Ultra-sensitive and rapid detection of nucleic acids and microorganisms in body fluids using single-molecule tethering

Abstract

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL−1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT’s clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.

An Ultra-Stable and Dense Single-Molecule Click Platform for Sensing Protein-DNA Interactions

“…For medical applications, single-molecule biomarker monitoring and detection methods based on TPM have been demonstrated previously. 11–14 The high-stability and fouling resistance of the scTPM platform can be used to produce biomarker detection sensors that remain functional for weeks to months at a time and under a range of environmental conditions. The TCO click sensing surface forms a flexible platform for the attachment of nucleotides, proteins and enzymes, and the scTPM surface can be readily functionalized with the required ligands for biomarker capture or the biomarker analog for a competition assay.…”

“…Recently, picomolar concentrations of nucleic acids were able to be continuously monitored, over several hours, in blood plasma by Biosensing based on Particle Mobility (BPM), 11–13 while fM detection sensitivity to nucleic acids in bodily fluid samples was demonstrated by Single Molecule Tethering (SMOLT). 14…”

An Ultrastable and Dense Single‐Molecule Click Platform for Sensing Protein–Deoxyribonucleic Acid Interactions

“…For medical applications, single‐molecule biomarker monitoring and detection methods based on TPM have been demonstrated previously. [ 11–14 ] The high‐stability and low non‐specific interactions of the scTPM platform can be used to produce biomarker detection sensors that remain functional for weeks to months at a time and under a range of environmental conditions. The TCO click sensing surface forms a flexible platform for the attachment of nucleotides, proteins, and enzymes, and the scTPM surface can be readily functionalized with the required ligands for biomarker capture or the biomarker analog for a competition assay.…”

“…Recently, picomolar concentrations of nucleic acids were able to be continuously monitored, over several hours, in blood plasma by biosensing based on particle mobility, [ 11–13 ] while f m detection sensitivity to nucleic acids in bodily fluid samples was demonstrated by single‐molecule tethering. [ 14 ]…”

Biosensors and Diagnostics for Fungal Detection

“…C. albicans DNA was detected in human blood utilising a real-time PCR-based microfluidic platform [ 146 ], with other reported methods including the use of AuNPs, peptide nucleic acid, Au-nanowire, nanoparticles and colloidal gold and silver [ 147 , 148 , 149 , 150 ]. Recently, a polymerase free assay was reported for C. albicans detection in clinical samples [ 151 ]. The proposed method was based on Single MOLecule Tethering (SMOLT), displacement of the beads tethered by DNA probes generated the signal, where the proposed method was able to detect 1 CFU/mL in blood sample.…”