Laser ablation-based one-step generation and bio-functionalization of gold nanoparticles conjugated with aptamers

Background: 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. Increased yields are required, particularly considering the conjugation of cost-intensive biomolecules like RNA aptamers. Results: Using 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. Conclusions: In 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.

Transcription of TIR1-Controlled Genes Can be Regulated within 10 Min by an Auxin-Induced Process. Can TIR1 be the Receptor?

ABP1 and TIR1/AFBs are known as auxin receptors. ABP1 is linked to auxin responses several of which are faster than 10 min. TIR1 regulates auxin-induced transcription of early auxin genes also within minutes. We use transcription of such TIR1-dependent genes as indicator of TIR1 activity to show the rapid regulation of TIR1 by exogenous auxin. To this end, we used quantification of transcription of a set of fifteen early auxin-induced reporter genes at t = 10 and t = 30 min to measure this as a TIR1-dependent auxin response. We conducted this study in 22 mutants of auxin transporters (pin5, abcb1, abcb19, and aux1/lax3), protein kinases and phosphatases (ibr5, npr1, cpk3, CPK3-OX, d6pk1, d6pkl1-1, d6pkl3-2, d6pkl1-1/d6pkl2-2, and d6pkl1-1/d6pkl3-2), of fatty acid metabolism (fad2-1, fad6-1, ssi2, lacs4, lacs9, and lacs4/lacs9) and receptors (tir1, tir1/afb2, and tir1/afb3) and compared them to the wild type. After 10 min auxin application, in 18 out of 22 mutants mis-regulated expression of at least one reporter was found, and in 15 mutants transcription of two-to-three out of five selected auxin reporter genes was mis-regulated. After 30 min of auxin application to mutant plants, mis-regulation of reporter genes ranged from one to 13 out of 15 tested reporter genes. Those genes chosen as mutants were themselves not regulated in their expression by auxin for at least 1 h, excluding an influence of TIR1/AFBs on their transcription. The expression of TIR1/AFB genes was also not modulated by auxin for up to 3 h. Together, this excludes a feedback or feedforward of these mutant genes/proteins on TIR1/AFBs output of transcription in this auxin-induced response. However, an auxin-induced response needed an as yet unknown auxin receptor. We suggest that the auxin receptor necessary for the fast auxin-induced transcription modulation could be, instead, ABP1. The alternative hypothesis would be that auxin-induced expression of a protein, initiated by TIR1/AFBs receptors, could initiate these responses and that this unknown protein regulated TIR1/AFB activities within 10 min.

Investigations of the Toxic Effect of Silver Nanoparticles on Mammalian Cell Lines

Silver nanoparticles are widely used for many applications. In this study silver nanoparticles have been tested for their toxic effect on fibroblasts (NIH-3T3), on a human lung adenocarcinoma epithelial cell line (A-549), on PC-12-cells, a rat adrenal pheochromocytoma cell line, and on HEP-G2-cells, a human hepatocellular carcinoma cell line. The viability of the cells cultivated with different concentrations of silver was determined by the MTT assay, a photometric method to determine cell metabolism. Dose-response curves were extrapolated and IC50, total lethal concentration (TLC), and no observable adverse effect concentration (NOAEC) values were calculated for each cell line. As another approach, ECIS (electric-cell-substrate-impedance-sensing) an automated method to monitor cellular behavior in real-time was applied to observe cells cultivated with silver nanoparticles. To identify the type of cell death the membrane integrity was analyzed by measurements of the lactate dehydrogenase releases and by determination of the caspase 3/7 activity. To ensure that the cytotoxic effect of silver nanoparticles is not traced back to the presence of Ag+ ions in the suspension, an Ag+ salt (AgNO3) has been examined at the same concentration of Ag+ present in the silver nanoparticle suspension that is assuming that the Ag particles are completely available as Ag+ ions.