Oligonucleotide and Polymer Functionalized Nanoparticles for Amplification-Free Detection of DNA

Abstract

Sensitive and quantitative nucleic acid testing from complex biological samples is now an important component of clinical diagnostics. Whereas nucleic acid amplification represents the gold standard, its utility in resource-limited and point-of-care settings can be problematic due to assay interferants, assay time, engineering constraints, and costs associated with both wetware and hardware. In contrast, amplification-free nucleic acid testing can circumvent these limitations by enabling direct target hybridization within complex sample matrices. In this work, we grew random copolymer brushes from the surface of silica-coated magnetic nanoparticles using azide-modified and hydroxyl oligo ethylene glycol methacrylate (OEGMA) monomers. The azide-functionalized polymer brush was first conjugated, via copper-catalyzed azide/alkyne cycloaddition (CuAAC), with herpes simplex virus (HSV)-specific oligonucleotides and then with alkyne-substituted polyethylene glycol to eliminate all residual azide groups. Our methodology enabled control over brush thickness and probe density and enabled multiple consecutive coupling reactions on the particle grafted brush. Brush- and probe-modified particles were then combined in a 20 min hybridization with fluorescent polystyrene nanoparticles modified with HSV-specific reporter probes. Following magnetic capture and washing, the particles were analyzed with an aggregate fluorescence measurement, which yielded a limit of detection of 6 pM in buffer and 60 pM in 50% fetal bovine serum. Adoption of brush- and probe-modified particles into a particle counting assay will result in the development of diagnostic assays with significant improvements in sensitivity.

Toward Non-Enzymatic Ultrasensitive Identification of Single Nucleotide Polymorphisms by Optical Methods

“…Using synthetic nanoparticles prepared by click chemistry between azido groups of oligo ethylene glycol mecrylate (OEGMA) polymer on the surface of silica-coated magnetic nanoparticles with a gene-specific probe, the DNA of herpes simplex (HSV) virus was detected without amplification at low picomolar range (i.e., 6 pM in aqueous buffer and 60 pM in fetal bovine serum) by means of fluorescence ( Figure 4A) [44]. The reported methodology of preparation allows control over both OEGMA polymer -brush‖ thickness and immobilized probe density.…”

“…After 20 min hybridization with HSV target and the second pool of nanoparticles containing specific HSV probes and fluorescent dye, a wash step and analysis were subsequently performed. Authors mentioned that further development of a particle-counting enzyme-free methodology will result in the development of diagnostic assays with significant improvements in sensitivity [44].…”

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

“…As an example latex macrofluorophores, available from LifeTechnologies, have been documented by imaging techniques to be taken up into the cell [39]. Ihara, et al, and Thomson, et al, employed these techniques recently to demonstrate successful examples of signal enhancement using fluorescent organic polymers with a p53 SNP sensitivity assay for DNA detection and an HSV DNA detection at sub-picomolar levels, respectively [40,41]. An alternative approach to signal enhancement using fluorophores is the use of nucleic acid intercalating and groove binding dyes [42,43].…”

In vivo monitoring of rat macrophages labeled with poly(l‐lysine)‐iron oxide nanoparticles

“…1 Biomedical applications benefit from the fact that the superparamagnetic particles can be easily manipulated and/ or detected using an external magnetic field. The applications using manipulations include immunoassays, 2 separation of microorganisms and biomolecules, such as viruses, 3 hormones, 4 oligonucleotides, 5 DNA, 6 proteins 7,8 and cells, 9 targeted drug and gene delivery. 10 However, the use of particles for detection has concentrated on cell tracking, [11][12][13] tagging and imaging [by magnetic resonance imaging (MRI)] 14 as well as on cancer treatment by hyperthermia.…”