Interleukin-6 (IL-6) is a biomarker that is increasingly being used as an indicator of inflammation, as an early warning sign for a range of diseases from asthma to cancer. Biomarker IL-6, but also other biomarkers, can bind to specific aptamer molecules. Aptamers that are immobilized on a sensing surface thus make for efficient and sensitive biosensors that can be utilized for point-of-care diagnostics or in biochemical laboratories to detect a wide panel of medical conditions.

Aptamers immobilized on graphene, and in particular graphene field-effect transistors (GFETs), have become a well-known detection platform in biosensing of proteins, bacteria, viruses and chemicals. Nevertheless, the conventional two-step process of immobilizing aptamers on a graphene surface requires the use of aggressive organic solvents that may attack vulnerable components of a biosensing chip, such as for example polymers, passivation layers, and microfluidic tubing, which in turn may lead to device damage or fluid leakage. In addition, some of the organic solvents typically used are toxic to humans. Consequently, two-step aptamerization of graphene complicates device fabrication, negatively impacting the potential for wide-scale usage of graphene-based biosensors.

Image: Graphene-based biosensors. From Khan and Song, Sensors 2021, 21(4), 1335.

Now, researchers Khan and Song from the University of New Hampshire have discovered a method of single-step aptamerization of graphene that does not require the use of organic solvents, which enables a wider portfolio of sensor device fabrication and integration methods. The scientists did that using the Graphenea GFET-S20 product that is designed with biosensing in mind. The research was published in a special issue of the journal Sensors, dedicated to Field Effect Transistor (FET)-Based Biosensors.

Anchoring an aptamer to graphene requires an intermediary pyrene derivative that binds to graphene on one side and to the aptamer on the other side.  Unfortunately, pyrene-based crosslinkers require organic solvents such as DMF or DMSO in order to be well-dispersed in a solution and bind appropriately. In their novel approach, Khan and Song first link the aptamers to pyrene, creating pyrene-tagged DNA aptamers (PTDA), and then disperse PTDA onto graphene in a non-aggressive aqueous buffer solution, thus bypassing the potentially harmful fabrication step. Although such a process would naturally form a limited aptamer cover on the surface of graphene, the researchers came up with an innovative step to improve surface coverage. Making use of the fact that PTDA is negatively charged, they drew PTDA towards the graphene surface by setting a voltage between a built-in electrode on the GFET and a second electrode immersed in the PTDA solution.

The effectiveness of the one-step technique in GFET-based biosensor implementation was demonstrated by detecting IL-6 as a representative target analyte. The sensor was confirmed to be selective to IL-6 and sensitive to concentrations down to 100 picomoles. This is higher than that achieved with standard two-step immobilization of aptamers, however the single-step method offers fabrication advantages. The limit of detection was improved to 8 picomoles when the pH of the buffer solution was adjusted down to 3.6 from the physiological 7.4.

Although performed in lab conditions, the proposed technology has the potential to be used in monitoring IL-6 from real physiologically relevant fluid samples such as sweat, serum and cerebrospinal fluid.