A Sensitive DNA Bowtie Sensor for Calibration-Free Sex Hormone Detection Using Analyte-DNA Conjugates and Multiple Redox Labels.

Using a modular and reconfigurable DNA-based bowtie sensor structure, several advancements in electrochemical (EC) biosensing are reported. Raw current and sensitivity were enhanced up to 13-fold using a novel redox reporter design, where a DNA strand was decorated with five methylene blue molecules (MB5) instead of the typical single label (MB), and flexible spacers further improved signal. Leveraging the bowtie sensor's modularity, custom synthesized analyte-DNA conjugates (testosterone-DNA, estradiol-DNA, and progesterone-DNA) and a control strand (digoxigenin-DNA) were incorporated into the complete DNA structure on electrode surfaces by enzymatic ligation. Upon binding antibodies, each of these sensors showed enhanced current with the MB5 label, and responses consistently followed a Langmuir curve. A previously inaccessible signal-on responsesustained current at longer timeswas observed at low square wave voltammetry frequencies using MB5, allowing new differential signal corrections. The limit of detection (LOD) for direct antibody sensing was as low as 0.08 nM (80 pM) for the antidigoxigenin antibody (control system). A mathematical model was devised based on uncoupled equilibria among the analytes, antibodies, and the electrode surface. After training the model on binding curves, calibration-free measurements of small molecule hormones were possible, with observed LODs for testosterone, estradiol, and progesterone at 0.2, 0.8, and 1 nM, respectively, extending within human clinical ranges. For example, the testosterone sensor's range was 200 pM to 70 nM, covering the clinical range in both females (0.52 to 2.43 nM) and males (9.0 to 34.7 nM), and the sensor functions with 90% recovery in 50% human serum. This work expands the usage of the EC bowtie sensor, employing custom synthetic organic methods to introduce new analyte-DNA conjugates and further validate the sensor's generalizability, alongside developing a unique model of the binding mechanism. Beyond this, the newly introduced MB5 label should be functional with a range of other DNA-based EC sensors or aptamer-based sensors.