Harnessing Effective Molarity to Design an Electrochemical DNA‐based Platform for Clinically Relevant Antibody Detection

Abstract

Easy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibodies. We show a highly selective, signal‐on DNA‐based sensor that takes advantage of antibody‐binding‐induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.

Accelerated Electron Transfer in Nanostructured Electrodes Improves the Sensitivity of Electrochemical Biosensors

“…[ 5 , 6 , 7 , 8 , 9 , 10 , 11 ] Sensitivity is a key consideration for many biomedical applications, because many clinical biomarkers are present at nanomolar to picomolar concentrations, and the biosensor must achieve sufficient sensitivity in a complex background of interferent molecules. [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ] Unfortunately, due to noise limitations in existing electronic measurement systems, the signal‐to‐noise ratio of conventional electrochemical biosensors degrades precipitously when they are miniaturized to the micron scale, [ 21 ] reducing their sensitivity and making meaningful measurements of analyte concentrations challenging or even impossible in many cases. [ 22 , 23 ]…”

Nucleic-Acid Driven Cooperative Bioassays Using Probe Proximity or Split-Probe Techniques

“…The products of the toehold displacement reaction formed a heterodimer which included two copies of the surface hybridization strand, thereby generating bivalent affinity to the surface which stabilized the binding for EC readout. Recently, Porchetta and co-workers, referring to the proximity effect as “effective molarity”, carefully tuned their system for rapid antibody detection. As shown in Figure A (top), a low affinity hybridization interaction between the capture strand and the output strand could be enhanced by a bivalent antibody mimicking strand (Ab-mimic) similar to DNA loops developed previously. ,, This experimental model clearly shows the enhancement given by the proximity effect, where the response curve was shifted to lower concentrations in the presence of the Ab-mimic (red curve).…”

“…The researchers then applied the concept to quantify bivalent antibodies, as shown in Figure A (bottom), to give a modular and rapid EC approach based on the proximity effect. Both IgG and IgE antibody classes could be detected, and the sensor was validated with four different clinically relevant antibodies in bodily fluids …”

“…However, this is an oversimplification of the true nature of the complexes, which are often present with DoC values in the range of 0 to 4 (Figure B). Most theoretical analyses and even experimental studies that leverage antibody mimicking strands assume DoC = 1 for the model systems, ,,, and this undoubtedly limits the predictive power of such systems. With DoC > 1, background complexes (see Figure ) will be more stable, and it is hypothesized that as DoC increases, the stabilities of background and signal complexes will converge, rendering most proximity assays useless.…”

“…Yet if the length of the complementary strands is too short, the stability of both signal and background complexes will suffer. It is therefore important to evaluate the signal-to-background ratios to determine the optimized probe length for the assay being designed. − ,,,, …”