Yiqiu Xia

Point-of-Care Microdevices for Blood Plasma Analysis in Viral Infectious Diseases

Each year, outbreaks of viral infections cause illness, disability, death, and economic loss. As learned from past incidents, the detrimental impact grows exponentially without effective quarantine. Therefore, rapid on-site detection and analysis are highly desired. In addition, for high-risk areas of viral contamination, close monitoring should be provided during the potential disease incubation period. As the epidemic progresses, a response protocol needs tobe rapidly implemented and the virus evolution fully tracked. For these scenarios, point-of-care microdevices can provide sensitive, accurate, rapid and low-cost analysis for a large population, especially in handling complex patient samples, such as blood, urine and saliva. Blood plasma can be considered as a mine of information containing sources and clues of biomarkers, including nucleic acids, immunoglobulin and other proteins, as well as pathogens for clinical diagnosis. However, blood plasma is also the most complicated body fluid. For targeted plasma biomarker detection or untargeted plasma biomarker discovery, the challenges can be as difficult as identifying a needle in a haystack. A useful platform must not only pursue single performance characteristics, but also excel at multiple performance parameters, such as speed, accuracy, sensitivity, selectivity, cost, portability, reliability, and user friendliness. Throughout the decades, tremendous progress has been made in point-of-care microdevices for viral infectious diseases. In this paper, we review fully integrated lab-on-chip systems for blood analysis of viral infectious disease.

A Spontaneous 3D Bone-On-a-Chip for Bone Metastasis Study of Breast Cancer Cells

Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone-on-a-chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co-culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone-on-a-chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis.

Size-based separation methods of circulating tumor cells

Abstract Circulating tumor cells (CTCs) originate from the primary tumor mass and enter into the peripheral bloodstream. Compared to other “liquid biopsy” portfolios such as exosome, circulating tumor DNA/RNA (ctDNA/RNA), CTCs have incomparable advantages in analyses of transcriptomics, proteomics, and signal colocalization. Hence, CTCs hold the key to understanding the biology of metastasis and play a vital role in cancer diagnosis, treatment monitoring, and prognosis. Size‐based enrichment features are prominent in CTC isolation. It is a label‐free, simple and fast method. Enriched CTCs remain unmodified and viable for a wide range of subsequent analyses. In this review, we comprehensively summarize the differences of size and deformability between CTCs and blood cells, which would facilitate the development of technologies of size‐based CTC isolation. Then we review representative size‐/deformability‐based technologies available for CTC isolation and highlight the recent achievements in molecular analysis of isolated CTCs. To wrap up, we discuss the substantial challenges facing the field, and elaborate on prospects. Graphical abstract Figure. No Caption available.

Label-Free Virus Capture and Release by a Microfluidic Device Integrated with Porous Silicon Nanowire Forest

Viral diseases are perpetual threats to human and animal health. Detection and characterization of viral pathogens require accurate, sensitive, and rapid diagnostic assays. For field and clinical samples, the sample preparation procedures limit the ultimate performance and utility of the overall virus diagnostic protocols. This study presents the development of a microfluidic device embedded with porous silicon nanowire (pSiNW) forest for label-free size-based point-of-care virus capture in a continuous curved flow design. The pSiNW forests with specific interwire spacing are synthesized in situ on both bottom and sidewalls of the microchannels in a batch process. With the enhancement effect of Dean flow, this study demonstrates that about 50% H5N2 avian influenza viruses are physically trapped without device clogging. A unique feature of the device is that captured viruses can be released by inducing self-degradation of the pSiNWs in physiological aqueous environment. About 60% of captured viruses can be released within 24 h for virus culture, subsequent molecular diagnosis, and other virus characterization and analyses. This device performs viable, unbiased, and label-free virus isolation and release. It has great potentials for virus discovery, virus isolation and culture, functional studies of virus pathogenicity, transmission, drug screening, and vaccine development.

Aptamer-Conjugated Extracellular Nanovesicles for Targeted Drug Delivery

Extracellular nanovesicles (ENV) released by many cells contain lipids, proteins, and nucleic acids that contribute to intercellular communication. ENVs have emerged as biomarkers and therapeutic targets but they have also been explored as drug delivery vehicles. However, for the latter application, clinical translation has been limited by low yield and inadequate targeting effects. ENV vectors with desired targeting properties can be produced from parental cells engineered to express membrane-bound targeting ligands, or they can be generated by fusion with targeting liposomes; however, neither approach has met clinical requirements. In this study, we demonstrate that mechanical extrusion of approximately 107 cells grafted with lipidated ligands can generate cancer cell-targeting ENV and can be prepared in approximately 1 hour. This rapid and economic approach could pave the way for clinical implementation in the future.Significance: A new and rapid method for production of drug-targeting nanovesicles has implications for cancer treatment by chimeric antigen receptor T cells and other therapies. Cancer Res; 78(3); 798-808. ©2017 AACR.

Preoccupation of Empty Carriers Decreases Endo-/Lysosome Escape and Reduces the Protein Delivery Efficiency of Mesoporous Silica Nanoparticles

Endo-/lysosome escape is a major challenge in nanoparticle-based protein delivery for cancer therapy. To enhance the endo-/lysosomal escape and increase the efficacy of protein delivery, current strategies mainly focus on destroying endo-/lysosomes by employing modified nanoparticles, such as pH-sensitive polyplexes, cell-penetrating peptides, and photosensitive molecules. Herein, we hypothesize that pretreatment with empty nanocarriers might make endo-/lysosomes occupied and affect the endo/lysosomal escape of protein subsequently delivery by nanocarriers. We first treated breast carcinoma MDA-MB-231 cells with a high concentration of empty nanocarriers, mesoporous silica nanoparticles (MSN), to occupy the endo-/lysosome. After 2 h, we treated the cells with a lower concentration of fluorescein isothiocyanate-labeled MSN (MSN-FITC) and investigated the intracellular spatial and temporal distribution of MSN-FITC and their colocalization with endo-/lysosomes. We discovered the preoccupation of endo-/lysosomes by the empty nanocarriers did exist, mainly through changing the spatial distribution of the subsequently introduced nanocarriers. Furthermore, for the protein delivery, we observed reduced MSN-saporin delivery after endo-/lysosome preoccupation by MSN empty carriers. A similar result is observed for the delivery of cytochrome C by MSN but not for the small-molecule anticancer drug doxorubicin. The results show that the empty nanocarriers inhibit the endo-/lysosome intracellular trafficking process and decrease the endo-/lysosome escape of proteins subsequently delivered by the nanocarriers. This new discovered phenomenon of declined endo-/lysosome escape after endo-/lysosome preoccupation indicates that repeated treatment by nanomaterials with low protein-loading capacity may not yield a good cancer therapeutic effect. Therefore, it provides a new insightful perspective on the role of nanomaterial carriers in intracellular protein delivery.

A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection

Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 103 EID50 mL−1 (EID50: 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme‐linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off‐chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low‐cost and convenient biomarker detection.

A mcirofluidic device of biodegradable porous silicon nanowires for size based capturing and releasing viruses

A microfluidic device of porous silicon nanowire (PSNW) channel walls was fabricated as a point-of-care platform to capture nanoparticles (NPs) like viruses based on their sizes. PSNWs were synthesized on the bottom and sidewalls of the micro channels with metal-assisted etching. The space between PSNWs was controlled to capture NPs with a specific size. To enhance the capture rate, meandering channels were designed to increase the interaction between NPs and PSNWs by the Dean flow. With this system, we demonstrated label-free N5H2 avian influenza virus capture. In addition, virus can be released later and collected at the outlet by degrading PSNWs in PBS for 24 hours.

Nucleus of Circulating Tumor Cell Determines Its Translocation Through Biomimetic Microconstrictions and Its Physical Enrichment by Microfiltration

The mechanism of cells passing through microconstrictions, such as capillaries and endothelial junctions, influences metastasis of circulating tumor cells (CTCs) in vivo, as well as size-based enrichment of CTCs in vitro. However, very few studies observe such translocation of microconstrictions in real time, and thus the inherent biophysical mechanism is poorly understood. In this study, a multiplexed microfluidic device is fabricated for real-time tracking of cell translocation under physiological pressure and recording deformation of the whole cell and nucleus, respectively. It is found that the deformability and size of the nucleus instead of the whole cell dominate cellular translocation through microconstrictions under a normal physiological pressure range. More specifically, cells with a large and stiff nucleus are prone to be blocked by relatively small constrictions. The same phenomenon is also observed in the size-based enrichment of CTCs from peripheral blood of metastatic cancer patients. These findings are different from a popular viewpoint that the size and deformability of a whole cell mainly determine cell translation through microconstrictions, and thus may elucidate interactions between CTCs and capillaries from a new perspective and guide the rational design of size-based microfilters for rare cell enrichment.

Zinc oxide nanorod integrated microdevice for multiplex virus detection

Technology development for point-of-care viral pathogen detection is critical for early diagnosis of infectious diseases, and rapid and effective disease intervention. In this paper, we present the development of a zinc oxide nanorod-integrated microdevice for highly sensitive and specific detection of avian influenza virus. This multiplexed immunofluorescence platform takes two advantages of the zinc oxide nanorods. On one hand, the 3D morphology of zinc oxide nanorods efficiently increases the effective surface area for monoclonal antibodies and decreases the diffusion distance between antibody and pathogens. On the other hand, the unique optical property of the translucent randomly ordered zinc oxide nanorod surface enhances fluorescence detection by 30–70%. We demonstrated the detection limit of the H5N2 avian influenza virus could be lowered down to 3.6×103 EID50/mL (EID50: 50% embryo infectious dose), which was about 22 times more sensitive than conventional ELISA assay tested under the same conditions. We further designed the microfluidic biosensor platform to detect multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 avian influenza virus can be released by simply dissolving zinc oxide nanorods under slightly acidic environment for subsequent off-chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low-cost and convenient biomarker detection.