Next-Gen Biosensing: Unraveling Molecular Mysteries at Unprecedented Sensitivity

An unplugged electronic device may operate, but it delivers far superior sound when connected to an amplifier. Likewise, environmental toxins and other minute molecules at minimal concentrations may produce faint signals that go unnoticed without specialized laboratory technology.

Now, aided by a “clever technique in biochemistry” that repurposes a sensing platform already utilized by Northwestern researchers to assess toxins in drinking water, scientists have gained the ability to identify and even quantify chemicals at low enough concentrations for applications beyond the laboratory. By integrating circuitry similar to a volume control to “amplify” subtle signals, the team has paved the way for disease detection and monitoring within the human body concerning nucleic acids such as DNA and RNA, along with bacteria like E. coli.

The findings, which outline a system that is tenfold more sensitive than earlier cell-free sensors developed by the team, were published today (Jan. 13) in the journal Nature Chemical Biology.

Biosensors adapted from nature can potentially detect a wide array of pollutants and indicators of human health, although they often lack sufficient sensitivity in their current state. By incorporating genetic circuitry that functions as an amplifier, we can enhance this biosensing platform to achieve the sensitivity required for environmental and human health monitoring.”

Julius Lucks, corresponding author and Northwestern synthetic biologist

Lucks serves as a professor of chemical and biological engineering at Northwestern’s McCormick School of Engineering and co-director of the Center for Synthetic Biology.

Engineering a ‘pregnancy test for water’

ROSALIND’s initial model could detect 17 distinct contaminants in just a single drop of water, glowing green when a contaminant surpassed the standards set by the U.S. Environmental Protection Agency. A subsequent model empowered the platform to calculate varying concentrations of pollutants, resulting in something far more advanced than merely a “pregnancy test for water.”

Lucks and his team employed a method known as cell-free synthetic biology to develop ROSALIND, which entails extracting molecular machinery—such as DNA, RNA, and proteins—from cells and then reprogramming it to fulfill new functions.

A useful flaw in the system

Synthetic biologists engaged with DNA and RNA frequently encounter an uncooperative adversary known as T7 RNA polymerase enzyme, which Lucks likens to the battery of a radio for its function of producing output signals. For many, this enzyme can also act as a flaw, consuming RNA fragments it shouldn’t, thus causing disruption in nucleic acid circuits. However, Lucks speculated if it could instead be harnessed to their benefit.

Lucks uses the evolution of the transistor radio to illustrate progress in the sensing system developed by his team, aptly named ROSALIND (in honor of renowned chemist Rosalind Franklin, and stands for “RNA output sensors activated by ligand induction”).

“You could assemble the first transistor radio in your Electronics 101 class, and it can receive a signal, but it comes with numerous issues,” Lucks commented. “If you walk behind a tree, the signal drops, and if you move closer to the source, it becomes louder. In the future versions of that radio, they integrated additional electronic circuits to rectify those problems. This version is essentially adding a volume dial to the radio.”

By using a signal amplification technique from DNA nanotechnology that enables a circuit to recycle and retransmit its input, the researchers discovered a way to enhance the signal of an input molecule. When a signal is produced, the “bug” consumes and reprocesses it, leading to another signal being generated. This innovation allowed the team to identify molecules—such as antibiotics and heavy metals—at significantly lower concentrations than previous versions.

“We established a new system to enhance signals within ROSALIND,” noted first author Jenni Li, a Ph.D. candidate in the Lucks lab. “Through a clever trick in biochemistry, we improved the system’s sensitivity to detect compounds at reduced levels without altering the actual biosensor protein. This is achieved within nucleic acid ‘circuits.’ ROSALIND 3.0 is now more responsive and can identify nucleic acids even when it could only detect small molecule compounds previously.”

ROSALIND in action

Past versions of ROSALIND have already been deployed in practical settings, including an ongoing field study in the Chicago area that is monitoring lead levels in drinking water. According to Lucks, the new elements from their “3.0” model can be seamlessly integrated into this and additional initiatives.

“We are also working on ROSALIND to identify human health indicators, food quality markers, and agricultural substances, thus broadening the potential applications of this platform technology,” Lucks said. “This new sensitization method is versatile, suggesting we will be able to expedite the development of sensors capable of detecting compounds at actionable levels in the coming years.”

The research was supported by a National Institutes of Health training grant (T32GM008449) through Northwestern’s Biotechnology Training Program, a National Science Foundation Synthesizing Biology Across Scales training program (2021900), and Northwestern’s Graduate School Cluster in Biotechnology, System and Synthetic Biology. Additional backing comes from Army Contracting Command (W52P1J-21-9-3023), the Defense Advanced Research Projects Agency (DARPA) (N660012324041), and the National Science Foundation (2310382).

Northwestern’s startup company Stemloop is commercializing the ROSALIND technology. Lucks has financial interests and affiliations with Stemloop. Northwestern holds financial stakes (equity, royalties) in Stemloop.