Third-Generation Direct Electron Transfer (DET) Enzymes

Introduction to Third-Generation Direct Electron Transfer (DET) Enzymes

A third-generation glucose sensor is one where the enzyme hands electrons straight to the electrode. No oxygen and no added go-between molecule does the carrying. Researchers call this direct electron transfer, or DET, and describe it as the most elegant design for an enzyme sensor, even though only a few glucose enzymes can do it [2]. The numbering follows history: first-generation sensors use oxygen as the electron acceptor, and second-generation sensors add electron mediators, small redox molecules that ferry electrons from the enzyme to the electrode [2].

The two CGM sensors whose chemistry we traced in primary documents fit those older categories. FDA's 2018 review of the Dexcom G6 describes a glucose oxidase sensor that produces hydrogen peroxide, and the peroxide generates the current the device measures [6]. Dexcom says it will no longer manufacture the G6 after July 1, 2026, and is moving users to its G7 sensors [7]. In a 2017 paper, Abbott scientists described the FreeStyle Libre sensor of that time as using Wired Enzyme chemistry, in which an osmium-complex mediator is crosslinked with glucose oxidase inside a polymer [5]. In a DET sensor, the electron starts at the enzyme's redox center, a cofactor such as FAD (flavin adenine dinucleotide) or PQQ (pyrroloquinoline quinone). It reaches the electrode by electron tunneling, a quantum effect that depends on how far the electron has to travel [1][3].

Key Principles of DET Enzymes

Here's what surprised us: the cofactor usually isn't the part touching the electrode. Glucose oxidase, FAD-dependent glucose dehydrogenase (FAD-GDH) and PQQ-dependent glucose dehydrogenase (PQQ-GDH) show no useful DET on their own, and efficient DET is hard to get without an extra heme group built into the enzyme [1]. In the enzymes that do it well, the catalytic domain is connected to a heme-containing cytochrome domain. Electrons run from the active site through the heme and out to the electrode [1].

One of the best-known glucose examples is a bacterial FAD-GDH complex with three subunits, one of which exists to transfer electrons [2]. Position matters as well. Work on other redox enzymes shows that orienting the enzyme so its electron exit faces the electrode shortens the tunneling distance and raises the current [3].

Advantages of DET Enzymes

DET offers two main advantages over its predecessors:

• Enhanced Selectivity: Without a mediator, a DET sensor can run at a potential close to the enzyme's own redox potential, which cuts down on reactions from other substances in the sample [1]. A DET glucose sensor built on an FAD-GDH complex gave a signal unaffected by acetaminophen or ascorbic acid (vitamin C) in lab testing [4]. That matters because FDA's G6 review notes that acetaminophen caused significant interference in the earlier peroxide-sensing Dexcom G4 and G5 systems [6]. Low-voltage operation isn't unique to DET, though. The Wired Enzyme sensor in Abbott's 2017 paper runs its working electrode at only 40 mV, which also limits interference [5]. Our CGM interference profiles comparison covers what current labeling says about specific substances.
• Simplified Design: Dropping the mediator removes one reagent from the sensor, along with a molecule that can drive nonspecific reactions of its own, so the device is easier to build [1]. A DET sensor also doesn't rely on oxygen as the electron acceptor, the way first-generation sensors do [2].

Challenges and Limitations

DET is still hard to turn into a sensor you can wear, for two reasons:

• Low Signal Strength: Electrons tunnel efficiently only when the enzyme sits close to the electrode in the right orientation [3]. Much of the published DET work is about modifying electrode surfaces to improve that electron transfer [1].
• Enzyme Instability: Enzymes adsorbed directly onto an electrode can unfold or lose their cofactor. Milton and Minteer caution that FAD signals reported from adsorbed glucose oxidase are increasingly attributed to cofactor that has come loose from the enzyme, not to working enzyme [3]. Staying stable for a full wear period is the real test for a CGM. One DET sensor ran continuously for more than 9 days in the lab without significant change in signal, sensitivity or range [4], but that was a bench result, not a device worn on the body.

Innovations and Future Directions

Nanomaterials and enzyme engineering are the two main tools researchers use against these limits. Electrodes built with carbon nanotubes or gold nanoparticles appear repeatedly in DET glucose biosensors based on cellobiose dehydrogenase, an enzyme with its own heme domain [1]. On the engineering side, one group fused a cytochrome domain from a PQQ-dependent alcohol dehydrogenase onto PQQ-GDH, and the fused protein passed electrons from its heme to the electrode [1]. FAD-GDH is the other major target. A 2020 review called fungal versions the most popular enzymes in second-generation test strips, and the DET-capable bacterial complex is being engineered for sensor use [2].

Conclusion

DET takes oxygen and mediators out of the sensing chain, which is why researchers keep returning to it. What still separates the lab from a wearable sensor is a current strong enough to measure reliably and an enzyme that stays active on the electrode for the whole wear period [1][3]. As of October 2026, the DET glucose sensors we found in the literature are research prototypes like the 9-day bench sensor above [4]. We could not confirm whether any CGM on the market uses DET.