RESUMEN
Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent "bloom" events for positive samples, in an approach called "Spatial LAMP" (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17-32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 102 copies per µl, and a gamma-irradiated virus of 103 virus particles in a single 12.5 µl droplet blood sample.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Microfluídica , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , Sensibilidad y Especificidad , Teléfono Inteligente , Instrumentos Quirúrgicos , Virus Zika/genética , Infección por el Virus Zika/diagnósticoRESUMEN
Zinc germanate doped with Mn2+ (Zn2GeO4:Mn2+) is known to be a persistent luminescence green phosphor with potential applications in biosensing and bioimaging. Such applications demand nanoparticulated phosphors with a uniform shape and size, good dispersibility in aqueous media, high chemical stability, and surface-functionalization. These characteristics could be major bottlenecks and hence limit their practical applications. This work describes a one-pot, microwave-assisted hydrothermal method to synthesize highly uniform Zn2GeO4:Mn2+ nanoparticles (NPs) using polyacrylic acid (PAA) as an additive. A thorough characterization of the NPs showed that the PAA molecules were essential to realizing uniform NPs as they were responsible for the ordered aggregation of their building blocks. In addition, PAA remained attached to the NPs surface, which conferred high colloidal stability to the NPs through electrostatic and steric interactions, and provided carboxylate groups that can act as anchor sites for the eventual conjugation of biomolecules to the surface. In addition, it was demonstrated that the as-synthesized NPs were chemically stable for, at least, 1 week in phosphate buffer saline (pH range = 6.0-7.4). The luminescence properties of Zn2GeO4 NPs doped with different contents of Mn2+ (0.25-3.00 mol %) were evaluated to find the optimum doping level for the highest photoluminescence (2.50% Mn) and the longest persistent luminescence (0.50% Mn). The NPs with the best persistent luminescence properties were photostable for at least 1 week. Finally, taking advantage of such properties and the presence of surface carboxylate groups, the Zn2GeO4:0.50%Mn2+ sample was successfully used to develop a persistent luminescence-based sandwich immunoassay for the autofluorescence-free detection of interleukin-6 in undiluted human serum and undiluted human plasma samples. This study demonstrates that our persistent Mn-doped Zn2GeO4 nanophosphors are ideal candidates for biosensing applications.
Asunto(s)
Luminiscencia , Nanopartículas , Humanos , Nanopartículas/química , Resinas Acrílicas , Zinc/químicaRESUMEN
Sepsis is a life-threatening dysfunction of organ systems caused by a dysregulated immune system because of an infectious process. It remains one of the leading causes of hospital mortality and of hospital readmissions in the United States. Mortality from sepsis increases with each hour of delayed treatment, therefore, diagnostic devices that can reduce the time from the onset of a patient's infection to the delivery of appropriate therapy are urgently needed. Likewise, tools that are capable of high-frequency testing of clinically relevant biomarkers are required to study disease progression. Electrochemical biosensors offer important advantages such as high sensitivity, fast response, miniaturization, and low cost that can be adapted to clinical needs. In this review paper, we discuss the current state, limitations, and future directions of electrochemical-based point-of-care detection platforms that contribute to the diagnosis and monitoring of sepsis.