Increasing biosensor sensitivity with Affimer® proteins

Academic researchers at the University of Leeds have developed a biosensor that can rapidly detect the inflammatory biomarker, IL-8, at clinically relevant concentrations. With emphasis in the clinic placed on early diagnosis and individualised patient care, diagnostic assays that offer rapid point-of-care analysis of a patient’s condition are increasingly important. This proof-of-concept study (link is external) uses the Affimer scaffold, which contains two peptide loops that bind selectively and with high affinity to IL-8, in an electrochemical-impedance based assay to provide sample results within just 15 minutes.

IL-8 is associated with acute inflammation, making it an important biomarker in many disease profiles. During injury or infection it is involved in recruiting neutrophils to damaged tissue to promote angiogenesis. Elevated levels of IL-8 have been linked to chronic diseases, such as rheumatoid arthritis, atherosclerosis, chronic liver disease and cancer.

Schematic illustration of the biosensor assembly. A monothiol-alkane-PEG acid SAM is first assembled on a gold electrode. The carboxylic acid groups of the SAM are then activated with EDC/NHS to which the non-antibody capture proteins are attached.

Although approved diagnostic assays for the detection of IL-8 already exist, the current standard tests are based on ELISA. These assays are too time-intensive to allow point-of-care diagnosis, with waiting times of 2-3 hours for a result. Also, while basal levels of IL-8 in healthy patients are typically 5-10 pg/ml, most of the available tests have sensitivities in the range of 15 ± 5 pg/ml, so reproducible and reliable identification of small increases for early diagnosis can be difficult with the current tests.

As a label-free technique for the sensitive measurement of target analytes, electrochemical impedance spectroscopy-based detection is gaining interest. It uses small AC potentials across a large frequency range to detect changes in the electrochemical impedance caused by the binding of target analytes to capture proteins immobilized on the surface of the electrodes. Antibodies are the most commonly used capture reagent for target analytes, but their immobilisation onto solid supports has been shown in some cases to affect their stability, impacting upon their specificity and affinity. Affimer proteins are known to be stable when attached to solid supports, and so the choice of this affinity protein as a capture reagent offers reassurance that specificity and affinity for the target will be retained.
The research team at the University of Leeds, led by Professors Christoph Wälti and Mike McPherson, attached an Affimer protein with high affinity for IL-8 to the electrochemical-impedance sensor surface. The sensor yielded a linear response from 900 fg/ml to 900 ng/ml IL-8 in full serum and a time-to-result of 15 minutes, demonstrating increases of an order of magnitude in IL-8 sensitivity compared to the standard ELISA tests. The sensors showed no significant response even when exposed to high concentrations of BSA and IL-6, indicating the high specificity of the Affimer capture protein in this assay format. 

Prior to this work electrochemical-impedance-based assays have been used to detect IL-6, IL-2 and hCG at sub-pg/ml concentrations, though this work was carried out in buffer solutions rather than against a serum background, reducing any potential cross-reactivity. Tests for IL-8 with sensitivity below 5-10 pg/ml in clinical samples have not been reported. Electrochemical biosensors constitute a promising group of sensing devices that allow increased sensitivities, low cost, low analysis times, affordability, and miniaturized platforms. This most recent study demonstrates that the Affimer affinity proteins can be used for target analyte detection at clinically relevant concentrations, producing results within minutes, not hours, to enable accurate and rapid point-of-care biomarker detection to improve early diagnosis.