Biosensors that detect proteins are crucial for diagnostics, therapeutics, and biotechnology. Traditional ways to detect or measure the concentrations of proteins are time-consuming, labor-intensive, require specialized instrumentation and facilities.

Many diseases have specific protein signatures but developing cost-effective and rapid sensors for measuring these signatures is a challenge. Patients typically have to go to clinics and send samples to labs and wait days to weeks for results. Imagine rapid, at-home tests, much like the COVID-19 self-diagnostic kits, but for much more complex diseases like cancer and autoimmune disease. To get there we needed way of quickly building sensors for many different proteins of clinical relevance, as well as the ability to program in recognition of multi-input protein signatures and amplification.

That has led Johns Hopkins engineer Rebecca Schulman, core researcher at the Institute for NanoBioTechnology, associate professor of Chemical and Biomolecular Engineering, and the Kent Gordon Croft Investment Management Faculty Scholar, to investigate designing molecular biosensors that could generate a response to a target ligand without manual intervention, could be read without specialized equipment, and could easily be programmed to produce a variety of functional responses. Their research was published in Nature Communications,

Biosensors detect proteins are crucial for diagnostics, therapeutics, and biotechnology. Traditional ways to detect or measure the concentrations of proteins are time-consuming, labor-intensive, require specialized instrumentation and facilities.

The platform, named ARTIST (Aptamer-Regulated Transcription for In vitro Sensing and Transduction) by the team, uses aptamer-protein binding to regulate transcription of a DNA template, like how transcription factors regulate gene expression in cells. The DNA template produces an RNA output that reacts with a fluorescent reporter, meaning  the level of fluorescence would be determined by the concentration of proteins that react with the DNA template.

Molecular biosensors require coupling a recognition element such as an allosteric protein switch or aptamers with molecular circuits, but often require extensive reengineering for each target protein or desired output. This makes it difficult to rapidly develop biosensors against other targets or to adopt existing biosensors for additional functionalities. ARTIST is designed such that the recognition element and the molecular circuit are independent from one another. By decoupling the input and output domains of the biosensors, the team can decide upon which protein the biosensor detects, or what kind of output the biosensor produces.

“We wanted to develop a platform that enables rapid construction of protein biosensors that can detect diverse targets and easily integrate with downstream circuits for programmable responses,” fifth year PhD student in the Schulman lab, Heonjoon Lee said.

Diseases are often characterized by specific patterns of protein expression levels, requiring a method capable of measuring multiple proteins simultaneously, and ARTIST offers a promising and straightforward workflow for designing arrays of protein biosensors. This approach can be used to identify distinct protein expression patterns, enabling accurate diagnosis of patients with various diseases.

“One could imagine the developing ARTIST into inexpensive home test kits, much like the COVID-19 kits, but kits that could diagnose complex diseases like auto-immune diseases, inflammatory diseases, or cancer by detecting patterns of protein expression. These tests could serve as a great screening tool both at home and in the clinic,” said Samuel Schaffter, Chemical Engineer at the National Institute of Standards and Technology who contributed to the study and is a former Johns Hopkins PhD student and member of the Schulman lab.

One challenge is getting the sensors to detect the low concentrations of proteins associated with these diseases. They have demonstrated the ability to amplify the output signal of the biosensors, so the hope is to be able to increase the sensitivity to clinically relevant concentrations, Schaffter said.

The sensitivity of ARTIST suggests that it could be used for detecting the proteins of clinical interest. For example, they have built molecular biosensors that can detect cytokines often linked to autoimmune diseases. Since the outputs of these biosensors are molecular, they can be integrated into other molecular circuits for therapeutic applications, such as triggering the release of drug molecules or of mRNA-based vaccines.

“We are trying to implement ARTIST in hydrogels to demonstrate continuous measurement of molecules in cell culture. This could be very useful for dynamically measuring the secretion of proteins and small molecules from cells. We are currently collaborating with other labs in Hopkins to explore potential use in tissue engineering and biomanufacturing,” Lee said.