Caption: The microfluidic sorting device removes inactive and unwanted compounds, dumping the drops into a “bad egg” bin, and guides the others into a “keep” container. Specifically, as the drops flow through the channels they eventually encounter a junction (a two-channel fork). The device identifies the desired drops by using a laser focused on the channel before the fork to read a drop’s fluorescence level. The drops with greater intensity of fluorescence (those exhibiting the highest levels of activity) are pulled towards the keep channel by the application of an electrical force, a process known as dielectrophoresis. Credit: Courtesy of Jeremy Agresti, Harvard School of Engineering and Applied Sciences.
Harvard researchers and a team of international collaborators demonstrated a new microfluidic sorting device that rapidly analyzes millions of biological reactions. Smaller than an iPod Nano, the device analyzes reactions a 1,000-times faster and uses 10 million-fold less volumes of reagent than conventional state-of-the-art robotic methods.
The scientists anticipate that the invention could reduce screening costs by 1 million-fold and make directed evolution, a means of engineering tailored biological compounds, more commonplace in the lab.
These systems will be key to speeding up current biotechnology research and to enable the scaling of synthetic biology and DNA nanotechnology. Also, when molecular nanotechnology is developed microfluidics will likely be an early step in scaling that up as well.
The new microfluidic system bypasses conventional limitations through the use of drop-based microfluidics, squeezing tiny capsules of liquid through a series of intricate tubes, each narrower than a single human hair.
“Each microscopic drop can trap an individual cell and thus it becomes like a miniature test tube,” explains Amy Rowat, a postdoctoral fellow at SEAS. “The drops are coated with a surfactant, or stabilization molecule, that prevents the drops from coalescing with each other and also prevents the contents from sticking to the wall of the drops.”
To sort, the system removes inactive and unwanted compounds, dumping the drops into a “bad egg” bin, and guides the others into a “keep” container. Specifically, as the drops flow through the channels they eventually encounter a junction (a two-channel fork). Left alone, the drops will naturally flow towards the path of least fluidic resistance, or the waste channel.
The device identifies the desired drops by using a laser focused on the channel before the fork to read a drop’s fluorescence level. The drops with greater intensity of fluorescence (those exhibiting the highest levels of activity) are pulled towards the keep channel by the application of an electrical force, a process known as dielectrophoresis.
“Our concept was to build a miniature laboratory for performing biological experiments quickly and efficiently,” explains collaborator Adam Abate, a postdoctoral fellow in applied physics at SEAS. “To do this we needed to construct microfluidic versions of common bench-top tasks, such as isolating cells in a compartment, adding reagents, and sorting the good from the bad. The challenge was to do this with microscopic drops flowing past at thousands per second.”
“The sorting process is remarkably efficient and fast. By shrinking down the reaction size to 10 picoliters of volumes, we increased the sorting speed by the same amount,” adds Agresti. “In our demonstration with horseradish peroxidase, we evolved and improved an already efficient enzyme by sorting through 100 million variants and choosing the best among them.”
In particular, the researchers were struck by the ability to increase the efficiency of an already efficient enzyme to near its theoretical maximum, the diffusion limit, where the enzyme can produce products as quickly as a new substrate can bump into it.
Using conventional means, the sorting process would have taken several years. Such a dramatic reduction of time could be a boon for the burgeoning field of synthetic biology. For example, a biofuels developer could use the device to screen populations of millions of organisms or metabolic pathways to find the most efficient producer of a chemical or fuel. Likewise, scientists could speed up the pace of drug development, determining the best chemical candidate compounds and then evolving them based upon desired properties.
“The high speed of our technique allows us to go through multiple cycles of mutation and screening in a very short time,” says Agresti. “This is the way evolution works best. The more generations you can get through, the faster you can make progress.”
The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼10^7. After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼10^8 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost