Advances in Concentration Gradient Generation Approaches in a Microfluidic Device for Toxicity Analysis

Abstract

This systematic review aimed to analyze the development and functionality of microfluidic concentration gradient generators (CGGs) for toxicological evaluation of different biological organisms. We searched articles using the keywords: concentration gradient generator, toxicity, and microfluidic device. Only 33 of the 352 articles found were included and examined regarding the fabrication of the microdevices, the characteristics of the CGG, the biological model, and the desired results. The main fabrication method was soft lithography, using polydimethylsiloxane (PDMS) material (91%) and SU-8 as the mold (58.3%). New technologies were applied to minimize shear and bubble problems, reduce costs, and accelerate prototyping. The Christmas tree CGG design and its variations were the most reported in the studies, as well as the convective method of generation (61%). Biological models included bacteria and nematodes for antibiotic screening, microalgae for pollutant toxicity, tumor and normal cells for, primarily, chemotherapy screening, and Zebrafish embryos for drug and metal developmental toxicity. The toxic effects of each concentration generated were evaluated mostly with imaging and microscopy techniques. This study showed an advantage of CGGs over other techniques and their applicability for several biological models. Even with soft lithography, PDMS, and Christmas tree being more popular in their respective categories, current studies aim to apply new technologies and intricate architectures to improve testing effectiveness and reduce common microfluidics problems, allowing for high applicability of toxicity tests in different medical and environmental models.

Keywords: CGG; concentration gradient generator; drug screening; microdevice; microdevice gradient generator; microfluidic device; toxicity.

Figure 1

Schematic representation of the process for articles’ identification, screening, and eligibility for inclusion in this systematic review following the PRISMA guidelines.

Figure 2

Schematic diagram of microfluidic devices with CGG system for toxicological analysis, representing some of the studies selected in this systematic review. (A) Representation of device layers, gradient generator structure, details of fluid mixing units and air bubble valves. Adapted with permission from [44], Biosensors and Bioelectronics. (B) Project showing a physical map of the CGG system and the photomicrograph of Pyramimonas sp. and Chlorella. Adapted with permission from [23], Sensors (Switzerland). (C) A schematic showing the design of a μFSCD with a concentration gradient generator. It exposed the structures, dimensions, and characteristics of the two layers, adapted with permission from [34], Molecules. (D) Construction of the Sphero Chip system proving the measurement principle of the experimental scheme and results of the computational modeling of a CGG structure. Adapted with permission from [41], Lab on a Chip. (E) The microdevice contains eight sets of C-Chambers, which can simultaneously enable eight sets of noninterfering ASTs with each other. Antibiotics can be preincorporated into the C chambers with a specific mass gradient. AST and MIC results can be obtained by comparing the fluorescence intensities between each set of C-Chambers. Adapted with permission from [16], Biosensors and Bioelectronics. (F) CGG microdevice used for toxicity tests based on marine phytoplankton motility containing four units connected to a central removable outlet. Shown is the enlarged image of the single-frame unit containing an upstream CGG and downstream diffusible cameras. Motility signals can be collected in real time. Adapted with permission from [25], Marine Pollution Bulletin. (G) Schematic design of the CGG microfluidic chip with cell chambers (top panel) and the chip manufactured with pumping machine (bottom panel). Chamber-diffused Rh-123 (green) and morphological characteristics of A549 cells with or without CAF matrix are shown. Adapted with permission from [37], PLoS ONE. (H) CGG containing four parallel operational modules including inputs CSE: 18 parallel cell chambers and 6 cell inputs. A CSE concentration gradient is shown from entry one to six, adapted with permission from [40], Journal of Thoracic Oncology. (I) Schematic overview of the microfluidic device with a CGG and chambers with passive hydrodynamic cell trap arrays. It shows details of branching and diffusional mixing of two fluorescent fluids with different concentrations and optical micrograph of cell traps in PDMS. Adapted with permission from [42], Lab on a Chip. (J) Schematic drawing of the CGG device, illustrating cross-section and theoretical profiles of Ciprofloxacin concentration in the observation channel. Antibiotic solutions with 3× MIC (blue curve) or 6× MIC (orange curve). Adapted with permission from [17], Frontiers in Microbiology. Abbreviations: CGG: concentration gradient generator; μFSCD: microfluidic spheroid culture device; ASTs: antibiotic susceptibility tests; Rh−123: Rhodamine; A549: adenocarcinoma human alveolar basal epithelial cell line; CAF: cancer-associated fibroblasts; CSE: cigarette smoke extracts; PDMS: polydimethylsiloxane; MIC: minimal inhibitory concentration.

Figure 3

The systematic review identified 4 main types of organisms used for toxicity analysis using the CGG system in microfluidic devices: microalgae, zebrafish embryo, tumor cells and other models, and microorganisms. The figure shows the main important aspects (as percentages) regarding the microfluidic device material, manufacturing technology, gradient system, culture environment, culture site, biological model, and toxicity condition. Abbreviations: PDMS: polydimethylsiloxane; SC: serpentine channel; CGG: concentration gradient generator; RM: red microalgae; CDDP: Cisplatin; 5-FU: 5-Fluorouracil; DOX: Doxorubicin; SM: silicon; SPD-CGG: static-pressure-driven CGG; NR: not reported; Sac-Cer: Saccharomyces cerevisiae; PTX: Paclitaxel; AMP: Ampicillin; TAC: Tetracycline; CIPRO: Ciprofloxacin.