DNA-DISK: Automated end-to-end data storage via enzymatic single-nucleotide DNA synthesis and sequencing on digital microfluidics.

In the age of information explosion, the exponential growth of digital data far exceeds the capacity of current mainstream storage media. DNA is emerging as a promising alternative due to its higher storage density, longer retention time, and lower power consumption. To date, commercially mature DNA synthesis and sequencing technologies allow for writing and reading of information on DNA with customization and convenience at the research level. However, under the disconnected and nonspecialized mode, DNA data storage encounters practical challenges, including susceptibility to errors, long storage latency, resource-intensive requirements, and elevated information security risks. Herein, we introduce a platform named DNA-DISK that seamlessly streamlined DNA synthesis, storage, and sequencing on digital microfluidics coupled with a tabletop device for automated end-to-end information storage. The single-nucleotide enzymatic DNA synthesis with biocapping strategy is utilized, offering an ecofriendly and cost-effective approach for data writing. A DNA encapsulation using thermo-responsive agarose is developed for on-chip solidification, not only eliminating data clutter but also preventing DNA degradation. Pyrosequencing is employed for in situ and accurate data reading. As a proof of concept, DNA-DISK successfully stored and retrieved a musical sheet file (228 bits) with lower write-to-read latency (4.4 min of latency per bit) as well as superior automation compared to other platforms, demonstrating its potential to evolve into a DNA Hard Disk Drive in the future.

Digital microfluidics-engaged automated enzymatic degradation and synthesis of oligosaccharides.

Glycans are an important group of natural biopolymers, which not only play the role of a major biological energy resource but also as signaling molecules. As a result, structural characterization or sequencing of glycans, as well as targeted synthesis of glycans, is of great interest for understanding their structure-function relationship. However, this generally involves tedious manual operations and high reagent consumptions, which are the main technical bottlenecks retarding the advances of both automatic glycan sequencing and synthesis. Until now, automated enzymatic glycan sequencers or synthesizers are still not available on the market. In this study, to promote the development of automation in glycan sequencing or synthesis, first, programmed degradation and synthesis of glycans catalyzed by enzymes were successfully conducted on a digital microfluidic (DMF) device by using microdroplets as microreactors. In order to develop automatic glycan synthesizers and sequencers, a strategy integrating enzymatic oligosaccharide degradation or synthesis and magnetic manipulation to realize the separation and purification process after enzymatic reactions was designed and performed on DMF. An automatic process for enzymatic degradation of tetra-N-acetyl chitotetraose was achieved. Furthermore, the two-step enzymatic synthesis of lacto-N-tetraose was successfully and efficiently completed on the DMF platform. This work demonstrated here would open the door to further develop automatic enzymatic glycan synthesizers or sequencers based on DMF.

Digital microfluidic platform assembled into a home-made studio for sample preparation and colorimetric sensing of S-nitrosocysteine.

Digital microfluidics (DMF) is a versatile lab-on-a-chip platform that allows integration with several types of sensors and detection techniques, including colorimetric sensors. Here, we propose, for the first time, the integration of DMF chips into a mini studio containing a 3D-printed holder with previously fixed UV-LEDs to promote sample degradation on the chip surface before a complete analytical procedure involving reagent mixture, colorimetric reaction, and detection through a webcam integrated on the equipment. As a proof-of-concept, the feasibility of the integrated system was successfully through the indirect analysis of S-nitrosocysteine (CySNO) in biological samples. For this purpose, UV-LEDs were explored to perform the photolytic cleavage of CySNO, thus generating nitrite and subproducts directly on DMF chip. Nitrite was then colorimetrically detected based on a modified Griess reaction, in which reagents were prepared through a programable movement of droplets on DMF devices. The assembling and the experimental parameters were optimized, and the proposed integration exhibited a satisfactory correlation with the results acquired using a desktop scanner. Under the optimal experimental conditions, the obtained CySNO degradation to nitrite was 96%. Considering the analytical parameters, the proposed approach revealed linear behavior in the CySNO concentration range between 12.5 and 400 µmol L-1 and a limit of detection equal to 2.8 µmol L-1. Synthetic serum and human plasma samples were successfully analyzed, and the achieved results did not statistically differ from the data recorded by spectrophotometry at the confidence level of 95%, thus indicating the huge potential of the integration between DMF and mini studio to promote complete analysis of lowmolecular weight compounds.

Design of a Hand-Held and Battery-Operated Digital Microfluidic Device Using EWOD for Lab-on-a-Chip Applications.

Microfluidics offer many advantages to Point of Care (POC) devices through lower reagent use and smaller size. Additionally, POC devices offer the unique potential to conduct tests outside of the laboratory. In particular, Electro-wetting on Dielectric (EWOD) microfluidics has been shown to be an effective way to move and mix liquids enabling many PoC devices. However, much of the research surrounding these microfluidic systems are focused on a single aspect of the system capability, such as droplet control or a specific new application at the device level using the EWOD technology. Often in these experiments the supporting systems required for operation are bench top equipment such as function generators, power supplies, and personal computers. Although various aspects of how an EWOD device is capable of moving and mixing droplets have been demonstrated at various levels, a complete self-contained and portable lab-on-a-chip system based on the EWOD technology has not been well demonstrated. For instance, EWOD systems tend to use high voltage alternating current (AC) signals to actuate electrodes, but little consideration is given to circuitry size or power consumption of such components to make the entire system portable. This paper demonstrates the feasibility of integrating all supporting hardware and software to correctly operate an EWOD device in a completely self-contained and battery-powered handheld unit. We present results that demonstrate a complete sample preparation flow for deoxyribonucleic acid (DNA) extraction and isolation. The device was designed to be a field deployable, hand-held platform capable of performing many other sample preparation tasks automatically. Liquids are transported using EWOD and controlled via a programmable microprocessor. The programmable nature of the device allows it to be configured for a variety of tests for different applications. Many considerations were given towards power consumption, size, and system complexity which make it ideal for use in a mobile environment. The results presented in this paper show a promising step forward to the portable capability of microfluidic devices based on the EWOD technology.

Designing Splicing Digital Microfluidics Chips Based on Polytetrafluoroethylene Membrane.

As a laboratory-on-a-chip application tool, digital microfluidics (DMF) technology is widely used in DNA-based applications, clinical diagnosis, chemical synthesis, and other fields. Additional components (such as heaters, centrifuges, mixers, etc.) are required in practical applications on DMF devices. In this paper, a DMF chip interconnection method based on electrowetting-on-dielectric (EWOD) was proposed. An open modified slippery liquid-infused porous surface (SLIPS) membrane was used as the dielectric-hydrophobic layer material, which consisted of polytetrafluoroethylene (PTFE) membrane and silicone oil. Indium tin oxide (ITO) glass was used to manufacture the DMF chip. In order to test the relationship between the splicing gap and droplet moving, the effect of the different electrodes on/off time on the minimum driving voltage when the droplet crossed a splicing gap was investigated. Then, the effects of splicing gaps of different widths, splicing heights, and electrode misalignments were investigated, respectively. The experimental results showed that a driving voltage of 119 V was required for a droplet to cross a splicing gap width of 300 µm when the droplet volume was 10 µL and the electrode on/off time was 600 ms. At the same time, the droplet could climb a height difference of 150 µm with 145 V, and 141 V was required when the electrode misalignment was 1000 µm. Finally, the minimum voltage was not obviously changed, when the same volume droplet with different aqueous solutions crossed the splicing gap, and the droplet could cross different chip types. These splicing solutions show high potential for simultaneous detection of multiple components in human body fluids.

A Low-Cost, Disposable and Portable Inkjet-Printed Biochip for the Developing World.

Electrowetting on dielectric-based digital microfluidic platforms (EWOD-DMF) have a potential to impact point-of-care diagnostics. Conventionally, EWOD-DMF platforms are manufactured in cleanrooms by expert technicians using costly and time consuming micro-nanofabrication processes such as optical lithography, depositions and etching. However, such high-end microfabrication facilities are extremely challenging to establish in resource-poor and low-income countries, due to their high capital investment and operating costs. This makes the fabrication of EWOD-DMF platforms extremely challenging in low-income countries, where such platforms are most needed for many applications such as point-of-care testing applications. To address this challenge, we present a low-cost and simple fabrication procedure for EWOD-DMF electrode arrays, which can be performed anywhere with a commercial office inkjet printer without the need of expensive cleanroom facilities. We demonstrate the utility of our platform to move and mix droplets of different reagents and physiologically conductive buffers, thereby showing its capability to potentially perform a variety of biochemical assays. By combining our low-cost, inkjet-printed EWOD-DMF platform with smartphone imaging technology and a compact control system for droplet manipulation, we also demonstrate a portable and hand-held device which can be programmed to potentially perform a variety of biochemical assays.