RESUMEN
Herein, we report an ATP-responsive nanoparticle (GroEL NP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroEL NP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroEL NP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable GroEL NP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroEL NP per GroEL was 4.8 and 4.0 times greater than those of precursor cys GroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that GroEL NP could be iteratively extended to double-layered ( GroEL ) 2 ${{^{({\rm GroEL}){_{2}}}}}$ NP.
Asunto(s)
Adenosina Trifosfato , Chaperoninas , Chaperoninas/metabolismo , Adenosina Trifosfato/metabolismo , Chaperonina 60/química , Pliegue de ProteínaRESUMEN
It has been recently reported that the fluorescence of some DNA-templated silver nanoclusters (AgNCs) can be significantly enhanced upon by hybridizing with a partially complementary DNA containing a G-rich overhang near the AgNCs. This discovery has found a number of analytical applications but many fundamental questions remain to be answered. In this work, the photostability of these activated AgNCs is reported. After adding the G-rich DNA activator, the fluorescence intensity peaks in â¼1 h and then starts to decay, where the decaying rate is much faster with light exposure. The lost fluorescence is recovered by adding NaBH4, suggesting that the bleaching is an oxidative process. Once activated, the G-rich activator can be removed while the AgNCs still maintain most of their fluorescence intensity. UV-vis spectroscopy suggests that new AgNC species are generated upon hybridization with the activator. The base sequence and length of the template DNA have also been varied, leading to different emission colors and color change after hybridization. G-rich aptamers can also serve as activators. Our results indicate that activation of the fluorescence by G-rich DNA could be a convenient method for biosensor development since the unstable NaBH4 is not required for the activation step.
Asunto(s)
ADN/química , Guanina/química , Nanoestructuras/química , Plata/química , Secuencia de Bases , Fluorescencia , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fotoblanqueo , Espectrometría de FluorescenciaRESUMEN
DNA oligomers are particularly interesting templates for making silver nanoclusters (AgNCs) as different emission colors can be obtained by varying the DNA sequence. Many AgNCs have been used as Hg²âº sensors since Hg²âº induces fluorescence quenching. From an analytical chemistry standpoint, however, these 'light off' sensors are undesirable. In this work, taking advantage of the fact that some AgNCs are not as effectively quenched by Hg²âº, we design a sensor with AgNCs containing two emission peaks. The red peak is strongly quenched by Hg²âº while the green peak shows a concomitant increase, producing an orange-to-green visual fluorescence transformation. Using this AgNC, we demonstrate ratiometric detection with a detection limit of 4 nM Hg²âº. This sensor is further immobilized in a hydrogel matrix and this gel is also capable of detecting Hg²âº with a visual response.
Asunto(s)
Técnicas Biosensibles/métodos , ADN/química , Hidrogeles/química , Mercurio/análisis , Nanoestructuras/química , Plata/química , Cationes Bivalentes/análisis , Fluorescencia , Límite de DetecciónRESUMEN
Few-atom noble metal nanoclusters have attracted a lot of interest due to their potential applications in biosensor development, imaging and catalysis. DNA-templated silver nanoclusters (AgNCs) are of particular interest as different emission colors can be obtained by changing the DNA sequence. A popular analytical application is fluorescence quenching by Hg(2+), where d(10)-d(10) metallophilic interaction has often been proposed for associating Hg(2+) with nanoclusters. However, it cannot explain the lack of response to other d(10) ions such as Zn(2+) and Cd(2+). In our effort to elucidate the quenching mechanism, we studied a total of eight AgNCs prepared by different hairpin DNA sequences; they showed different sensitivity to Hg(2+), and DNA with a larger cytosine loop size produced more sensitive AgNCs. In all the cases, samples strongly quenched by Hg(2+) were also more easily photobleached. Light of shorter wavelengths bleached AgNCs more potently, and photobleached samples can be recovered by NaBH4. Strong fluorescence quenching was also observed with high redox potential metal ions such as Ag(+), Au(3+), Cu(2+) and Hg(2+), but not with low redox potential ions. Such metal induced quenching cannot be recovered by NaBH4. Electronic absorption and mass spectrometry studies offered further insights into the oxidation reaction. Our results correlate many important experimental observations and will fuel the further growth of this field.