Circulating Disease Biomarker Detection in Complex Matrices: Real-Time, In Situ Measurements of DNA/miRNA Hybridization via Electrochemical Impedance Spectroscopy

Abstract

The quantification of significant amounts of disease biomarkers circulating in the bloodstream represents one of the challenging frontiers in biomedicine. The complexity of blood composition has opened the quest for novel detection technologies, capable of discerning small amount of specific biomarkers from other blood proteins/oligonucleotides and of reliably measuring them. In this context, we have developed a label-free device based on differential double-layer capacitance readout at microfabricated gold electrodes and demonstrated its detection performance in real biosample volumes. By means of electrochemical impedance spectroscopy (EIS) measurements in a three electrodes setup, we first calibrated the system following the in situ hybridization of a self-assembled monolayer of single-stranded, short oligonucleotides on the gold microelectrode through the measurement of differential capacitance changes as a function of time, for different concentrations of complementary DNA in a saline buffer. Based on this calibration we used the device to quantify the presence of microRNAs (miRNAs) in human plasma. We demonstrated that our device is fast, sensitive, reusable, reproducible, and perfectly suited to detect biomarkers in complex matrices, as cell lysate, serum, and blood. We put forward the possibility to apply this platform to the bioaffinity detection of protein biomarkers as well as circulating drugs in blood, for therapeutic drug monitoring applications.

Advances in liquid biopsy on-chip for cancer management: Technologies, biomarkers, and clinical analysis

“…The recognition element is the piece that allows for analyte specificity, and can be immune recognition, size or physical parameter based, or based on electrochemical cues[14], [20]. The choice of recognition element is generally based upon the biomarker analyte desired and the required specificity of the system.…”

Miniaturized Aptamer-Based Assays for Protein Detection

“…As already explained in [11,12], at each frequency we collect 200 complete periods from which we compute the root mean squared value of the measured current, I rms = 2πfV rms C d , and the relative uncertainties using error propagation analysis. We also proved that the device is reusable up to several times and the data are reproducible on different sensors with a standard deviation of only a few percent.…”

“…Low density monolayers were chosen to avoid steric hindrance limitations to the hybridization efficiency. In this regime DNA hybridization follows Langmuir-like kinetics [12,27], while electrostatic charges are largely screened allowing for fast hybridization kinetics and reducing the limit of detection of the device. After each functionalization step the devices were rinsed with the proper DNA/protein buffer solution, prior to capacitance measurements.…”

“…Gold electrode functionalization with ssDNA molecules was carried out using the well-established procedures for creating DNA SAMs on gold [24][25][26]. Initially the electrodes were wetted for 15 min with a drop of a high-ionic-strength buffer, TE NaCl 1 M, containing cF9-SH to create a low density DNA SAM (2.1 × 10 12 molecules/cm 2 ) [12]. Low density monolayers were chosen to avoid steric hindrance limitations to the hybridization efficiency.…”

“…On such active DNA nanospots, the aptamer-cDNA conjugate is then loaded through Watson-Crick base-pairing, in a process known as DNA-directed immobilization [9]. On the other side, an electrochemical impedance-based biosensor, with a three-electrode design developed in our group for the recognition of nucleic acids [11,12], are tested towards protein detection through modified aptamers. The working micro-electrode is fully covered by a functional, thiolated, single-stranded DNA SAM, on which the aptamer-cDNA conjugate is then loaded via DDI.…”