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Accelerating Synthetic Biology Using Picodroplets

An interview with Xin Li, Associate Director of Science at Sphere Fluidics




Using ESI-Mine™, researchers can now sort and analyze up to 200,000 samples a day. Providing an ultra-high-throughput mass spectrometry screening method to transform synthetic biology workflows.

In this blog post, we spoke with Dr. Xin Li, Associate Director of Science at Sphere Fluidics, to explore how picodroplet technology can overcome the current synthetic biology workflow challenges. We also discussed some of his latest R&D work at Sphere Fluidics.



Overcoming challenges in Synthetic Biology workflows

Mass spectrometry (MS) is a powerful, routine detection method for identifying compounds and analyzing their structure and reactivity and for the qualitative and quantitative analysis of various compounds. However, its screening throughput (5,000 – 15,000 samples per day) is currently a significant bottleneck in the field of synthetic biology research.

Analyzing engineered cell libraries using traditional plate-based approaches remains incredibly time-consuming although recent advances have greatly improved the screening throughput. For example, Agilent RapidFire 400 can run one cycle of sample injection per 8 seconds. Another example is acoustic droplet ejection (ADE) technology assisted sampling and ionization developed by Labcyte Inc, which is now part of Beckman Coulter Life Sciences, that has an MS analyzing power of 1-2 samples per second reaching to a screening throughput 100,000 samples per day.

ESI-Mine™ interfaces with MS instruments to simplify the process of screening pooled libraries of synthetic enzymes using high-throughput mass spectrometry and picodroplet microfluidics. Picodroplets, picolitre volume droplets, increase the per day throughput by orders of magnitude. Thus, enabling researchers to sort and analyze up to 200,000 samples a day.

As well as the enhanced throughput, miniaturized picodroplets also reduce the required sample and reagent volumes, from 100 L using standard plate-based sample preparation platforms to 1 mL (for analyzing 1 million mutants) in picodroplets. They also eliminate the need for expensive equipment such as colony pickers, centrifuges, and incubators, resulting in massive cost savings. By implementing picodroplet technologies, one industrial partner stated that they could achieve £500,000 savings in consumable costs (Figure 1). As a result, single cell analysis methods become considerably more cost-efficient, not even including the significant reduction in labor costs by increasing throughput.


Figure 1. Cost savings achieved with the implementation of picodroplet technologies compared to a microplate approach.



The significant time and consumable savings are only some of the benefits of picodroplets. Perhaps the most notable feature is ESI-Mine™'s unique microfluidic workflow that enables researchers to analyze samples and retrieve replicates (based on the MS analysis result) for further analysis.

MS is a destructive analysis method, preventing the direct sorting of 'hit' molecules or samples of interest. We circumvent this problem by implementing picodroplet splitting and sorting into the microfluidic design. Using ESI-Mine™, picodroplets generated from a sample are split on the biochip, one sibling picodroplet is used for the MS analysis, and the other is sorted based on the result (Figure 2).


Application of ESI-Mine™ in Synthetic Biology

Sphere Fluidics has collaborated with a Top 10 Pharma Company to combine picodroplet technology with MS to monitor enzyme reactions. The project involved using synthetic biology to engineer an E. coli cell capable of producing different variants of a particular enzyme. The enzyme variants interacted with a common substrate and produced product at different speeds. ESI-Mine™ was used to analyze the array results and screen for enhanced attributes, enabling researchers to find the highest concentration of the product molecule.

Figure 2. Complete Synthetic Biology workflow using ESI-Mine™


Cells are encapsulated into picodroplets in a one-cell-one-droplet (OCOD) manner, creating a library of picodroplets that can express and retain novel proteins or other hydrophilic entities. The picodroplets are incubated to enable cell growth, protein expression, and metabolic processes to occur. They are then reinjected into the proprietary microfluidic emitter biochip on the ESI-Mine™ platform for label-free screening followed by real-time sorting based on MS analysis results. Each picodroplet is asymmetrically split at this stage, producing two daughter picodroplets from the same parent picodroplet. ESI-MS analyzes the larger daughter picodroplet. Its smaller sibling picodroplet is processed through microfluidic channels and sorted based on the MS signal. Hits are then collected as a pool for further downstream analysis.


Ongoing research and development at Sphere Fluidics

A major driving force behind our research is the possibility of improving screening throughput even further. We are currently operating at 30 Hz MS scan rate, limited by the speed of data processing and transfer. As we unlock these limitations and achieve a proportional increase, ESI-Mine™ will potentially offer sample analysis throughput (beyond the current 3 Hz) of at least 10 Hz. This is significantly faster than any existing MS analysis method. Alongside advancements in screening, our team is working on several complex challenges such as generating novel surfactants and trapping a broader range of molecules. This is to expand the application scope to include drug screening, metabolism profiling and diagnosis.

Sphere Fluidics is actively exploring new applications of ESI-Mine™ technology. We have currently demonstrated the ability to use ESI-Mine™ with samples containing small molecules and proteins. But researchers can potentially use the ESI-Mine™ platform in any MS-based screening application. We foresee a broader class of molecules and scientific challenges that could be transformed using ESI-Mine™, ranging from environmental monitoring to gene expression profiling to clinical analysis. Within the clinical field, ESI-Mine™ could be used for drug analysis. Mass spectrometry coupled to liquid chromatography (LC-MS) is a popular drug screening method, but it remains labor-intensive and has limited throughput despite integration with automated robots. These limitations can easily be overcome using ESI-Mine™.

We are also collaborating extensively with Manchester Institute of Biotechnology on coupling microdroplet microfluidics with different MS platforms. They have a wide range of MS systems from different vendors, providing a valuable platform/hub for new research and development. Findings from this work can be found in the paper entitled 'Coupling Droplet Microfluidics with Mass Spectrometry for Ultrahigh-Throughput Analysis of Complex Mixtures up to and above 30 Hz', by Kempa et al.



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