An Ultrasensitive Method for Protein and DNA Detection at the Single Molecule Level

We demonstrate a general single molecule method for ultrasensitive detection of macromolecules such as DNA, proteins and biomarkers. The method is based on conjugation of two nanoparticles to target macromolecules followed by imaging of the specific dimeric structures formed using electron microscopy.

Detection of macromolecular biomarkers such as disease-specific DNA and proteins provides essential information that allows early diagnosis, prognosis and management of diseases. Today biomarkers detection is limited in sensitivity and therefore detection often comes at a late disease stage. Therefore new techniques are required to enable rapid and immediate diagnosis from physiological samples. Critically, such a system must be capable of detecting very low levels of biomarkers, as many of them are present at minute concentrations during early disease phases. These methods should be generic for a wide range of macromolecules and based on affordable detection tools.

The proposed method presents ultrasensitive detection of macromolecules at the single molecule level using NPs. The method is based on conjugation of two different ligands, which bind the target macromolecule at two different binding sites, to two NPs with different sizes. The formed complex, in which each macromolecule is flanked by two easily recognizable NPs (a dimer), is then detected and characterized using EM. The surface concentration of the dimers can be calibrated to the concentration of the macromolecule in the sample.

We will use model systems to validate the general proposed concept of dimer formation and highly sensitive detection. The first model system will be a target DNA, which is detected using two single NPs each bearing a complementary ssDNA. The second model system will be a target protein, which is detected using two NPs covered with conjugated peptides that bind specifically to two different sites in the protein. Following the implementation of those model systems, we will utilize the proposed method for various macromolecules and biomarkers detection.