Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

Anhui University of Technology

Circulating tumor cells (CTCs) detection, enumeration and characterization with microfluidic chips has critical significance in cancer prognosis offering a non-invasive "liquid biopsy". Based on physical differences of size and deformability, we explore micro-ellipse filters consisting of microfuidic slits in series gradually narrowed. Slender tunnels sensitively capture tumor cells with slim chance to escape. Tumor cells could reside at capture sites organized by arrays of micro-ellipse microposts enduring less stress. Circular elliptical microstructures produce smooth flow minimally reducing any damage. "Air Suction" could extremely shorten capture. Capture efficiency comes out to be a robust yield of 90% and percentage obeys Gaussian distribution at various stages. With rare number accurately enumerated, micro-Ellipse filters have been tested high efficiently capturing tumor cells in both whole and lysed blood. To clinically validate the device, the microfluidic chip was utilized to identify and capture CTCs from metastatic breast, colon and non-small-cell lung (NSCLC) cancer patients. CTCs were detected positive in all samples with 4 patients having more than 20 CTCs. Those sensitive results are consistent with theoretical expectation. Efficient micro-ellipse filters enable clinical enumeration of metastasis, on-chip anti-cancer drug responses and biological molecular analysis.

Highly-sensitive capture of circulating tumor cells using micro-ellipse filters.

Inertial and deformability- based particles separations gradually attract more significant attentions. In this work, we present a hybrid chip by combining the advantages of inertial and deformability -based principle. The chip is a triplet parallelizing spiral inertial microfluidic chip interconnected with numerable tilted slits (Spiral-Slits Chip) for continuous separation of circulating tumor cells. Utilizing the inertial lift and viscous drag forces, different sized particles achieve different equilibrium at distinct streamlines of the spiral microchannel. Numerable tilted slits are organized along the flow direction. They frequently transport segregated streamline particles into a paralleled smaller microchannel. These frequent dragging results in the amount of certain sized particles in the original microchannel gradually and dramatically reduced. Inertial separation of distinct sized particles could be achievable. Two arrays of numerable tilted slits function as bridges. This Spiral-Slits Chip could substitute for Red Blood Cells Lysis (RBCL) and is most effective for ultra-high throughput. The overall arrangement of this triplet parallelizing spiral inertial microfluidic reflects stable streamlines distribution in the first main microchannel. Combining with Ellipse filters, robust and reproducible capture of CTCs could be achieved at high flow rates. Optical absorption detection has been tentatively tested, and this could simplify the process.

A Triplet Parallelizing Spiral Microfluidic Chip for Continuous Separation of Tumor Cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Author Correction: Highly-sensitive capture of circulating tumor cells using micro-ellipse filters.

Fibroblasts have significant involvement in cancer progression and are an important therapeutic target for cancer. Here, we present a microfluidic non-contact co-culture device to analyze interactions between tumor cells and fibroblasts. Further, we investigate myofibroblast behaviors induced by lung tumor cells as responses to gallic acid and baicalein. Human lung fibroblast (HLF) and lung cancer cell line (A549) cells were introduced into neighboring, separated regions by well-controlled laminar flows. The phenotypic behavior and secretion activity of the tumor cells indicate that fibroblasts could become activated through paracrine signaling to create a supportive microenvironment for cancer cells when HLF is co-cultured with A549. Furthermore, both gallic acid (GA) and baicalein (BAE) could inhibit the activation of fibroblasts. In situ analysis of various cell communications via the paracrine pathway could be realizable in this contactless co-culture single device. This device facilitates a better understanding of interactions between heterotypic cells, thus exploring the mechanism of cancer, and performs anti-invasion drug assays in a relatively complex microenvironment.

In Situ Analysis of Interactions between Fibroblast and Tumor Cells for Drug Assays with Microfluidic Non-Contact Co-Culture.

As a "liquid biopsy," circulating tumor cell (CTC) enumeration with microfluidic chips has great significance in cancer prognosis. CTCs carry significant information as the original tumor. Integrated microfluidic chips are combining with affinity- and physical-based such as wave chip offers a new way to segregate CTCs. In this work, we further study capturing clinical applications of CTCs with wave chip. When cell suspension moves across the microposts array, CTCs squeeze out from narrow gaps organized by microposts. This movement renders CTCs to obtain a tilted velocity to fluid direction. This tilted velocity would direct CTCs to be captured by the smaller neighboring gaps next array. Simultaneously, interaction or friction time is longer due to barrier of modified microposts. These microposts would be effective for realizing binding of antigen and antibody. Therefore, both antibody-coated and physical-based isolations could be combined in isolating CTCs. Capture percentage concentrated on the first several arrays is shown theoretically and experimentally. Efficient capture could be obtained for artificial patient blood. Clinically, CTCs were tested positive for three metastatic human breast cancer patient samples. This wave chip is prospectively to be a valid tool for clinical enumeration of CTCs, carrying out anti-cancer drug assay.

Capturing and Clinical Applications of Circulating Tumor Cells with Wave Microfluidic Chip.

Pipette tip-based solid phase extraction (PT-SPE) has been proved to be an effective and user-friendly separation technique due to its miniaturized procedure and practical convenience. However, the vast majority of existing PT-SPE devices consist of a filter-sorbents-filter sandwich structure, which may suffer the unforeseen risk of sorbents leakage caused by the looseness of filters. More importantly, many high-capacity nanosorbents with particle size smaller than pore size of filters are unavailable. Thus, sorbent packing-free and sample low-consumption PT-SPE could be a more robust strategy for separation and detection, but such a possibility has not been explored yet. Herein we report a tubing reshaping strategy for facile fabrication of sorbent packing-free PT-SPE devices. Three types of reshaped PTs, namely stretched tube-like, self-crimping and filter in-built PTs, were fabricated via simple heating and stretching operations. The reshaped PTs exhibited flexible surface chemical post-modification. The SPE process was directly performed in reshaped PTs with an obviously enhanced extraction efficiency compared to once-shaping PTs while no need of packing sorbents. Extraction of nucleosides from human urine by boronic acid-functionalized reshaped PTs was demonstrated. Our findings technically renovate the structural composition of PT-SPE devices. As PTs are inexpensive and high-plasticity, the sorbent packing-free SPE scheme presented herein could find more promising applications and provides a new perspective for design and fabrication of novel sorbent packing-free SPE devices.

Reshaping of pipette tip: A facile and practical strategy for sorbent packing-free solid phase extraction.

Circulating tumor cells (CTCs) are tumor cells detached from the original lesion and getting into the blood and lymphatic circulation systems. They potentially establish new tumors in remote areas, namely, metastasis. Isolation of CTCs and following biological molecular analysis facilitate investigating cancer and coming out treatment. Since CTCs carry important information on the primary tumor, they are vital in exploring the mechanism of cancer, metastasis, and diagnosis. However, CTCs are very difficult to separate due to their extreme heterogeneity and rarity in blood. Recently, advanced technologies, such as nanosurfaces, quantum dots, and Raman spectroscopy, have been integrated with microfluidic chips. These achievements enable the next generation isolation technologies and subsequent biological analysis of CTCs. In this review, we summarize CTCs' separation with microfluidic chips based on the principle of immunomagnetic isolation of CTCs. Fundamental insights, clinical applications, and potential future directions are discussed.

Immunomagnetic separation of circulating tumor cells with microfluidic chips and their clinical applications.

Transparent glass ceramics (GCs) containing BaYbF:Er nanocrystals were successfully fabricated by a traditional melt-quenching method. The formation of BaYbF nanocrystals was confirmed by X-ray diffraction, transmission electron microscopy, and selected area electron diffraction. Compared with the precursor glass, the enhanced emission intensity and lifetime of GCs indicate that the Er ions incorporate into the BaYbF nanocrystals after crystallization. The color tuning properties with doping under 980 nm excitation have been systematically discussed. It was found that the red/green ratio increased with Er ion doping and the corresponding color changed from greenish-yellow to yellow-green. Furthermore, the temperature-dependent luminescence properties were studied in detail by the fluorescence intensity ratio (FIR) technique. The monotonic change of FIR with temperature indicates that this material is suitable for temperature sensing. At a temperature of 450 K, the relative sensitivity of the prepared sample reached its maximal value of 0.20% K. The results show that the GCs containing BaYbF:Er nanocrystals are candidate materials for temperature sensing.

Luminescence properties of BaYbF:Er nanocrystals embedded in glass ceramics for optical thermometry.

A filter paper-based colorimetric strategy for instrument-independent visual detection of artificial food colorants (FCs) was developed in this study. Indicator papers were prepared via the one-step polycondensation of silane coupling agents onto glass microfiber filter papers, and colorimetric cards with a fine one-to-one correspondence between their colors and FCs concentrations were straightforward obtained by the extraction of FCs with indicator papers by virtue of electrostatic interaction and hydrophobic effect. Filter papers post-modified via such a simple way were proved to be of improved binding class selectivity and colorimetric sensitivity, allowing for in-situ colorimetric assay of FCs in an unprecedently wide range of applicable pH (1.0-12.0) with high reliability and fine versatility. Finally, the semiquantitative naked-eye determination of FCs (Allura red, brilliant blue and sunset yellow) in real-world drink samples was experimentally confirmed to be feasible by comparison with the findings of UV-vis absorption spectra, HPLC and mass spectra.

Filter paper-based colorimetric analysis: An instrument-free strategy for semiquantitative naked-eye detection of food colorants.

The existing strategies for the determination of synthetic food colorants (FCs) in manufactured foods are highly relied on specialized instruments and skilled personnel which are limited by the high technical threshold and instrumentation cost. Herein, highly branched pipette tips (PTs) were fabricated as a robust all-in-one device for high-performance extraction and visual detection of FCs via handy aspiration and dispensing procedures of pipette controller. The density of extraction groups and inner specific surface area of PTs greatly increased after facile physical coating and subsequent layer-by-layer branching reactions, and the maximum increment in binding capacity of PTs was exceeded 300 times at 8-10 iterations of branching layers, enabling the PTs to be colored just by short-time extraction of FCs and to achieve the instrument-independent visual detection of FCs by virtue of their outstanding PT-SPE performance. As a proof-of-concept, the in-situ PT-based solid phase extraction (PT-SPE) with high recoveries (from 91.73 ± 4.76% to 99.90 ± 4.14%) and semiquantitative naked-eye detection of FCs (Allura red and brilliant blue) in real beverages were experimentally demonstrated to be highly feasible by comparison with classical techniques like spectrophotometry, HPLC, and mass spectrometry.

Semiquantitative naked-eye detection of synthetic food colorants using highly-branched pipette tip as an all-in-one device.

Altering the local symmetry of an activator by lattice tuning is considered an effective strategy to optimize the luminescence performance of phosphors. Herein, the novel Mn-activated double perovskite phosphor LaMgTaO (LMTO) with A-site defects was successfully prepared. Benefiting from the random occupation of the nearest A-site by cations and vacancies, the distorted [MnO] octahedra lack the inversion center. The LMTO:0.4 mol%Mn phosphor has a significant zero-phonon line intensity with a high internal quantum efficiency (IQE) value of 62.97% after the Laporte selection rule is broken. Furthermore, local lattice tuning was performed by increasing the randomness of the A-site and the distortion index of the [MnO] octahedron by co-doping with Ca or Sr ions. Importantly, the IQE value of the samples was enhanced from 62.97% to 72.65% and the activation energy increased from 0.497 eV to 0.548 eV, which can be well applied in the fields of plant cultivation and warm white light-emitting diodes. These studies provide valid fundamental insights for the selection of excellent luminescent matrices to obtain efficient Mn-activated phosphors  local lattice tuning.

Enhancing the luminescence performance of an LED-pumped Mn-activated highly efficient double perovskite phosphor with A-site defects  local lattice tuning.

Circulating tumor cells (CTCs) are cancer cells that are released from the original tumor and circulate in the blood vessels, carrying greatly similar constituents as the original tumor. Therefore, CTCs have a significant value in cancer prognosis, early diagnosis, and anti-cancer therapy. However, their rarity and heterogeneity make the isolation of CTCs an arduous task. In the present research, we propose a double spiral chip-embedded micro-trapezoid filter (SMT filter) for the sensitive isolation of the CTCs of prostate cancer by spectral detection. SMT filters were elongated to effectively capture CTCs and this distinctive design was conducive to their isolation and enrichment. The SMT filters were verified with tumor cells and artificial patient blood with a capture efficiency as high as 94% at a flow rate of 1.5 mL h. As a further validation, the SMT filters were validated in isolating CTCs from 10 prostate cancers and other cancers in 4 mL blood samples. Also, the CTCs tested positive for each patient blood sample, ranging from 83-114 CTCs. Significantly, we advanced hyperspectral imaging to detect the characteristic spectrum of CTCs both captured  on SMT filters and enriched after isolation. The CTCs could be positively identified by hyperspectral imaging with complete integrity of the cell morphology and an improved characteristic spectrum. This represents a breakthrough in the conventional surface-enhanced Raman scattering (SERS) spectroscopy of nanoparticles. Also, the characteristic spectrum of the CTCs would be highly beneficial for distinguishing the cancer type and accurate for enumerating tumor cells with varied intensities. Furthermore, a novel integrated flower-shaped microfilter was presented with all these aforementioned merits. The success of both the SMT filters and characteristic spectral detection indicated their feasibility for further clinical analysis, the evaluation of cancer therapy, and for potential application.

Hongmei Chen

School of Microelectronics and Data Science

Anhui University of Technology

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Hongmei Chen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Microelectronics and Data Science

Anhui University of Technology

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

School of Microelectronics and Data Science