Here we describe a new hybrid fluorescent nanoprobe composed of a nuclease-resistant molecular beacon (MB) backbone, CdSe-ZnS core-shell quantum dots (QDs) as donors, and gold nanoparticles (Au NPs) as quenchers, for the real-time visualization of virus replication in living cells. By using a Au NP-MB to QD ratio of 6 : 1, a 7.3-fold increase in fluorescent signal was achieved upon target binding. For living cell experiments, a hexahistidine-appended Tat peptide was self-assembled onto the QD surface to provide nearly 100% non-invasive delivery of the QD-MB-Au NP probes within 2 h. By directly visualizing the fluorescent complexes formed with the newly synthesized viral RNA, this QD-MB-Au NP probe provided sensitive and real-time detection of infectious viruses as well as the real-time visualization of cell-to-cell virus spreading.
Rapid and efficient detection of viral infection is crucial for the prevention of disease spread during an outbreak and for timely clinical management. In this paper, the utility of Tat peptide-modified molecular beacons (MBs) as a rapid diagnostic tool for the detection of virus-infected cells was demonstrated. The rapid intracellular delivery mediated by the Tat peptide enabled the detection of infected cells within 30 s, reaching saturation in signal in 30 min. This rapid detection scheme was coupled with flow cytometry (FC), resulting in an automated, high-throughput method for the identification of virus-infected cells. Because of the 2-order-of-magnitude difference in fluorescence intensity between infected and uninfected cells, as few as 1% infected cells could be detected. Because of its speed and sensitivity, this approach may be adapted for the practical diagnosis of multiple viral infections.
Thermal dissipation is an important issue for power devices. In this work, the impact of thermal effects on the performance of Cu electroplated GaN-based high-electron-mobility transistors (HEMTs) are considered. Electrical, thermometry and micro-Raman characterization techniques were used to correlate the effects of improved heat dissipation on device performance for GaN HEMTs with different thicknesses of Si substrate (50, 100, 150 μm), with and without an additional electroplated Cu layer. GaN HEMTs on electroplated Cu on Si (≤50 μm) demonstrate an enhanced on/off current ratio compared to bare Si substrate by a factor of ~400 (from 9.61 × 105 to 4.03 × 108). Of particular importance, surface temperature measurements reveal a much lower channel temperature for thinner HEMT devices with electroplated Cu samples compared to those without.
In this study, we describe the use of nuclease-resistant molecular beacons (MBs) for the real-time detection of coxsackievirus B6 replication in living Buffalo green monkey kidney (BGMK) cells via Tat peptide delivery. A nuclease-resistant MB containing 2'-O-methyl RNA bases with phosphorothioate internucleotide linkages was designed to specifically target an 18-bp 5' noncoding region of the viral genome. For intracellular delivery, a cell-penetrating Tat peptide was conjugated to the MB by using a thiol-maleimide linkage. Presence of the Tat peptide enabled nearly 100% intracellular delivery within 15 min. When the conjugate was introduced into BGMK cell monolayers infected with coxsackievirus B6, a discernible fluorescence was observed at 30 min after infection, and as few as 1 infectious viral particle could be detected within 2 h. The stability and the intracellular delivery properties of the modified MBs enabled real-time monitoring of the cell-to-cell spreading of viral infection. These results suggest that the Tat-modified, nuclease-resistant MBs may be powerful tools for improving our understanding of the dynamic behavior of viral replication and for therapeutic studies of antiviral treatments.
Waterborne transmitted viruses pose a public health threat due to their stability in aquatic environments and their ease of transmission with high morbidity rates at low infectious doses. The ability to detect infectious viruses is of critical importance for environmental health and safety. Current methods to assess the presence of infectious viruses are based on detecting the production of cytopathic effects from mammalian cell culture and can take up to weeks before positive identification. Improved methods for rapid and reliable detection and population quantification of infectious viruses are needed for public health assessments. Molecular beacons (MBs), which produce fluorescence upon target binding, provide a simple and separation-free scheme for rapid and sensitive detection of infectious viruses. However, for real-time studies in living cells, the durability of MBs is affected by the intracellular nuclease degradation. Additionally, cell fixation and permeabilization are required to maintain the cellular structure before introducing MBs. In this study, we developed several FRET (fluorescence resonance energy transfer)-based MBs combined with fluorescence microscopy to directly visualize the fluorescent hybrids with newly synthesized viral RNA as an indication of viral infection and to subsequently follow virus spread among cells in situ/in vivo. To prevent nucleolytic degradation, we designed MBs containing 2'-O-methyl RNA bases with phosphorothioate internucleotide linkages, which specifically target the 5' noncoding region of the viral genome. A cell-penetrating Tat peptide was appended to the FRET probes to facilitate non-invasive entry into host cells. To further improve the probe sensitivity in providing real-time and long-term detection of viral replication, MBs composed of quantum dot and gold nanoparticles were generated. Confluent cell monolayers were incubated with the probes followed by infection with virus dilutions and the fluorescence intensity was monitored in real time. Sensitivity experiments showed that 1 PFU detection limit could be achieved within one replicative cycle. The illumination of fluorescent cells increased in a dose-responsive manner and enabled the direct quantification of infectious viral doses. By introducing the modified MBs into the host cell population prior to viral infection and tracking the change of fluorescence signals, we observed cell-to-cell spread when progeny virions infected new host cells in which the infectious cycle could be repeated. The specific nature of these probes enable their utility for rapid diagnostics of viral infections and real-time viral detection in vivo provides sufficient information regarding multiple steps in infection processes at the subcellular level, which will be valuable for the prevention, control, and understanding of viral infection.
Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5' noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.
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