NEW YORK – A team led by researchers from the University of California, Santa Cruz and Brigham Young University has developed a programmable chip that combines nanopores, optofluidics, and an electronic feedback control circuit to select, sort, and analyze single molecules and particles.
The researchers have also launched a Santa Clara, California-based startup called Fluxus to commercialize a platform based on the optofluidic chip for downstream molecular analysis.
UCSC researcher Holger Schmidt explained that his team sought to develop a tool that could select target molecules — such as ribosomes, DNA, bacteria, or viruses — and hold them in place for downstream experiments. The group therefore created a thumbnail-sized silicon chip that can deliver specific molecules and allow researchers to perform tests within the microfluidic system based on customized parameters.
Originally developed by Schmidt and his team in 2014, the thumbnail-sized chip now includes a new feedback-control circuit, which helps automate molecule movement and extraction. Schmidt noted that the tool's "smart-gate" can now deliver up to three molecules into the microchannel based on the parameters set for each test.
In a proof-of-concept study published in Nature Communications today, Schmidt and his team used the optofluidic device to manipulate the delivery of individual molecules — bacterial 70S ribosomes and DNA — into a liquid-based channel on the chip.
"You give the circuit the parameters for what a blockade should look like, and it recognizes that and turns everything on and off automatically and much faster than what we could do by hand," Schmidt explained. "The old version just regulated the flow, in that it put in the molecule one by one, but there was no way to really say how many you wanted and then stop, other than turning the device off completely."
The optofluidic chip primarily contains a microfluidic channel that acts as a liquid-core optical waveguide that connects to solid-core waveguides, allowing fiber-optic access to and from the chip. As part of the electrical feedback system, which includes a microcontroller and solid-state relay, researchers can introduce a sample into the waveguide by applying a voltage across attached fluidic reservoirs at the end of each channel.
According to Schmidt, the tool's microcontroller performs real-time analysis of the current through the nanopore. When a current wave rises above a customized threshold value and causes a peak height signal, the microcontroller registers the molecule's translocation. The researchers then use the solid-state relay to shut off the nanopore driving voltage after a determined number of particles has passed through the nanopore.
While the team typically pushes very little liquid through the channel for each run, Schmidt said that the platform can ultimately cycle anywhere from a few microliters to milliliters of a sample to collect targeted molecules.
In the study, the nanopore's dimension relative to the size of the ribosome only allowed one ribosome at a time in the pore. The voltage gating therefore allowed capture of a single target particle inside the analysis region by preventing further molecular insertion.
While the study tested single-biomolecule extraction on strands of DNA, viral proteins, ribosomes, and cellulose molecules, Schmidt said that the tool can also examine biomolecules derived from small organisms like bacteria. He noted that the team had previously used the chip as part of a multiplex influenza panel that examined six different viral biomolecules.
The study authors also noted that the system's infrastructure allows the user to modify experimental parameters as part of collecting the particles, including altering the liquid's pH, temperature, and buffer composition. Schmidt and his team could program the control algorithm to translocate a desired number of particles before shutting off the pore, and later reopening the pore after a set time following a translocation event.
Schmidt also noted that his group could use the feedback system to quickly deliver large amounts of individual molecules into the channel. He said that the automated tool can track and deliver single molecules at more than 500 particles/min, which he believes is one of its major strengths.
"Once the molecules move through the nanopore and get into the microfluidic channels, everything is controlled by the pressure and the flow speed in the channel," Schmidt explained. "A researcher could select hundreds of molecules and do an experiment on each one in a well-controlled environment."
In addition, Schmidt highlighted that the system can also separate a target molecule from a larger heterogenous mixture. In the study, the researchers selectively gated single DNA particles mixed with bacterial 70S ribosomes by identifying the different electric translocation signals including amplitude and dwell times — time spent in a stationary position —specific to the different molecule types.
According to the study authors, the feedback control system had an accuracy of nearly 95 percent when distinguishing between the 70S ribosomes and DNA passing through the channel. In addition, the authors noted that they could alter the microcontroller to specifically voltage-gate ribosomes by choosing events with longer dwell times and larger differential current changes.
The researchers then demonstrated that they could use the system for optical detection of a specific number of biomolecules. In the study, the group introduced a DNA molecule into the chip, closed the nanopore by turning off the appropriate voltage, and then fluorescently detect the targeted molecule.
By using both the electrical and optical signals in the combined system, the researchers could calculate the flow velocity of the DNA in the channel. They also believe that the optofluidic system's dual-mode electro-optical ability could guarantee particle screening when almost simultaneous translocations from multiple markers might create questionable current blockades.
In order to develop the combined electrical and optofluidic platform, Schmidt noted that the researchers dealt with challenges such as figuring out the correct nanopore hole depth needed to achieve the highest rate of particle collection.
"We had to drill into the thick wall over the channel, but we had to drill into it so we had a very small membrane," Schmidt said. "To optimize the process, we had to stop at the right point without [piercing] through it and making a big hole, which required additional work."
Schmidt also noted that his team ran into limitations because of time delays related to the current version of the feedback system. The feedback control circuit's ability to pass single molecules requires at least 1.5 ms to reopen the pore, which reduces the number of molecules the team could look at over during a designated period of time.
The researchers therefore plan to combine the feedback control system with field programmable gate arrays, which Schmidt explained are more deeply integrated and faster versions of the circuit. The study authors expect to apply anti-Brownian electrokinetic (ABEL) trapping, a method that uses wavelengths to trap single molecules in an aqueous solution.
While the study only tested buffer solutions containing targeted biomolecules, Schmidt noted that the group has also started a new project to detect targeted molecules in blood and urine samples.
The team is now using the optofluidic chip to investigate the ribosome's functions. By capturing the ribosome with an optical based method such as ABEL, the group aims to learn how the ribosome dynamically produces proteins.
"We want to capture a ribosome as it translates RNA through its machinery and makes a protein," Schmidt said. "However, these are really tiny things to capture and hold, so we'd need to optimize the optical and wave-trapping parts … to perform these experiments."
In addition to the current patents the UCSC team has for the optofluidic chip and its wave-guiding technology, Schmidt said that his group plans to file for IP regarding the feedback control circuit through the university in the near future.
As part of the team's plans to improve the optofluidic chip, Schmidt and BYU researcher Anthony Hawkins founded a startup called Fluxus. Spun out of UCSC in 2017, Fluxus is now commercializing a sample prep platform based on the optofluidic and nanopore patents the firm is licensing from the university.
According to Schmidt, the startup has been fully funded from angel and investors outside UCSC and now aims to apply the platform for downstream, ultra-sensitive detection of molecules in a variety of liquid samples.