"The advantages of diamond in the field of biosensors are enormous: chemical stability, biocompatibility, easy surface modification, largest electrochemical potential window and so on," Jose Garrido told nanotechweb.org. "We have demonstrated that it is possible to immobilize enzymes on the surface of nanocrystalline diamond thin films using our existing knowledge of organic chemistry. And what is more important, the enzymes are still active despite being immobilized."

Garrido and colleagues used a photochemical process to attach amino groups to the surface of a hydrogen-terminated nanocrystalline diamond film. The diamond, which had an average grain size of about 20 nm, was grown by hot-wire chemical vapour deposition from a mixture of hydrogen and methane. The researchers then covalently bonded molecules of green fluorescent protein (GFP) to the amino groups. Using GFP enabled them to employ an inverted fluorescence microscope to confirm that the protein was attached and still functional.

To make an enzyme-based amperometric biosensor, the team attached catalase to the surface of a nanocrystalline diamond film and added some electrical contacts.

"Direct electron transfer from the enzyme redox centre to the [diamond] electrode surface was reported, as well as activity in the presence of hydrogen peroxide," said Garrido. "Thus, nanocrystalline diamond electrodes provide both a perfect substrate for biomolecule immobilization and also a unique set of electrochemical properties."

Although other groups have attached proteins to gold surfaces using self-assembled monolayers of thiols and disulphides, these have shown poor stability in the presence of oxidizing agents such as ozone and hydrogen peroxide. According to the researchers, the strength of the carbon-carbon covalent bond at the diamond/biolayer interface in their films provides an important advantage over metal electrodes such as gold.

"The potential applications of biosensors in daily life are tremendous: from environmental and water analysis to personalized healthcare, from quality food control to home patient monitoring," said Garrido. "In addition, other aspects with strong impact on public healthcare - such as drug screening and genetic disease control - will certainly benefit from biosensors."

Now the researchers are trying to immobilize other enzymes, for example glucose oxidase and horseradish peroxidase, on the surface of both nanocrystalline and monocrystalline diamond films. "One of our goals is the development of diamond-based amperometric biosensors using multilayers of proteins immobilized via covalent bonds to the diamond surface," said Garrido. "These electrodes can be designed in a microarray structure in order to achieve parallel detection of several metabolites, for instance glucose, urea, lactate and cholesterol."

Besides these catalytic biosensors, the team is also developing affinity biosensors that use impedimetric detection to study substrate-protein binding, as well as DNA hybridization. "Affinity interaction can be used for the design of immunosensors," explained Garrido. "In addition, we are working on the immobilization of enzymes on the surface of diamond ion field-effect transistors (ISFETs) in order to develop enzyme field-effect transistors (ENFETs)."

The researchers reported their work in Nature Materials.