Facile and Rapid Generation of Large-Scale Microcollagen Gel Array for Long-Term Single-Cell 3D Culture and Cell Proliferation Heterogeneity Analysis

Microfabricated devices are suitable for single-cell analysis due to their high throughput, compatible dimensions and controllable microenvironment. However, existing devices for single-cell culture and analysis encounter some limitations, such as nutrient depletion, random cell migration and complicated fluid shear influence. Moreover, most of the single-cell culture and analysis devices are based on 2D cell culture conditions, even though 3D cell culture methods have been demonstrated to better mimic the real cell microenvironment in vivo. To solve these problems, herein we develop a microcollagen gel array (μCGA) based approach for high-throughput long-term single-cell culture and single-cell analysis under 3D culture conditions. Type-I collagen, a well-established 3D cell culture medium, was used as the scaffold for 3D cell growth. A 2 × 2 cm PDMS chip with 10 000 μCGA units was fabricated to encapsulate thousands of single cells in less than 15 min. Single cells were able to be confined and survive in μCGA units for more than 1 month. The capability of large-scale and long-term single-cell 3D culture under open culture conditions allows us to study cellular proliferation heterogeneity and drug cytotoxicity at the single-cell level. Compared with existing devices for single-cell analysis, μCGA solves the problems of nutrient depletion and random cellular migration, avoids the influence of complicated fluid shear, and mimics the real 3D growth environment in vivo, thereby providing a feasible 3D long-term single-cell culture method for single-cell analysis and drug screening

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol–gel switching property of agarose enable formation of stable beads by chilling the droplet array at −20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol–gel switching property of agarose enable formation of stable beads by chilling the droplet array at −20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis

A Cell-Surface-Anchored Ratiometric Fluorescent Probe for Extracellular pH Sensing

Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid–DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid–DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid–DNA probe showed sensitive and reversible response to pH change in the range of 6.0–8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid–DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid–DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies

Integrating Target-Responsive Hydrogel with Pressuremeter Readout Enables Simple, Sensitive, User-Friendly, Quantitative Point-of-Care Testing

Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation, is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the cross-linking DNA for selective target recognition. The hydrogel response to the target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H₂O₂ to O₂. Then, the generated O₂ in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A, and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings