Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells.

Recently, stretchable electrochemical sensors have stood out as a powerful tool for the detection of soft cells and tissues, since they could perfectly comply with the deformation of living organisms and synchronously monitor mechanically evoked biomolecule release. However, existing strategies for the fabrication of stretchable electrochemical sensors still face with huge challenges due to scarce electrode materials, demanding processing techniques and great complexity in further functionalization. Herein, we report a novel and facile strategy for one-step preparation of stretchable electrochemical biosensors by doping ionic liquid and catalyst into a conductive polymer (poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate), PEDOT:PSS). Bis(trifluoromethane) sulfonimide lithium salt as a small-molecule plasticizer can significantly improve the stretchability and conductivity of the PEDOT:PSS film, and cobalt phthalocyanine as an electrocatalyst endows the film with excellent electrochemical sensing performance. Moreover, the functionalized PEDOT:PSS retained good cell biocompatibility with two extra dopants. These satisfactory properties allowed the real-time monitoring of stretch-induced transient hydrogen peroxide release from cells. This work presents a versatile strategy to fabricate conductive polymer-based stretchable electrodes with easy processing and excellent performance, which benefits the in-depth exploration of sophisticated life activities by electrochemical sensing.

Correction: Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells.

[This corrects the article DOI: 10.1039/D1SC04138J.].

Near-Infrared Light-Responsive Hydrogel for Specific Recognition and Photothermal Site-Release of Circulating Tumor Cells.

Isolation of single circulating tumor cells (CTCs) from patients is a very challenging technique that may promote the process of individualized antitumor therapies. However, there exist few systems capable of highly efficient capture and release of single CTCs with high viability for downstream analysis and culture. Herein, we designed a near-infrared (NIR) light-responsive substrate for highly efficient immunocapture and biocompatible site-release of CTCs by a combination of the photothermal effect of gold nanorods (GNRs) and a thermoresponsive hydrogel. The substrate was fabricated by imprinting target cancer cells on a GNR-pre-embedded gelatin hydrogel. Micro/nanostructures generated by cell imprinting produce artificial receptors for cancer cells to improve capture efficiency. Temperature-responsive gelatin dissolves rapidly at 37 °C; this allows bulk recovery of captured CTCs at physiological temperature or site-specific release of single CTCs by NIR-mediated photothermal activation of embedded GNRs. Furthermore, the system has been applied to capture, individually release, and genetically analyze CTCs from the whole blood of cancer patients. The multifunctional NIR-responsive platform demonstrates excellent performance in capture and site-release of CTCs with high viability, which provides a robust and versatile means toward individualized antitumor therapies and also shows promising potential for dynamically manipulating cell-substrate interactions in vitro.

High-Efficiency Capture of Individual and Cluster of Circulating Tumor Cells by a Microchip Embedded with Three-Dimensional Poly(dimethylsiloxane) Scaffold.

Effective isolation of circulating tumor cells (CTCs) has great significance for cancer research but is highly challenged. Here, we developed a microchip embedded with a three-dimensional (3D) PDMS scaffold by a quadratic-sacrificing template method for high-efficiency capture of CTCs. The microchip was gifted with a 3D interconnected macroporous structure, strong toughness, and excellent flexibility and transparency, enabling fast isolation and convenient observation of CTCs. Especially, 3D scaffold chip perfectly integrates the two main strategies currently used for enhancement of cell capture efficiency. Spatially distributed 3D scaffold compels cells undergoing chaotic or vortex migration in the channel, and the spatially distributed nanorough skeleton offers ample binding sites, which synergistically and significantly improve CTCs capture efficiency. Our results showed that 1-118 CTCs/mL were identified from 14 cancer patients' blood and 5 out of these cancer patients showed 1-14 CTC clusters/mL. This work demonstrates for the first time the development of microchip with transparent interconnected 3D scaffold for isolation of CTCs and CTC clusters, which may promote in-depth analysis of CTCs.

Determination of DNA and RNA Methylation in Circulating Tumor Cells by Mass Spectrometry.

DNA methylation (5-methylcytosine, 5-mC) is the best characterized epigenetic mark that has regulatory roles in diverse biological processes. Recent investigation of RNA modifications also raises the possible functions of RNA adenine and cytosine methylations on gene regulation in the form of "RNA epigenetics." Previous studies demonstrated global DNA hypomethylation in tumor tissues compared to healthy controls. However, DNA and RNA methylation in circulating tumor cells (CTCs) that are derived from tumors are still a mystery due to the lack of proper analytical methods. In this respect, here we established an effective CTCs capture system conjugated with a combined strategy of sample preparation for the captured CTCs lysis, nucleic acids digestion, and nucleosides extraction in one tube. The resulting nucleosides were then further analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). With the developed method, we are able to detect DNA and RNA methylation (5-methyl-2'-deoxycytidine, 5-methylcytidine, and N(6)-methyladenosine) in a single cell. We then further successfully determined DNA and RNA methylation in CTCs from lung cancer patients. Our results demonstrated, for the first time, a significant decrease of DNA methylation (5-methyl-2'-deoxycytidine) and increase of RNA adenine and cytosine methylations (N(6)-methyladenosine and 5-methylcytidine) in CTCs compared with whole blood cells. The discovery of DNA hypomethylation and RNA hypermethylation in CTCs in the current study together with previous reports of global DNA hypomethylation in tumor tissues suggest that nucleic acid modifications play important roles in the formation and development of cancer cells. This work constitutes the first step for the investigation of DNA and RNA methylation in CTCs, which may facilitate uncovering the metastasis mechanism of cancers in the future.

Biotin-triggered decomposable immunomagnetic beads for capture and release of circulating tumor cells.

Isolation of rare, pure, and viable circulating tumor cells (CTCs) provides a significant insight in early cancer diagnosis, and release of captured CTCs without damage for ex vivo culture may offer an opportunity for personalized cancer therapy. In this work, we described a biotin-triggered decomposable immunomagnetic system, in which peptide-tagged antibody designed by chemical conjugation was specifically immobilized on engineered protein-coated magnetic beads. The interaction between peptide and engineered protein can be reversibly destroyed by biotin treatment, making capture and release of CTCs possible. Furthermore, the peptide could mediate multiple antibodies' coimmobilization on engineered protein-coated magnetic beads, by which capture efficiency for CTCs was obviously improved. Quantitative results showed that 70% of captured cells could be released by biotin addition, and 85% of released cells remained viable. In addition, 79% of cancer cells spiked in human whole blood were captured and could also be successfully released for culture. Finally, immunomagnetic beads simultaneously loaded with anti-EpCAM, anti-HER2, and anti-EGFR were successfully applied to isolate and detect CTCs in 17 cancer patients' peripheral blood samples, and 2-215 CTCs were identified with high purity. These results suggest that our method is reliable and has great potential in CTC detection for CTC-based molecular profiling, diagnosis, and therapy.

Real-time monitoring of H2O2 release from single cells using nanoporous gold microelectrodes decorated with platinum nanoparticles.

Here, we report a self-supported nanoporous gold microelectrode decorated with well-dispersed and tiny platinum nanoparticles as an electrochemical nonenzymatic hydrogen peroxide biosensor. Nanoporous gold was fabricated by electrochemical alloying/dealloying and then small-sized platinum nanoparticles were electrodeposited uniformly on them. This novel hybrid nanostructure endows the sensor with high sensitivity and selectivity towards the reduction of hydrogen peroxide with a low detection limit of 0.3 nM. The sensor has been successfully applied for the measurement of H2O2 release from a single isolated human breast cancer cell, demonstrating its great potential for further physiological and pathological applications.

Photoresponsive immunomagnetic nanocarrier for capture and release of rare circulating tumor cells.

Isolation, release and culture of rare circulating tumor cells (CTCs) may, if implemented, promote the progress of individualized anti-tumor therapies. To realize the release of CTCs without disruption of their viability for further culture and analysis, we designed an effective photocontrolled CTC capture/release system by combination of photochemistry and immunomagnetic separation. 7-Aminocoumarin was synthesized as the phototrigger to bridge the connection between the anti-EpCAM antibody and the magnetic beads. The coumarin moieties produced cleavage of a C-O bond under both ultraviolet (UV) and near-infrared (NIR) light illumination, breaking the bridge and releasing CTCs from the immunomagnetic beads. Compared with conventional immunomagnetic separation systems, the negative influence of absorbed immunomagnetic beads on further CTCs culture and analysis was effectively eliminated. The system can specifically recognize 102 MCF-7 cells in 1 mL of human whole blood samples with 90% efficiency and 85% purity. Under the irradiation of UV and NIR light, 73 ± 4% and 52 ± 6% of captured cells were released with a viability of 90% and 97%, respectively. Furthermore, this technique has been used to detect CTCs from whole blood of cancer patients with high purity. This study demonstrates that the photochemical-based immunomagnetic separation method for isolating, releasing and culturing CTCs from clinic patients may provide new opportunities for cancer diagnosis and personalized therapy.

Engineering interconnected 3D vascular networks in hydrogels using molded sodium alginate lattice as the sacrificial template.

Engineering 3D perfusable vascular networks in vitro and reproducing the physiological environment of blood vessels is very challenging for tissue engineering and investigation of blood vessel function. Here, we engineer interconnected 3D microfluidic vascular networks in hydrogels using molded sodium alginate lattice as sacrificial templates. The sacrificial templates are rapidly replicated in polydimethylsiloxane (PDMS) microfluidic chips via Ca⁺²-crosslinking and then fully encapsulated in hydrogels. Interconnected channels with well controlled size and morphology are obtained by dissolving the monolayer or multilayer templates with EDTA solution. The human umbilical vein endothelial cells (HUVECs) are cultured on the channel linings and proliferated to form vascular lumens. The strong cell adhesion capability and adaptive response to shear stress demonstrate the excellent cytocompatibility of both the template and template-sacrificing process. Furthermore, the barrier function of the endothelial layer is characterized and the results show that a confluent endothelial monolayer is fully developed. Taken together, we develop a facile and rapid approach to engineer a vascular model that could be potentially used in physiological studies of vascular functions and vascular tissue engineering.