Graphene oxide aptasensor droplet assay for detection of metabolites secreted by single cells applied to synthetic biology.

Synthetic biology harnesses the power of natural microbes by re-engineering metabolic pathways to manufacture desired compounds. Droplet technology has emerged as a high-throughput tool to screen single cells for synthetic biology, while the challenges in sensitive flexible single-cell secretion assay for bioproduction of high-value chemicals remained. Here, a novel droplet modifiable graphene oxide (GO) aptasensor was developed, enabling sensitive flexible detection of different target compounds secreted from single cells. Fluorophore-labeled aptamers were stably anchored on GO through π-π stacking interactions to minimize the non-specific interactions for low-background detection of target compounds with high signal-to-noise ratios. The assay's versatility was exhibited by adapting aptamer sequences to measure metabolic secretions like ATP and naringenin. To show the case, engineered E. coli were constructed for the bioproduction of naringenin. The high signal-to-noise ratio assay (∼2.72) was approached to precisely measure the naringenins secreted from single E. coli in the droplets. Consequently, secretory cells (Gib) were clearly distinguished from wild-type (WT) cells, with a low overlap in cell populations (∼0%) for bioproduction.

Development of Paper Microfluidics with 3D-Printed PDMS Barriers for Flow Control.

Paper microfluidics has been extensively exploited as a powerful tool for environmental and medical detection applications. Both flow delay and compatibility with either polar or non-polar reagents are indispensable for the automation of detections requiring multiple reaction steps. This article reports the systematic studies of a 3D-printing protocol, characterization, and application of both the partially and fully penetrated polydimethylsiloxane (PDMS) barriers for flexible flow control in paper microfluidics. The physical parameters of PDMS barriers printed using a simple liquid dispenser were found related to the printing pressure, speed, diffusion time after printing, baking temperature, and PDMS viscosity. The capability of PDMS barriers to confine the flow of non-polar solvents was demonstrated using oil flow in both wax- and PDMS-surrounded channels. It was identified that the minimum width of channels to prevent leakage was 470 ± 54 µm, which was as narrow as that fabricated using stamps from lithography. Both the partially penetrated barriers (PPBs) and constriction channels were of the capability to delay flow in paper microfluidics. Additionally, an in silico investigation led to the further understanding that the reduction of channel cross-section resulting from PPBs was the primary reason for flow delay. Our results suggest that increasing the penetration depth of the barriers is more efficient in delaying flow than increasing the PPB length. Finally, devices with four inlet channels and 0-6 PPBs across each channel were successfully applied in flow delay for sequential fluid delivery. These results improve the understanding of the major factors, affecting the 3D PDMS barrier fabrication and the resulting flow control in paper microfluidics, providing practical implications for applications in various fields.

Size-Based Sorting and In Situ Clonal Expansion of Single Cells Using Microfluidics.

Separation and clonal culture and growth kinetics analysis of target cells in a mixed population is critical for pathological research, disease diagnosis, and cell therapy. However, long-term culture with time-lapse imaging of the isolated cells for clonal analysis is still challenging. This paper reports a microfluidic device with four-level filtration channels and a pneumatic microvalve for size sorting and in situ clonal culture of single cells. The valve was on top of the filtration channels and used to direct fluid flow by membrane deformation during separation and long-term culture to avoid shear-induced cell deformation. Numerical simulations were performed to evaluate the influence of device parameters affecting the pressure drop across the filtration channels. Then, a droplet model was employed to evaluate the impact of cell viscosity, cell size, and channel width on the pressure drop inducing cell deformation. Experiments showed that filtration channels with a width of 7, 10, 13, or 17 µm successfully sorted K562 cells into four different size ranges at low driving pressure. The maximum efficiency of separating K562 cells from media and whole blood was 98.6% and 89.7%, respectively. Finally, the trapped single cells were cultured in situ for 4-7 days with time-lapse imaging to obtain the lineage trees and growth curves. Then, the time to the first division, variation of cell size before and after division, and cell fusion were investigated. This proved that cells at the G1 and G2 phases were of significantly distinct sizes. The microfluidic device for size sorting and clonal expansion will be of tremendous application potential in single-cell studies.

Automatic flow delay through passive wax valves for paper-based analytical devices.

Microfluidic paper-based analytical devices (µPADs) have been widely explored for point-of-care testing due to their simplicity, low cost, and portability. µPADs with multiple-step reactions usually require precise flow control, especially flow-delay. This paper reports the numerical, mathematical, and experimental studies of flow delay through wax valves surrounded by PDMS walls on paper microfluidics. The predried surfactant in the sample zone diffuses into the liquid sample which can therefore flow through the wax valves. The delay time is automatically regulated by the diffusion of the surfactant after sample loading. The numerical study suggested that both the elevated contact angle and the reduced porosity and pore size in the wax printed region could effectively prevent water but allow liquids with lower contact angles (e.g., surfactant solutions) to flow through. The PDMS walls fabricated using a low-cost liquid dispenser effectively prevented the leakage of surfactant solutions. By controlling the quantity, diffusion distance, and type of the surfactant predried on the chip, the system successfully achieved a delay time ranging from 1.6 to 20 minutes. A mathematical model involving the above parameters was developed based on Fick's second law to predict the delay time. Finally, the flow-delay systems were applied in sequential mixing and distance-based detection of either glucose or alcohol. Linear ranges of 1-100 mg dL-1 and 1-40 mg dL-1 were achieved for glucose and alcohol, respectively. The lower limit detection (LOD) of glucose and alcohol was 1 mg dL-1. The LOD of glucose was only 1/11 of that detected using µPADs without flow control, indicating the advantage of controlling fluid flow. The systematic findings in this study provide critical guidelines for the development and applications of wax valves in automatic flow delay for point-of-care testing.