Tiny molecules such as serotonin, testosterone, and many hormones or neurotransmitters are too small to fit two antibodies at once.
Their findings, published in Nature Chemistry, show how specially engineered DNA molecules can create a sandwich-style test for small molecules for the first time.
Small molecules do not.
To demonstrate the idea, the team worked with aptamers designed to recognize four small molecules: Serotonin, Dopamine, Testosterone, and Deoxycorticosterone 21-glucoside (DOG).
Unfortunately, most of these small molecules are still difficult to analyze outside of a specialized laboratory environment.
At-home COVID-19 tests work because they use a clever molecular trick known as a sandwich assay. One molecule grabs the virus, another molecule grabs the same virus from a different side, and together they form a detectable “sandwich” that produces a visible signal.
The approach is simple, cheap, and highly effective. But it has one major limitation: it only works well for large targets. Tiny molecules such as serotonin, testosterone, and many hormones or neurotransmitters are too small to fit two antibodies at once. Detecting them usually requires more complicated laboratory equipment and specialized sensors.
Now, researchers at EPFL’s Laboratory of Chemical Nanotechnology (CHEMINA) have developed a new strategy that could solve this long-standing problem. Their findings, published in Nature Chemistry, show how specially engineered DNA molecules can create a sandwich-style test for small molecules for the first time.
A typical sandwich assay is based on using two different binding sites. When a large molecule, such as a viral protein or a pregnancy hormone, has enough surface area, both antibodies can bind at the same time.
Small molecules do not. Because of their size, scientists have generally had to rely on more complex sensing technologies that detect tiny electrical or fluorescent changes when a target molecule binds.
These systems can work well, but often require laboratory instruments and complicated signal-processing methods. The EPFL team wanted a simpler solution.
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The researchers turned to aptamers, short synthetic strands of DNA designed to bind specific molecules. Normally, aptamer-based sensors use a single DNA strand that both recognizes the target and generates a signal.
The new approach separates those tasks. A primary aptamer first binds the target molecule.
Then a second DNA strand, called a secondary aptamer, recognizes not the target itself but the new three-dimensional shape the primary aptamer adopts after binding.
In effect, the target causes the first aptamer to change shape, and the second aptamer detects that change.
“Aptamer-based technologies have gained momentum in recent years, with a growing number of startups relying on them for human health applications,” said CHEMINA laboratory head Nako Nakatsuka.
“Instead of simply refining sensors, we wanted to rethink the molecular recognition architecture itself to address a long-standing gap in small-molecule sensing.”
A novel DNA nano-engine
The work introduces an unusual type of molecular recognition. Normally, DNA strands identify one another through matching genetic sequences. In this case, the secondary aptamer recognizes a three-dimensional structure instead.
“A sandwich test for small molecules is just one application. More broadly, this two-layer aptamer design lets us combine separate recognition steps rather than asking a single aptamer to do everything,” Nakatsuka said.
The approach adds a second verification step, potentially increasing both flexibility and accuracy.
To demonstrate the idea, the team worked with aptamers designed to recognize four small molecules: Serotonin, Dopamine, Testosterone, and Deoxycorticosterone 21-glucoside (DOG).
After allowing the primary aptamers to bind their targets, the researchers screened billions of DNA sequences to find candidates capable of recognizing the aptamers’ altered shapes. They successfully identified secondary aptamers for serotonin, testosterone, and DOG.
As a proof of concept, they then built a lateral-flow test for serotonin that produced a result visible to the naked eye, much like a home COVID test. The researchers were unable to identify a suitable secondary aptamer for dopamine, and understanding why is now one of the questions the team hopes to answer.
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The researchers believe the technology could eventually be used for a wide range of applications, from medical diagnostics to molecular computing. Because the system relies on two separate recognition events, it could potentially be adapted into biological logic circuits where molecular binding acts like an on-off switch.
“By allowing aptamers to recognize a molecule’s state rather than simply its identity, our work expands what DNA-based systems can detect, creating a foundation for more complex layers of molecular recognition,” Nakatsuka said.
Throughout the body, small molecules serve as vital components. They include hormones, neurotransmitters, and metabolites. Each category provides essential data concerning health, disease, and how well a patient is responding to treatment. Unfortunately, most of these small molecules are still difficult to analyze outside of a specialized laboratory environment.
Applying the sandwich assay to molecules previously thought to be too small may make it easier, cheaper, and more accessible to develop diagnostic assays.
One day, if developments are successful, we could detect substances like serotonin or testosterone almost as quickly as we can take a rapid COVID test.
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