Rapid and ultrasensitive electrochemical detection of circulating tumor DNA by hybridization on the network of gold-coated magnetic nanoparticles

Abstract

An accurate and robust method for quantifying the levels of circulating tumor DNA (ctDNA) is vital if this potential biomarker is to be used for the early diagnosis of cancer. The analysis of ctDNA presents unique challenges because of its short half-life and ultralow abundance in early stage cancers. Here we develop an ultrasensitive electrochemical biosensor for rapid detection of ctDNA in whole blood. The sensing of ctDNA is based on hybridization on a network of probe DNA modified gold-coated magnetic nanoparticles (DNA-Au@MNPs). This DNA-Au@MNPs biosensor can selectively detect short- and long-strand DNA targets. It has a broad dynamic range (2 aM to 20 nM) for 22 nucleotide DNA target with an ultralow detection limit of 3.3 aM. For 101 nucleotide ctDNA target, a dynamic range from 200 aM to 20 nM was achieved with a detection limit of 5 fM. This DNA-Au@MNPs based sensor provides a promising method to achieve 20 min response time and minimally invasive cancer early diagnosis.

Fig. 1. Schematic illustration of DNA-mediated reduction of potassium ferricyanide (K₃[Fe(CN)₆]) by methylene blue (MB). (a) Workflow for the measurement of target ctDNA. (b) Electrochemical sensing mechanism of the DNA sensor. (c) Background-subtracted square wave voltammograms (SWVs) in the absence and presence of target ctDNA (101 nucleotides). The frequency and pulse amplitude of square wave voltammograms were 2 Hz and 25 mV, respectively.

Fig. 2. Hybridization-induced change in background-subtracted SWVs and percentage change in current after exposing the sensor to 20 nM complementary target DNA (22 nucleotides) in five different systems as indicated by the cartoons. Electrolyte solution was 0.5 mM K₃[Fe(CN)₆] in phosphate buffered saline (pH 7.4). The frequency and pulse amplitude of SWVs were 2 Hz and 25 mV, respectively. Error bars represent standard deviations from at least 3 independent measurements.

Fig. 3. The dependence of hybridization-induced current change from SWVs upon complementary target 22 nucleotide DNA concentration (0 aM, 2 aM, 200 aM, 20 fM, 2 pM, 200 pM and 20 nM). Two dash lines are provided to indicate the levels of the average blank and the three times standard deviation above the averaged blank. Error bars represent standard deviations from 5 independent measurements.

Fig. 4. (a) Hybridization-induced change in the SWVs after exposing the sensor to different concentrations of complementary ctDNA target (101 nucleotides) wherein the probe DNA hybridized to the 3′ end (red data points) and the middle (black data points) of the ctDNA. (b) Effect of hybridization time after exposing the sensor to 20 nM complementary ctDNA target (101 nucleotides) on the SWV current change. (c) Hybridization-induced change in the SWVs after exposing the sensor to complementary and single mismatched circulating tumor DNA target sequences. (d) Hybridization-induced change in SWVs after exposing the sensor to different concentrations of complementary ctDNA target (101 nucleotides) spiked in phosphate buffered saline (black data points), human plasma (blue data points) and 50% human blood (red data points). Two dash lines are provided to indicate the levels of the average blank and the three times standard deviation above the averaged blank. Error bars represent standard deviations from at least 5 independent experiments.