Abstract
BackgroundBio-conjugated nanoparticles are important analytical tools with emerging biological and medical applications. In this context, in situ conjugation of nanoparticles with biomolecules via laser ablation in an aqueous media is a highly promising one-step method for the production of functional nanoparticles resulting in highly efficient conjugation. Increased yields are required, particularly considering the conjugation of cost-intensive biomolecules like RNA aptamers.ResultsUsing a DNA aptamer directed against streptavidin, in situ conjugation results in nanoparticles with diameters of approximately 9 nm exhibiting a high aptamer surface density (98 aptamers per nanoparticle) and a maximal conjugation efficiency of 40.3%. We have demonstrated the functionality of the aptamer-conjugated nanoparticles using three independent analytical methods, including an agglomeration-based colorimetric assay, and solid-phase assays proving high aptamer activity. To demonstrate the general applicability of the in situ conjugation of gold nanoparticles with aptamers, we have transferred the method to an RNA aptamer directed against prostate-specific membrane antigen (PSMA). Successful detection of PSMA in human prostate cancer tissue was achieved utilizing tissue microarrays.ConclusionsIn comparison to the conventional generation of bio-conjugated gold nanoparticles using chemical synthesis and subsequent bio-functionalization, the laser-ablation-based in situ conjugation is a rapid, one-step production method. Due to high conjugation efficiency and productivity, in situ conjugation can be easily used for high throughput generation of gold nanoparticles conjugated with valuable biomolecules like aptamers.
Highlights
Bio-conjugated nanoparticles are important analytical tools with emerging biological and medical applications
We show the functionalization of nanoparticles with aptamers during femtosecond-pulsed, laser-induced gold nanoparticle formation in an aqueous media using a DNA aptamer directed against streptavidin as a model system
We have found that the miniStrep aptamer requires an additional spacer placed between the aptamer and the substrate to show activity that is slightly higher when immobilized via its 3’ terminus
Summary
Bio-conjugated nanoparticles are important analytical tools with emerging biological and medical applications. In this context, in situ conjugation of nanoparticles with biomolecules via laser ablation in an aqueous media is a highly promising one-step method for the production of functional nanoparticles resulting in highly efficient conjugation. Gold nanoparticles (AuNPs) feature unique optical properties, including high surface plasmon resonance (SPR), enhanced absorbance and scattering with high quantum efficiency. In addition to their resistance against photobleaching, AuNPs perfectly fulfill requirements for use as colorimetric sensors and markers. Aptamer-conjugated AuNPs have already been successfully used for the detection of proteins in a dry-reagent strip biosensor, [9] for detection of thrombin on surfaces, [4] for colorimetric detection of platelet-derived growth factor, [5] for detection of adenosine and potassium ions in an agglomeration-based approach, [10] for detection of thrombin in a dot blot assay [11] and for targeting and therapy of cancerous cells [12,13]
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