Gold nanoprobe-based non-crosslinking hybridization for molecular diagnostics

Abstract

Non-crosslinking (NCL) approaches using DNA-modified gold nanoparticles for molecular detection constitute powerful tools with potential implications in clinical diagnostics and tailored medicine. From detection of pathogenic agents to identification of specific point mutations associated with health conditions, these methods have shown remarkable versatility and simplicity. Herein, the NCL hybridization assay is broken down to the fundamentals behind its assembly and detection principle. Gold nanoparticle synthesis and derivatization is addressed, emphasizing optimal size homogeneity and conditions for maximum surface coverage, with direct implications in downstream detection. The detection principle is discussed and the advantages and drawbacks of different NCL approaches are discussed. Finally, NCL-based applications for molecular detection of clinically relevant loci and potential integration into more complex biosensing platforms, projecting miniaturization and portability are addressed.

Gold aggregating gold: A novel nanoparticle biosensor approach for the direct quantification of hepatitis C virus RNA in clinical samples

“…This is due to electrostatic interaction affinity between the probes’ nitrogenous bases and the citrate AuNPs, which prevents hybridization between the RNA target and the probes (Li and Rothberg 2004b). A brief denaturation step was therefore conducted for the nanoprobe/RNA mixture and prior to the addition of the cationic AuNPs to allow accessibility of the probes to the RNA molecules (Larguinho et al, 2015), and thus allowing for the unfolding of the RNA tertiary structure and subsequent hybridization with its complementary probes. Incubation of the mixture at room temperature prior to the addition of the cationic AuNPs was essential.…”

“…Thus, AuNPs have been employed in many colorimetric assays for different biological molecules as proteins (Kim et al, 2009), and nucleic acids (Liandris et al, 2009). For example, Mirkin and co-workers were the first to develop a modified AuNPs cross-linking method for the direct detection of nucleic acids (Elghanian et al, 1997, Larguinho et al, 2015). Despite the high sensitivity and specificity of this method, it requires firm temperature control for precise target detection.…”

“…However, the desired results are only achieved if the target is of the same length as the probe. Baptista and co-workers improved this technique to detect long nucleic acids; however it still depends on either high ionic strength or the pH to induce AuNPs aggregation (Baptista et al, 2008, Larguinho et al, 2015). …”

Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes

“…Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) due to their unique optical proprieties have been widely used for nucleic acid detection with high sensibility and sensitivity at much lower costs when compared with conventional molecular methods. Disperse Aunanoprobe solutions show a characteristic surface plasmon resonance (SPR) band at around 525 nm (red color), which is red-shifted upon salt-induced aggregation (blue color); hybridization to a complementary nucleic acid target sequence prevents aggregation and the solution remains red [1]. This non-cross-linking colorimetric detection method has been efficiently applied to the detection of pathogens and other DNA sequences of interest, where hybridization to a specific complementary target leads to Au-nanoprobe stabilization and resistance to salt-induced aggregation [2,3].…”

“…Based on their size-dependent surface plasmon resonance (SPR) absorption and the high surface-to-volume ratio allowing for surface functionalization with a variety of molecules, gold nanoparticles (AuNPs) are frequently used as probes in biological detections [1,2]. Baptista and colleagues developed a proof-of-concept colorimetric Au-nanoprobe method for direct detection and quantification of a BCR-ABL1 e14a2 fusion suitable for identification of BCR-ABL targets in lab-controlled conditions [3].…”