Highly sensitive and rapid isolation of fetal nucleated red blood cells with microbead-based selective sedimentation for non-invasive prenatal diagnostics.

Non-invasive prenatal diagnostics (NIPD) has been an emerging field for prenatal diagnosis research. Carrying the whole genome coding of the fetus, fetal nucleated red blood cells (FNRBCs) have been pursued as a surrogate biomarker traveling around in maternal blood. Here, by combining a unique microbead-based centrifugal separation and enzymatic release, we demonstrated a novel method for FNRBC isolation from the blood samples. First, the gelatin-coated silica microbeads were modified with FNRBC-specific antibody (anti-CD147) to capture the target cells in the blood samples. Then, the density difference between microbead-bound FNRBCs and normal blood cells enables the purification of FNRBCs via an improved high-density percoll-based separation. The non-invasive release of FNRBCs can then be achieved by enzymatically degrading the gelatin film on the surface of the microbeads, allowing a gentle release of the captured target cells with as high as 84% efficiency and ∼80% purity. We further applied it to isolate fetal cells from maternal peripheral blood. The released cells were analyzed by real-time polymerase chain reaction to verify their fetal origin and fluorescent in situ hybridization to detect fetal chromosome disorders. This straightforward and reliable alternative platform for FNRBC detection may have the potential for realizing facile NIPD.

Red Blood Cell Membrane as a Biomimetic Nanocoating for Prolonged Circulation Time and Reduced Accelerated Blood Clearance.

For decades, poly(ethylene glycol) (PEG) has been widely incorporated into nanoparticles for evading immune clearance and improving the systematic circulation time. However, recent studies have reported a phenomenon known as "accelerated blood clearance (ABC)" where a second dose of PEGylated nanomaterials is rapidly cleared when given several days after the first dose. Herein, we demonstrate that natural red blood cell (RBC) membrane is a superior alternative to PEG. Biomimetic RBC membrane-coated Fe(3)O(4) nanoparticles (Fe(3)O(4) @RBC NPs) rely on CD47, which is a "don't eat me" marker on the RBC surface, to escape immune clearance through interactions with the signal regulatory protein-alpha (SIRP-α) receptor. Fe(3)O(4) @RBC NPs exhibit extended circulation time and show little change between the first and second doses, with no ABC suffered. In addition, the administration of Fe(3)O(4) @RBC NPs does not elicit immune responses on neither the cellular level (myeloid-derived suppressor cells (MDSCs)) nor the humoral level (immunoglobulin M and G (IgM and IgG)). Finally, the in vivo toxicity of these cell membrane-camouflaged nanoparticles is systematically investigated by blood biochemistry, hematology testing, and histology analysis. These findings are significant advancements toward solving the long-existing clinical challenges of developing biomaterials that are able to resist both immune response and rapid clearance.

Influence of PUB2 on the Leveling Effect of Chip Copper Connection Electroplating: Mechanism and Applications.

The copper connectivity technique is essential for achieving electrical interconnection in wafer level packaging (WLP), system in packaging (SiP), and 3D packaging. The essential processing material for copper connectivity is a copper sulfate electroplating solution in which organic additives play a crucial role in the regularity of copper electrodeposition. In this study, electrochemical tests, X-ray diffraction, 3D profiling, and scanning electron microscopy were used to investigate the leveling effect and mechanism of polyquaternary ammonium urea-containing polymer (PUB2) in the process of copper electrodeposition on-chip copper connections. PUB2 has excellent polarization ability on the target surface, remains unaffected by the sulfur additive SPS and poly(ethylene glycol), and displays a strong ability to regulate the copper deposition rate of through-holes and surface wiring. The waviness of the wafer surface wiring was reduced from 130 to approximately 70 nm after optimizing the PUB2 concentration, and the surface roughness was reduced from 10 to approximately 7 nm. The coating was dispersed evenly, and the rate of through-hole filling was improved by 57%. This study not only examined PUB2 leveling performance and mechanisms but also devised a research method and system for electroplating additives to facilitate the development and application of new electroplating additives.

Electroplated Copper Additives for Advanced Packaging: A Review.

Acid copper electroplating stands as a core technology in advanced packaging processes, facilitating the realization of metal interconnects, bumps, vias, and substrate wiring between transistors. The deposition quality of copper interconnect materials has a crucial impact on the final performance of chips, directly influencing their yield, reliability, and stability. In this intricate process, additives play a pivotal role in regulating the deposition quality and behavior of metal copper. This mini-review comprehensively summarizes the recent research progress in the field of electroplating copper additives for advanced packaging, both domestically and internationally, delving into the types and mechanisms of various additive molecules, including accelerators, inhibitors, and leveling agents. Through in-depth research on these additives, we gain a profound understanding of their specific roles in the electroplating process and the intricate interaction mechanisms among them, providing theoretical support for optimizing the electroplating process. Furthermore, this mini-review also delves into a thorough analysis of the current issues and challenges facing acid copper electroplating, exploring the key factors that constrain the further development of electroplating copper technology. Based on this analysis, we propose several potential solutions and research directions, offering crucial references for the development and application of electroplating copper additives in advanced packaging. In conclusion, this mini-review aims to provide a comprehensive perspective and profound understanding of the development and application of electroplating copper additives through a review and analysis of recent research progress, ultimately aiming to promote the further advancement of advanced packaging technology.

Biocompatible TiO2 nanoparticle-based cell immunoassay for circulating tumor cells capture and identification from cancer patients.

We demonstrate the isolation of circulating tumor cells (CTCs) with a biocompatible nano-film composed of TiO2 nanoparticles. Due to the enhanced topographic interaction between nano-film and cancer cell surface, cancer cells (HCT116) spiked into PBS and healthy blood can be recovered from the suspension, whose efficiencies were respectively 80 % and 50 %. Benifit from the biocompatibility of this nano-film, in-situ culture of the captured cancer cells is also available, which provides an alternative selection when the capture cell number was inadequate or the sample cannot be analyzed immediately. For the proof-of-concept study, we use this nano-film to separate the circulating tumor cells from the colorectal and gastric cancer patient peripheral blood samples and the captured CTCs are identified by a three-colored immunocytochemistry method. We investigated the cancer cells capture strength at the nano-bio interface through exposing the cells to fluid shear stress in microfluidic device, which can be utilized to increase the purity of CTCs. The result indicated that 50 % of the captured cells can be detached from the substrate when the fluid shear stress was 180 dyn cm(-2). By integration of this CTCs capture nano-film with other single cell analysis device, we expected to further explore their applications in genome sequencing based on the captured CTCs.

Non-invasive Prenatal Diagnosis of Chromosomal Aneuploidies and Microdeletion Syndrome Using Fetal Nucleated Red Blood Cells Isolated by Nanostructure Microchips.

Detection of detached fetal nucleated red blood cells (fNRBCs) in the maternal peripheral blood may serve as a prospective testing method competing with the cell-free DNA, in non-invasive prenatal testing (NIPT). Methods: Herein, we introduce a facile and effective lab-on-a-chip method of fNRBCs detection using a capture-releasing material that is composed of biotin-doped polypyrrole nanoparticles. To enhance local topographic interactions between the nano-components and fNRBC, a specific antibody, CD147, coated on the nanostructured substrate led to the isolation of fNRBCs from maternal peripheral blood. Subsequently, an electrical system was employed to release the captured cells using 0.8 V for 15 s. The diagnostic application of fNRBCs for fetal chromosomal disorders (Trisomy 13/21/18/X syndrome, microdeletion syndrome) was demonstrated. Results: Cells captured by nanostructured microchips were identified as fNRBCs. Twelve cases of chromosomal aneuploidies and one case of 18q21 microdeletion syndrome were diagnosed using the fNRBCs released from the microchips. Conclusion: Our method offers effective and accurate analysis of fNRBCs for comprehensive NIPT to monitor fetal cell development.

Gelatin Nanoparticle-Coated Silicon Beads for Density-Selective Capture and Release of Heterogeneous Circulating Tumor Cells with High Purity.

Background: Circulating tumor cells (CTCs) are a burgeoning topic in cancer biomarker discovery research with minimal invasive blood draws. CTCs can be used as potential biomarkers for disease prognosis, early cancer diagnosis and pharmacodynamics. However, the extremely low abundance of CTCs limits their clinical utility because of technical challenges such as the isolation and subsequent detailed molecular and functional characterization of rare CTCs from patient blood samples. Methods: In this study, we present a novel density gradient centrifugation method employing biodegradable gelatin nanoparticles coated on silicon beads for the isolation, release, and downstream analysis of CTCs from colorectal and breast cancer patients. Results: Using clinical patient/spiked samples, we demonstrate that this method has significant CTC-capture efficiency (>80%) and purity (>85%), high CTC release efficiency (94%) and viability (92.5%). We also demonstrate the unparalleled robustness of our method in downstream CTC analyses such as the detection of PIK3CA mutations. Conclusion: The efficiency and versatility of the multifunctional density microbeads approach provides new opportunities for personalized cancer diagnostics and treatments.

Effective cancer targeting and imaging using macrophage membrane-camouflaged upconversion nanoparticles.

Upconversion nanoparticles (UCNPs), with fascinating optical and chemical features, are a promising new generation of fluorescent probes. Although UCNPs have been widely used in diagnosis and therapy, there is an unmet need for a simple and effective surface engineering method that can produce cancer-targeting UCNPs. Here, we show that by coating particles with macrophage membranes, it becomes possible to utilize the adhesion between macrophages and cancer cells for effective cancer targeting. Natural macrophage membranes along with their associated membrane proteins were reconstructed into vesicles and then coated onto synthetic UCNPs. The resulting macrophage membrane-camouflaged particles (MM-UCNPs) exhibited effective cancer targeting capability inherited from the source cells and were further used for enhanced in vivo cancer imaging. Finally, the blood biochemistry, hematology testing and histology analysis results suggested a good in vivo biocompatibility of MM-UCNPs. The combination of synthetic nanoparticles with biomimetic cell membranes embodies a novel design strategy toward developing biocompatible nanoprobes for potential clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 521-530, 2017.

Fetal nucleated red blood cell analysis for non-invasive prenatal diagnostics using a nanostructure microchip.

Cell-free DNA has been widely used in non-invasive prenatal diagnostics (NIPD) nowadays. Compared to these incomplete and multi-source DNA fragments, fetal nucleated red blood cells (fNRBCs), once as an aided biomarker to monitor potential fetal pathological conditions, have re-attracted research interest in NIPD because of their definite fetal source and the total genetic information contained in the nuclei. Isolating these fetal cells from maternal peripheral blood and subsequent cell-based bio-analysis make maximal genetic diagnosis possible, while causing minimal harm to the fetus or its mother. In this paper, an affinity microchip is reported which uses hydroxyapatite/chitosan nanoparticles as well as immuno-agent anti-CD147 to effectively isolate fNRBCs from maternal peripheral blood, and on-chip biomedical analysis was demonstrated as a proof of concept for NIPD based on fNRBCs. Tens of fNRBCs can be isolated from 1 mL of peripheral blood (almost 25 mL-1 in average) from normal pregnant women (from the 10th to 30th gestational week). The diagnostic application of fNRBCs for fetal chromosome disorders (Trisomy 13 and 21) was also demonstrated. Our method offers effective isolation and accurate analysis of fNRBCs to implement comprehensive NIPD and to enhance insights into fetal cell development.

Autofluorescent gelatin nanoparticles as imaging probes to monitor matrix metalloproteinase metabolism of cancer cells.

In this paper, autofluorescent gelatin nanoparticles were synthesized as matrix metalloproteinase (MMP) responsive probes for cancer cell imaging. A modified two-step desolvation method was employed to generate these nanoparticles whose size was controllable and had stable autofluorescence. As glutaraldehyde was introduced as the crosslinking agent, the generation of Schiff base (CN) and double carbon bond (CC) between glutaraldehyde and gelatin endowed these gelatin nanoparticles distinct autofluorescence. Considering MMPs were usually overexpressed on the surface of cancer cells and they had degradation ability toward gelatin, we utilized these nanoparticles as imaging probes to responsively monitor the MMP metabolism of cancer cells according to the luminance change. As fluorescent probes, these nanoparticles had facile synthesis procedure and good biocompatibility, and provided a smart strategy to monitor cancer cell behaviors. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2854-2860, 2016.

Capture and Identification of Heterogeneous Circulating Tumor Cells Using Transparent Nanomaterials and Quantum Dots-Based Multiplexed Imaging.

BACKGROUND: Capture and identification of circulating tumor cells (CTCs) in the blood system can help guide therapy and predict the prognosis of cancer patients. However, simultaneous capture and identification of CTCs with both epithelial and mesenchymal phenotypes remains a formidable technical challenge for cancer research. This study aimed at developing a system to efficiently capture and identify these CTCs with heterogeneous phenotypes using transparent nanomaterials and quantum dots (QDs)-based multiplexed imaging. METHODS: Hydroxyapatite-chitosan (HA-CTS) nanofilm-coated substrates were modified based on our previous work to increase the capture efficiency of cancer cell lines by extending baking and incubating time. QDs-based imaging was applied to detect cytokeratin, epithelial cell adhesion molecule (EpCAM), and vimentin of cancer cells to demonstrate the feasibility of multiplexed imaging. And QDs-based multiplexed imaging of CD45, cytokeratin and vimentin was applied to detect CTCs from different cancer patients that were captured using HA-CTS nanofilm substrates. RESULTS: Comparisons of the capture efficiencies of cancer cells at different baking time of film formation and incubating time of cell capture revealed the optimal baking and incubating time. Optimal time was chosen to develop a modified CTCs capture system that could capture EpCAM-positive cancer cells at an efficiency > 80%, and EpCAM-negative cancer cells at an efficiency > 50%. QDs-based imaging exhibited comparable detection ability but higher photostability compared to organic dyes imaging in staining cells. In addition, QDs-based multiplexed imaging also showed the molecular profiles of cancer cell lines with different phenotypes well. The integrated CTCs capture and identification system successfully captured and imaged CTCs with different sub-phenotypes in blood samples from cancer patients. CONCLUSION: This study demonstrated a reliable capture and detection system for heterogeneous CTCs that combined enrichment equipment based on HA-CTS nanofilm substrates with QDs-based multiplexed imaging.

Efficient Purification and Release of Circulating Tumor Cells by Synergistic Effect of Biomarker and SiO2 @Gel-Microbead-Based Size Difference Amplification.

Microfluidics-based circulating tumor cell (CTC) isolation is achieved by using gelatin-coated silica microbeads conjugated to CTC-specific antibodies. Bead-binding selectively enlarges target cell size, providing efficient high-purity capture. CTCs captured can be further released non-invasively. This stratagem enables high-performance CTC isolation for subsequent studies.

Capture and Release of Cancer Cells by Combining On-Chip Purification and Off-Chip Enzymatic Treatment.

As "liquid biopsies", circulating tumor cells (CTCs) have been thought to hold significant insights for cancer diagnosis and treatment. Despite the advances of microfluidic techniques that improve the capture of CTCs to a certain extent, recovering the captured CTCs with enhanced purity at the same time remains a challenge. Here, by combining on-chip purification and off-chip enzymatic treatment, we demonstrate a two-stage strategy to enhance the purity of captured cancer cells from blood samples. The on-chip purification introduces a stirring flow to increase the capture sensitivity and decrease nonspecifically bounded cells. The off-chip enzymatic treatment enables the cancer cells to be released from the attached magnetic beads, further improving the purity and enabling next reculture. For the proof-of-concept study, spiked cancer cells are successfully obtained from unprocessed whole blood with high recovery rate (∼68%) and purity (∼61%), facilitating subsequent RNA expression analysis.

Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture.

Circulating tumor cells (CTCs) in the blood which have detached from both the primary tumor and any metastases may be considered as a "liquid biopsy" and are expected to replace tumor biopsies in the monitoring of treatment response and determining patient prognosis. Here, we introduce a facile and efficient CTC detection material made of hydroxyapatite/chitosan (HA/CTS), which is beneficial because of its transparency and excellent biological compatibility. Atomic force microscopy images show that the roughness of the HA/CTS nanofilm (HA/CTSNF) substrates can be controlled by changing the HA:CTS ratio. Enhanced local topographic interactions between nano-components on cancer cell membranes, and the antibody coated nanostructured substrate lead to improved CTC capture and separation. This remarkable nanostructured substrate has the potential for CTC culture in situ and merits further analysis. CTCs captured from artificial blood samples were observed in culture on HA/CTSNF substrates over a period of 14 days by using conventional staining methods (hematoxylin eosin and Wright's stain). We conclude that these substrates are multifunctional materials capable of isolating and culturing CTCs for subsequent studies.

Capture and release of cancer cells based on sacrificeable transparent MnO2 nanospheres thin film.

A CTCs detection assay using transparent MnO2 nanospheres thin films to capture and release of CTCs is reported. The enhanced local topography interaction between extracellular matrix scaffolds and the antibody-coated substrate leads to improved capture efficiency. CTCs captured from artificial blood sample can be cultured and released, represent a new functional material capable of CTCs isolation and culture for subsequent studies.