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

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.

Myoglobin (Mb) is an ideal biochemical marker for the diagnosis of certain diseases caused by damage to heart muscle or skeletal muscle. Nevertheless, serum myoglobin levels are usually very low while the interference components in real sample are extremely abundent. Hence, it is of great clinical significance to establish an effective method for Mb targeting. To obtain desired selectivity, targeting biomolecules like antibody and aptamer are essential to 'the state of the art'. However, such biomolecules suffer from many disadvantages, such as hard to prepare, susceptible to protease degradation, and high cost. Thus, novel alternatives that can overcome these issues are highly desirable. Herein, we pioneered a template-anchored controllable surface imprinting strategy for selective extraction of Mb from human serum via combining with facile magnetic separation of magnetic nanoparticles (MNPs). Mb-imprinted MNPs, as antibody-biomimetic materials, were prepared using amino group-modified MNPs as substrates and water-soluble self-polymerizable dopamine as imprinting monomer. The optimized imprinting time was 70 min, giving an optimal performance with high practical imprinting efficiency (up to 41%), high imprinting factor (4.2), high binding affinity (K=(2.05 ± 0.09) × 10 M), as well as excellent recognition selectivity. Moreover, compared to bare MNPs, Mb-imprinted MNPs possessed markedly better pH tolerance. Finally, the selective extraction of Mb from human serum sample by Mb-imprinted MNPs was experimentally confirmed and the recoveries of Mb in spiked serum ranged from (91.12 ± 6.81)% to (107.99 ± 7.76)%, indicating that the Mb-imprinted MNPs could be competent for the selective analysis of Mb in real bio-samples like human serum with high precision and reliability.

Selective extraction of myoglobin from human serum with antibody-biomimetic magnetic nanoparticles.

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.

Flavonoids have many positive pharmacological properties, such as antioxidant, antitumor, and anti-inflammatory activities. However, factors such as low water solubility and low dissolution rate limit their use. To overcome their poor solubility, carrier-free apigenin (API) microparticles and nanoparticles were prepared using three types of antisolvent precipitation technologies: supercritical antisolvent (SCF) technology, ultrasonic-assisted liquid antisolvent (UAL) technology, and high-pressure homogenization (HPH) technology. All three technologies can produce uniform tiny particles. However, the API particles obtained using these different techniques show subtle differences in terms of physical and chemical properties and biological activity. The preparation, characterization, and potential use of API microparticles and nanoparticles to improve in vitro release were studied. The resulting API particles were investigated and compared using Fourier-transform infrared spectroscopy, differential scanning calorimetry, X-ray powder diffraction, and scanning electron microscopy. We determined the optimum conditions for SCF, UAL, and HPH technologies to produce API microparticles and nanoparticles. The antioxidant and antitumor properties of the API particles were also investigated. The results demonstrated that the reduced particle size of the APIs prepared via SCF, UAL, and HPH technologies contributed to the enhanced dissolution rate, which in turn enhanced API bioactivity.

Fabrication of apigenin nanoparticles using antisolvent crystallization technology: A comparison of supercritical antisolvent, ultrasonic-assisted liquid antisolvent, and high-pressure homogenization technologies.

Herein, a strategy combining colorimetry and inner filter effect (IFE)-based fluorometry was developed for multimode visualization of food dyes (FDs) using CdTe quantum-dots-doped fluorescent indicator papers as a sample-to-answer device. Colorimetry was straightforwardly achieved by FDs extraction through electrostatic interaction and hydrophobic effect while fluorometry was implemented by IFE-induced fluorescence quenching. RGB/gray-scale values of colorimetry and fluorometry were furtherly picked by a smartphone application and applied to reconstruct color information-based digital image analysis for both direct alignments and linear regression analysis. The apparent color and fluorescence of FDs-bound indicator papers, together with their digitized color information, showed a good mapping to FDs concentrations in the range of 0-0.5 mg/mL for Sunset Yellow, 0-0.2 mg/mL for Allura Red, and 0-0.08 mg/mL for Brilliant Blue. As a proof of concept, the dosages of these FDs in real beverages and simulated dye effluents were deduced and cross-validated by different visualization modes, and finally double-checked by instrumental techniques such as spectrometric methods, high-performance liquid chromatography (HPLC), and mass spectroscopy (MS). The above findings concluded that (i) IFE mechanism is generally applicable to build fluorometric systems and (ii) cross validation of different visualization modes can markedly improve detection accuracy, which may provide references for design and fabrication of novel "lab-on-paper" devices for visualization applications with high reliability.

Colorimetry Combined with Inner Filter Effect-Based Fluorometry: A Versatile and Robust Strategy for Multimode Visualization of Food Dyes.

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.

Double spiral chip-embedded micro-trapezoid filters (SMT filters) for the sensitive isolation of CTCs of prostate cancer by spectral detection.

Herein, a multi-mode visualization platform was initiated for in-situ detection of food dyes (FDs) by combining colorimetry, fluorometry and smartphone‑based digital image analysis, in which water-dispersible quantum dots (QDs) were served as nanoprobes. Colorimetry was achieved by color comparison, while both fluorometry and fluorescence quantification were performed through inner filter effect (IFE)-induced fluorescence quenching, then color information (RGB & gray-scale values) of colorimetry and fluorometry was picked by a smartphone to reconstruct digitized alignments. Since IFE mechanism was concentration-dependent but did not rely on the interaction between fluorophore and quencher, the whole process of fluorescence response could be finished within 10 s, and both color gradients and fluorescence changes showed fine mappings to FDs concentrations in the range of 1.0 × 10∼0.035 mg/mL for brilliant blue, and 1.0 × 10∼0.1 mg/mL for Allura red and sunset yellow. As a proof-of-concept, the in-situ multi-mode visualization of these FDs in real beverages was experimentally proved to be highly feasible and reliable as compared with instrumental techniques like UV-vis/fluorescence spectrometry, along with HPLC. Finally, this strategy was extended to the multi-mode visualization of non-food dyes in three simulated wastewater samples with high credibility by contrast with the true additive amounts of model dyes.

Combination of colorimetry, inner filter effect-induced fluorometry and smartphone‑based digital image analysis: A versatile and reliable strategy for multi-mode visualization of food dyes.

Shuangshou Wang

School of Chemistry and Chemical Engineering

Anhui University of Technology

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

Shuangshou Wang

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

Anhui University of Technology

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

School of Chemistry and Chemical Engineering