Electrochemical Sensors for in Vitro Vascular Endothelial Growth Factor Measurement

Objectives: Periodontal disease progression affects soft tissue microvasculature. Vascular Endothelial Growth Factor (VEGF) is involved in the onset and progression of gingivitis and periodontitis, promoting microvascular permeability and angiogenesis, as observed during oral inflammation. New platforms to detect VEGF concentration under normal and disease states are necessary to better understand disease progression. The objective is to develop ratiometric label-free electrochemical biosensors to measure VEGF concentration in vitro.
Methods: We designed a ratiometric sensor with two redox probes, one specific to VEGF and the other serving as an internal standard, to minimize sensor-to-sensor variation and sensor degradation. Gold electrodes (sensors) were immersed in 2μM oligonucleotide (28 bases) solution in phosphate-buffered saline (PBS) for 4h and then in 20mM 6-mercaptohexanol for 12h. Oligonucleotides contained thiol and anthraquinone (AQ) functionalities at the 5’ end and methylene blue (MB) functionality at the 3’ end. Upon VEGF binding, the oligonucleotide folds, reducing the electrode-MB distance and increasing MB current, while the distance and current between AQ and electrode remain constant. Normalized change, S_corr, is calculated as a difference between MB and AQ current changes. A potentiostat was used to measure current from sensors immersed in test solutions. Data were compared to immunoassay tests.
Results: Peaks for AQ and MB appeared at −0.55V and −0.30V, indicating that electrodes were modified with oligonucleotides. Surface densities of thiol, AQ, and MB were 670±64, 194±32, and 1.78±0.45pmol/cm², respectively, indicating ≈30% of AQ and 0.3% of MB were electroactive. Sensors measured 125 to 1000pg/mL VEGF, demonstrated by a linear S_corr increase between 0.08±0.03 and 0.21±0.03 (n=3), which is comparable to the linear range of the immunoassay standard curve.
Conclusions: Sensors reproducibly measured VEGF and responded to varying low (pg/mL) concentrations of VEGF. The advantage of radiometric sensors is their decreased variability and increased reproducibility needed for studying inflammatory dental diseases.