with a broad range of applications in the fields of diagnosis and detection

Polymer Micro- and Nanofluidic Systems for In Vitro Diagnostics: Analyzing Single Cells and Molecules

Polymer micro- and nanofluidic systems, with their critical dimensions, offer a potential to outperform conventional analysis techniques and diagnostic methods by enhancing speed, accuracy, sensitivity and specificity. In this work, applications of microfluidics have been demonstrated to address the existing challenges in stroke diagnosis, by mRNA expression profiling from whole blood within \u3c20 min. A brief overview of various biomarkers for stroke diagnosis is given in chapter 1 followed by design and testing of individual microfluidic modules (chapter 2 and 3) required for the development of POC diagnostic strategy for stroke. We have designed and evaluated the performance of polymer microfluidic devices for the isolation of leukocyte subsets, known for their differential gene expression in the event of stroke. Target cells (T-cells and neutrophils) were selected from with greater purities, from 50 µl whole human blood by using affinity based capture in COC devices within a 6.6 min processing time. In addition, we have also demonstrated the ability to isolate and purify total RNA by using UV activated polycarbonate solid phase extraction platform. Polymer-based nanofluidic devices were used to study the effects of surface charge on the electrodynamic transport dynamics of target molecules. In this work, we report the fabrication of mixed-scale micro- and nanofluidic networks in poly(methylmethacrylate), PMMA, using thermal nanoimprint lithography using a resin stamp and surface modification of polymer nanoslits and nanochannels for the assessment of the associated electrokinetic parameters – surface charge density, zeta potential and electroosmotic flow. This study provided information on possible routes that can be adopted to engineer proper wall chemistry of polymer nanochannels for the enhancement or reduction of solute/wall interactions in a variety of relevant single-molecule studies