Quantification of cytokines involved in wound healing using surface plasmon resonance

Sensing of three cytokines related to chronic wound healing, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), with detection limits at or below 1 ng/mL in buffered saline solution and spiked cell culture medium (CCM) has been achieved. Fiber-optic surface plasmon resonance (SPR) sensors are coated with an antibody binding layer and antibodies specific to the cytokine of interest are covalently attached to this layer. To achieve such detection limits in a complex medium such as CCM, total protein content of 4 mg/mL, the use of a novel N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid (NHS-MHA) is necessary. A comparison of the detection limits for IL-6 using currently widely used CM-dextran and NHS-MHA shows an improvement by a factor of 3 using NHS-MHA. The detection limits for the monitoring of cytokines in spiked saline solutions and CCM were similar for TNF-alpha and slightly higher for IL-1 and IL-6. The detection of each cytokine in the presence of interfering agents resulted in concentration prediction well within the error of calibration. The SPR sensors are stable in CCM after 20 min of pretreatment in CCM, minimizing the reliance on a reference sensor to quantify the cytokines in complex media. This technique enables a major advancement in the field of real-time monitoring of biologically relevant molecules in complex biological fluids.