Due to steric hindrance, DNA hybridization on surfaces is influenced by target strand length. Previous work has found that longer DNA targets hybridize at a lower rate and form fewer probe-target duplexes than shorter targets. As such, the magnitude of the electrochemical response significantly reduces with increasing target length1,2. These findings lend to important considerations for assay design. First, the effect can be exploited to produce signal-OFF assays, as the capture of a bulky target on an electrode surface would suppress the sensor response compared to a negative control3. Conversely, steric hindrance limits nucleic acid detection in real-world samples, which can consist of long DNA fragments (ex. genomic DNA, PCR products)1,2. Therefore, this study presents methodologies for modulating steric effects in electrochemical DNA hybridization sensors. A 20 nucleotide (nt) thiolated single-stranded DNA (ssDNA) probe was immobilized on a gold electrode surface. Complementary strands were methylene blue labelled on the surface side and extended on the solution side using varying lengths of poly-T overhangs (T20, T40 and T80). DNA hybridization kinetics was monitored using real-time square wave voltammetry (SWV) measurements. When probe-target duplexes formed, electron transfer between the methylene blue reporter and the electrode surface increased the peak current magnitude. With both probe density and target size being sources of steric hindrance, the assay was conducted using a range of probe concentrations for each overhang length. DNA hybridization kinetics of the different targets was tested for five probe concentrations. Overall, increasing the overhang length resulted in a slower increase in current, indicating a lower hybridization rate. Furthermore, the signal magnitude was inversely related to overhang length, which signifies a reduction in probe-target duplexes formed. At low probe concentrations, the effect of overhang length was less prominent, as the difference between signal magnitudes for each length was diminished. The length dependence grew stronger with increased probe concentration and was maximized at the moderate probe concentration. Sensors with high probe concentrations could differentiate between overhang lengths with a sensitivity slightly lower than the moderate probe concentration, but significantly higher than the low probe concentrations. Adjusting probe concentration can also optimize sensor performance when the overhang length is held constant. In summary, the impact of steric hindrance from ssDNA target size can be controlled by assay conditions such as probe concentration. To add to this work, salt concentration, duplex length and electrode surface morphology will be varied to investigate their influence on steric hindrance. The insights from this study can inform the design of electrochemical DNA hybridization assays, leading to more versatile point-of-care diagnostic systems. D. K. Corrigan et al., Journal of Electroanalytical Chemistry, 732, 25–29 (2014).M. Riedel, J. Kartchemnik, M. J. Schöning, and F. Lisdat, Anal. Chem., 86, 7867–7874 (2014).S. S. Mahshid, F. Ricci, S. O. Kelley, and A. Vallée-Bélisle, ACS Sens., 2, 718–723 (2017). D. K. Corrigan et al., Journal of Electroanalytical Chemistry, 732, 25–29 (2014). M. Riedel, J. Kartchemnik, M. J. Schöning, and F. Lisdat, Anal. Chem., 86, 7867–7874 (2014). S. S. Mahshid, F. Ricci, S. O. Kelley, and A. Vallée-Bélisle, ACS Sens., 2, 718–723 (2017).