Siyang Zheng, Ph.D. - US grants

The objective of this program is to develop a new type of optoelectronic nano-hand by integrating fiber optics, electronics, and NEMS/MEMS (nano- or micro- electromechanical systems) in a unified platform. The PIs plan to carry out four core research tasks. First, they will demonstrate a prototype optoelectronic nanohand. Second, they will investigate its optical waveguiding properties. Third, they will investigate optical force exerted by laser beam delivered by the nanohand. Finally, they will investigate in situ electrical and optical characterization, and explore the application of the nanohand.

The intellectual merit is the demonstration and development of new capability to manipulate, control, and characterize individual nanoscale objects. The proposed nanohand has several unique features. It has a large aspect ratio and is free of substrate support. The nanohand has multiple independently controlled nano-actuators or fingers. An object can also be potentially controlled by auxiliary optical force. Finally, electric measurements and optical characterization can be performed to probe a single clenched nanoscale object in situ.

The broader impacts are as follows. The proposed nanohand represents a new class of multi-functional nano-manipulators and can have significant impact on several areas including NEMS/MEMS, optics, and bioengineering. It can open new possibilities for realizing nano-opto-electro-mechanical and nano-robotic systems, for assembling complex three-dimensional nanostructures, and for probing the properties of nanoscale systems and devices at a single nanoparticle or single nanowire level. The multi-disciplinary nature of the proposed research also makes this program an excellent opportunity to educate and train students.

DESCRIPTION (provided by applicant): Development of a flexible micro spring array device for viable circulating tumor cell enrichment, analysis, primary culture and ex vivo therapeutic response test Metastatic cancer accounts for over 90% of cancer related deaths. Cancer metastasis is the process by which malignant circulating tumor cells (CTCs) detach from a primary tumor, spread through peripheral blood, then invade and proliferate in distant organs. The fundamental challenge of detecting CTCs in blood samples is the fact that they are so rare, requiring detection sensitivity of only a few tumors cells in billions of blood cells. Since the majority of solid tumor cells are of epithelial origin, and are almost always significantly larger and more rigid than normal blood cells, size based separation has been demonstrated as an effective method for CTC capture. The first goal of this project is to characterize and optimize a new flexible micro spring array (FMSA) device for the enrichment of CTCs from peripheral blood samples. The novel spring structures incorporated into the device design minimize concentrated stresses experienced by the CTCs and encourage their health, survival and proliferation. We have optimized the gap size of the device to reliably achieve over 85% recovery, 104 enrichment against leukocytes, 80% viability, and throughput of 5mL fresh blood in EDTA tube in 5 minutes. This technology is portable, cheap, fast, and more effective than CellSearchTM, which is the only current FDA approved system for CTC enumeration in a limited variety of cancers. In our preliminary study, we also found the driving differential pressure is critical for ell viability and proliferability. The safe operation pressure is orders of magnitude lower than the pressure normally used for conventional cell filtration. Thus our second goal is to develop a MEMS enabled automatic low pressure control system to facilitate the FMSA device. Together with the FMSA device, the whole system can solve the over 40 years' problem that widely used conventional filtration can't enrich viable mammalian cells because of cell damage and device clogging. More importantly, the FMSA system is capable of the capture of viable tumor cells that are available for primary culture and ex vivo therapeutic drug efficacy test, which is the third goal of the proposed study. The successful capture of viable CTCs will prompt the establishment of systems and protocols for their growth into a primary culture. This will provide the basis for testing the tumor cells' response to a variety of anticancer drugs ex vivo. If proven to be effective, a more specific and personalized treatment plan may be developed without having to unnecessarily expose a patient to the cost and toxic effects of chemotherapy. With further optimization for device flexibility and primary cell culture, and establishment of an automated low pressure control prototype, the FMSA enrichment system can be readily adapted for clinical usage. A pilot study of clinical samples obtained from our clinical collaborators will evaluate CTC detection, primary culture, and ex vivo drug efficacy tests with the FMSA system. PUBLIC HEALTH RELEVANCE: The most deadly forms of cancer can release aggressive cells that travel through the bloodstream and spread throughout the body. This project will explore the use of a new microfabricated device to capture these invading cells and isolate them from a patient blood sample. The information obtained by growing and analyzing these cells can be used for improved detection, diagnosis, and development of personalized treatment plans.

DESCRIPTION (Provided by the applicant) Abstract: The ability of metastatic cancer to release circulating tumor cells (CTCs) that travel through the blood and invade different parts of the body accounts for over 90% of cancer related deaths. New techniques for improved diagnosis and therapeutic strategies are desperately needed to improve patient outcomes in late stage metastatic cancer patients. One such technique involves the analysis of these CTCs through isolation from patient blood samples. Since molecular profiles of CTCs can be quite different from those of the primary tumor and more similar to the metastatic tumors, CTCs are better suited for metastatic cancer prognosis and diagnosis. Current CTC technologies have serious difficulties and limitations for clinic applications due to poor sensitivity and selectivity, high cost, and long processing times. CellSearchTM, the only current FDA approved system for CTC analysis, is only used for detection of CTCs and is not capable of preserving viable cells. The fundamental challenge with obtaining CTCs from blood samples is the fact that they are so rare, with only a few tumors cells occurring among billions of blood cells. Since tumor cells are almost always significantly larger than normal blood cells, size based separation has been demonstrated as an effective method for CTC capture. We have taken a novel approach to established microfiltration technology by implementing an array of flexible microsprings and using a regulated low pressure flow system to minimize the mechanical stresses experienced by cells during the filtration process. This is an efficient and cost effective system that is capable of the enrichment of viable CTCs from a clinically relevant blood volume of 7.5 mL in only 10 minutes. Despite achieving greater than 104 enrichment against leukocytes, the purity of the enriched cells is not sufficient for obtaining clinically relevant genetic information. We propose the incorporation of a high throughput microfluidic system that will physically partition these cells for analysis on a single cell level.By exploiting this inherent advantage of microtechnology, a large volume of parallel reactions can be used to overcome the issues with purity. Genetic expression profiles and mutation detections may be used for improved diagnosis, and lead to the development of highly personalized therapy plans that are optimized for each patient. Furthermore, the use of microfluidics for multiwell partitioning of CTCs will be used to attempt the establishment of favorable conditions for the culture of CTCs, even at an initially low seeding number. Successful primary culture of CTCs will allow drug efficacy tests that may be used to assay potential drugs ex vivo without exposing a patient to the unnecessary cost or toxic effects of chemotherapy. These new approaches based on the analysis of viable CTCs represent a different approach that has not been proven. However, since this technological platform is applicable to almost every type of cancer, it could fairly quickly revolutionize the way that therapies are derived for metastatic cancer patients. Public Health Relevance: The most deadly forms of cancer can release aggressive cells that circulate through the bloodstream and spread throughout the body. Microfabrication technology has been developed to effectively isolate these cells from a patient blood sample, allowing a minimally invasive liquid biopsy that may be performed often for monitoring tumor progression. This project explores the integration of a high throughput approach to analysis that will make it possible to test various anticancer drugs on these cells at no risk to the patient, and obtain genetic information that will be crucial to developing a highly personalized treatment plan.

An award is made to the Pennsylvania State University to develop a microfabricated high-temperature flow control device for gas chromatography (GC) applications that will benefit agriculture and forestry to better target invasive species and pests.

Non Technical Abstract:
The device will enable a technique named chopper-modulated electroantennograph (EAG), which uses GC to separate volatile odorants, and insect antennae as detectors to identify specific compounds that are of importance to, and able to be sensed by the insect. As a result of millions of years of evolution, insect antennae are capable of detecting certain chemicals more sensitively and specifically than artificial chemical sensors. The chopper-modulated GC-EAG method will use the proposed flow control device to regulate gas flow in GC and improve the signal-to-noise ratio of the EAG, with a resulting several orders of magnitude improvement in detection sensitivity for behaviorally important volatile chemicals. The use of the proposed device in other GC applications can potentially contribute to the general chemical sensing community and benefit the society with more sensitive analysis for environmental monitoring, food safety, disease diagnostics and etc. The education plan includes student training and curriculum development. The instrument will also be integrated into various outreach activities including the annual PSU "Great Insect Fair", at which attendance routinely surpasses 8,000 people annually.

Technical Abstract:
The overall goal of the research is to develop a high temperature microelectromechanical system (MEMS) flow controller (HT-MEMS flow controller) whose purpose is to operate inside a conventional gas chromatography (GC) oven to enable chopper-modulated electroantennograph (EAG) − MEMS- GC-EAG as well as other GC applications. This project aims to develop and lead to commercialization of the HT-MEMS flow controller to (1) implement chopper stabilization for GC-EAG, which holds the promise of improving the sensitivity of the current GC-EAG system by orders of magnitude; (2) explore applications of the instrument in broader chemical sensing areas, including other GC detectors beyond EAG and two dimensional GC (2D-GC). Apart from resulting publications and presentations at workshops and meetings, the technology will be disseminated through a partnership with GC equipment manufacturers and distributors.

PROJECT SUMMARY Infectious diseases account for one quarter of global deaths and massive economic loss. Pathogen discovery is critical in combating infectious diseases. However, the causative agents in a large percentage of clinical infectious disease cases remain unidentified. Often occurring in resource-limited areas, major outbreaks were caused by newly emerging viruses, mostly wildlife-originated. The pressing challenges in viral infectious diseases call for a sample-to-answer virus discovery platform that can prepare viral samples from environmental and clinical samples for high throughput sequencing based virus discovery. In this proposed research, we will develop a new carbon nanotube 3D sample preparation and enrichment microdevice (CNT- 3D-SPEM) that employs engineered carbon nanotube material, as shown in our preliminary study, into a new continuous flow microfluidic design for virus particle enrichment. The new microfluidic design has a unique self- regulating flow mechanism to achieve high virus capture efficiency and high sample capacity and flow rate simultaneously. After enrichment, the captured viruses are lyzed inside the CNT-3D-SPEM. The viral genome will be purified and amplified in situ, which are required sample preparation steps for virus discovery through high throughput sequencing. Working with co-investigators together, we will conduct multiple pilot studies that use the developed technology for virus discovery from clinical and environmental samples. The ultimate goal is to develop the entire high-performance portable platform to advance virus discovery for the battle with viral infectious diseases.

PROJECT SUMMARY In this proposed research, we will develop a lipid nanoprobe (LNP) integrated microdevice, LiEVchip, for isolation of extracellular vesicles (EVs) from blood plasma of patients with non-small cell lung cancer (NSCLC). The proposed LiEVchip features high-throughput sample processing, one-step enrichment, and low cost. It is expected to isolate EVs from 1 ml of unprocessed plasma in 20 minutes with over 80% isolation efficiency. Furthermore, combination with duplex sequencing based ultrasensitive DNA mutation detection platform, the LiEVchip will significantly prompt EV-based cancer diagnostics in an invasive way. In our previous study, we developed a two-component LNP system for rapid EV isolation and comprehensively EV molecular analyses. Particularly, we successfully identified EV DNA mutations from four patients with advanced NSCLC. The LNP system overcomes low throughput, low purity and other common shortcomings in EV isolation, showing great potential for clinical use. Hence, we proposed to develop a highly integrated one- component LiEVchip for rapid and efficient EV isolation. Firstly, we will design and fabricate the proposed LiEVchip. The LiEVchip integrates the LNP to capture EVs based on their lipid membrane envelope. The EV isolation efficiency is boosted by the design of microchannel curvature to enhance flow-surface interaction by Dean flow, and the empowerment of electrokinetic enhanced EV isolation by the nanoelectrode array. Then, we will optimize its operational parameters to make a balance between EV isolation efficiency and purity. After comprehensive optimization, we will rigorously validate its performance with spike samples by analyzing EV content, including RNA, DNA, and proteins, and compare with those isolated by the prevalent EV isolation method, OptiPrep density gradient ultracentrifugation (odgUC). Subsequently, we will develop duplex sequencing based DNA mutation detection method with 3rd generation high-throughput sequencer (PacBio). The detection sensitivity of point/deletion mutations and complex structural variations (SVs) will be validated with spiked-in samples. Finally, in a cohort clinical study, we will first recruit 40 NSCLC patients at stage IV to validate the overall technology. EV from plasma will be isolated with LiEVchip, and a panel of 20 most common NSCLC mutated genes will be assayed simultaneously by duplex sequencing. We will further perform EV isolation and duplex sequencing in the additional 120 samples covering stage I-III NSCLC to investigate the potential of this technology in early NSCLC diagnosis. Besides, we will use the developed technology to monitor mutation status of 20 NSCLC patients undergoing targeted therapy to explore its clinical utility in treatment monitoring. The cohort clinical studies not only can testify whether LiEVchp combined with duplex sequencing can be routinely applied to detect diverse malignancies in NSCLC, but also can demonstrate the clinical diagnostic values of EV DNA. Altogether, the successful completion of this proposed project will pave the way for clinical translation of EV diagnostics.

PROJECT SUMMARY Pancreatic cancer is the deadliest solid malignancy with an abysmal overall 5-year survival less than 7%. Late stage diagnosis is regarded as the most important factor contributing to the bleak patient outcome. Since pancreatic neoplastic cells can take decades to metastasis, in theory, there is a large window for early diagnosis. However, current early diagnosis often fails due to lack of sensitive and specific pancreatic biomarkers and limitations on the spatial resolution of the imaging methods. Extracellular vesicles (EVs) are sub-micrometer vesicles released by almost all the cells and are emerging as an attractive non-invasive liquid biopsy target for clinical cancer diagnosis. Compared with other liquid biopsy materials, EVs are likely released by live cells and contains much longer DNA fragments than circulating tumor DNA (ctDNA), and are more abundant than circulating tumor cells (CTCs) to provide higher sensitivity and better clinical utility. Based on our preliminary study on using carbon nanotube (CNT) forest as an unprecedented material for sub-micrometer particle isolation, we propose a new CNT-integrated EV isolation and genome enrichment microdevice (CNT-iEVIGEM), which is a continuous flow microfluidic device integrated with CNT forest. The self-regulating continuous-flow design boosts the sample capacity and throughput of our previous CNT integrated device by 10 times and eliminates clogging, while making the device immune to local CNT forest defects and maintaining high EV capture efficiency. With additive fabrication of nanometer-thick functional zirconia on CNTs, the CNT-iEVIGEM can efficiently purify EV DNA in situ. For pancreatic cancer diagnosis, we will develop a multiplex digital droplet PCR (ddPCR) assay to detect KRAS mutations from EVs isolated from plasma by CNT-iEVIGEM. KRAS mutations are the most prevalent pancreatic cancer mutations with over 90% occurrence. It is a major driver oncogene and often the earliest event in pancreatic tumorigenesis. Using model samples of cancer-derived EVs spiked in healthy control plasma, we will determine the detection limit as the lowest mutant allele fraction that can be detected unambiguously. The assay will be validated with 75 plasma samples from stage IV pancreatic cancer patients and 75 age-/sex- matched healthy donors. Then we will use this assay to investigate diagnosis of pancreatic cancer at different stages, by recruiting 60 early stage (stage I + II) and 60 stage III pancreatic cancer patients. The sensitivity and specificity of the assay at different stages will be measured with the mutation status in tissue samples as the true values. The proposed study will pave the way for further clinical validation of the CNT- iEVIGEM technology for cancer diagnosis. The EV isolation platform can also be used for other DNA mutations, other EV cargos, and other cancer types as a non-invasive liquid biopsy technology.