RESUMO
Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.
Assuntos
DNA Bacteriano , DNA Fúngico , Teste em Amostras de Sangue Seco , Reação em Cadeia da Polimerase , Sepse , Antibacterianos/farmacologia , Candida albicans/genética , Candida albicans/isolamento & purificação , DNA Bacteriano/sangue , DNA Fúngico/sangue , Teste em Amostras de Sangue Seco/métodos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Heme/química , Humanos , Limite de Detecção , Meticilina/farmacologia , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Sepse/sangue , Sepse/diagnóstico , Sepse/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Células-TroncoRESUMO
The presence of numerous inhibitors in blood makes their use in nucleic acid amplification techniques difficult. Current methods for extracting and purifying pathogenic DNA from blood involve removal of inhibitors, resulting in low and inconsistent DNA recovery rates. To address this issue, a biphasic method is developed that simultaneously achieves inhibitor inactivation and DNA amplification without the need for a purification step. Inhibitors are physically trapped in the solid-phase dried blood matrix by blood drying, while amplification reagents can move into the solid nano-porous dried blood and initiate the amplification. It is demonstrated that the biphasic method has significant improvement in detection limits for bacteria such as Escherichia coli, Methicillin-resistant Staphylococcus aureus, Methicillin-Sensitive Staphylococcus aureus using loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Several factors, such as drying time, sample volume, and material properties are characterized to increase sensitivity and expand the application of the biphasic assay to blood diagnostics. With further automation, this biphasic technique has the potential to be used as a diagnostic platform for the detection of pathogens eliminating lengthy culture steps.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Reação em Cadeia da Polimerase , Técnicas de Amplificação de Ácido Nucleico/métodos , Staphylococcus aureus/genética , Escherichia coli/genética , Sensibilidade e EspecificidadeRESUMO
The advancement of point-of-care diagnostics is crucial to improving patient outcomes, especially in areas with low access to hospitals or specialized laboratories. In particular, rapid, sensitive, and multiplexed detection of disease biomarkers has great potential to achieve accurate diagnosis and inform high quality care for patients. Our Coulter counting and immunocapture based detection system has previously shown its broad applicability in the detection of cells, proteins, and nucleic acids. This paper expands the capability of the platform by demonstrating multiplexed detection of whole-virus particles using electrically distinguishable hydrogel beads by demonstrating the capability of our platform to achieve simultaneous detection at clinically relevant concentrations of hepatitis A virus (>2 × 103 IU mL-1) and human parvovirus B19 virus like particles (>106 IU mL-1) from plasma samples. The expanded versatility of the differential electrical counting platform allows for more robust and diverse testing capabilities.
Assuntos
Ácidos Nucleicos , Parvovirus B19 Humano , Humanos , Microfluídica , ProteínasRESUMO
The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per µL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.
Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Testes Imediatos/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , COVID-19 , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/normas , Mucosa Nasal/virologia , Pandemias , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , SARS-CoV-2 , SmartphoneRESUMO
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.
Assuntos
Infecção por Zika virus , Zika virus , Humanos , Microfluídica , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Sensibilidade e Especificidade , Smartphone , Instrumentos Cirúrgicos , Zika virus/genética , Infecção por Zika virus/diagnósticoRESUMO
The COVID-19 pandemic revealed fundamental limitations in the current model for infectious disease diagnosis and serology, based upon complex assay workflows, laboratory-based instrumentation, and expensive materials for managing samples and reagents. The lengthy time delays required to obtain test results, the high cost of gold-standard PCR tests, and poor sensitivity of rapid point-of-care tests contributed directly to society's inability to efficiently identify COVID-19-positive individuals for quarantine, which in turn continues to impact return to normal activities throughout the economy. Over the past year, enormous resources have been invested to develop more effective rapid tests and laboratory tests with greater throughput, yet the vast majority of engineering and chemistry approaches are merely incremental improvements to existing methods for nucleic acid amplification, lateral flow test strips, and enzymatic amplification assays for protein-based biomarkers. Meanwhile, widespread commercial availability of new test kits continues to be hampered by the cost and time required to develop single-use disposable microfluidic plastic cartridges manufactured by injection molding. Through development of novel technologies for sensitive, selective, rapid, and robust viral detection and more efficient approaches for scalable manufacturing of microfluidic devices, we can be much better prepared for future management of infectious pathogen outbreaks. Here, we describe how photonic metamaterials, graphene nanomaterials, designer DNA nanostructures, and polymers amenable to scalable additive manufacturing are being applied towards overcoming the fundamental limitations of currently dominant COVID-19 diagnostic approaches. In this paper, we review how several distinct classes of nanomaterials and nanochemistry enable simple assay workflows, high sensitivity, inexpensive instrumentation, point-of-care sample-to-answer virus diagnosis, and rapidly scaled manufacturing.
RESUMO
Biomedical diagnostics based on microfluidic devices have the potential to significantly benefit human health; however, the manufacturing of microfluidic devices is a key limitation to their widespread adoption. Outbreaks of infectious disease continue to demonstrate the need for simple, sensitive, and translatable tests for point-of-care use. Additive manufacturing (AM) is an attractive alternative to conventional approaches for microfluidic device manufacturing based on injection molding; however, there is a need for development and validation of new AM process capabilities and materials that are compatible with microfluidic diagnostics. In this paper, we demonstrate the development and characterization of AM cartridges using continuous liquid interface production (CLIP) and investigate process characteristics and capabilities of the AM microfluidic device manufacturing. We find that CLIP accurately produces microfluidic channels as small as 400 µm and that it is possible to routinely produce fluid channels as small as 100 µm with high repeatability. We also developed a loop-mediated isothermal amplification (LAMP) assay for detection of E. coli from whole blood directly on the CLIP-based AM microfluidic cartridges, with a 50 cfu/µL limit of detection, validating the use of CLIP processes and materials for pathogen detection. The portable diagnostic platform presented in this paper could be used to investigate and validate other AM processes for microfluidic diagnostics and could be an important component of scaling up the diagnostics for current and future infectious diseases and pandemics.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Escherichia coli/genética , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido NucleicoRESUMO
The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.
Assuntos
COVID-19 , Transcrição Reversa , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Pandemias , RNA Viral/genética , SARS-CoV-2 , Saliva , Sensibilidade e EspecificidadeRESUMO
Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response, leads the U.S in both mortality rate and cost of treatment. Sepsis treatment protocols currently rely on broad and non-specific parameters like heart and respiration rate, and temperature; however, studies show that biomarkers Interlukin-6 (IL-6) and Procalcitonin (PCT) correlate to sepsis progression and response to treatment. Prior work also suggests that using multi-parameter predictive analytics with biomarkers and clinical information can inform treatment to improve outcome. A point-of-care (POC) platform that provides information for multiple biomarkers can aid in the diagnosis and prognosis of potentially septic patients. Using impedance cytometry, microbead immunoassays, and biotin-streptavidin binding, we report a microfluidic POC system that correlates microbead capture to IL-6 and PCT concentrations. A multiplexed microbead immunoassay is developed and validated for simultaneous detection of both IL-6 and PCT from human plasma samples. Using the POC platform, we quantified plasma samples containing healthy, medium (~103pg/ml) and high (~105pg/ml) IL-6 and PCT concentrations with various levels of significance (P < 0.05-P < 0.00001) and validated the concept of this device as a POC platform for sepsis biomarkers.
Assuntos
Análise Química do Sangue/instrumentação , Interleucina-6/sangue , Dispositivos Lab-On-A-Chip , Testes Imediatos , Pró-Calcitonina/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Impedância Elétrica , Humanos , Sepse/sangue , Sepse/diagnóstico , Fatores de TempoRESUMO
Sepsis, a life-threatening syndrome that contributes to millions of deaths annually worldwide, represents a moral and economic burden to the healthcare system. Although no single, or even a combination of biomarkers has been validated for the diagnosis of sepsis, multiple studies have shown the high specificity of CD64 expression on neutrophils (nCD64) to sepsis. The analysis of elevated nCD64 in the first 2-6 hours after infection during the pro-inflammatory stage could significantly contribute to early sepsis diagnosis. Therefore, a rapid and automated device to periodically measure nCD64 expression at the point-of-care (POC) could lead to timely medical intervention and reduced mortality rates. Current accepted technologies for measuring nCD64 expression, such as flow cytometry, require manual sample preparation and long incubation times. For POC applications, however, the technology should be able to measure nCD64 expression with little to no sample preparation. In this paper, we demonstrate a smartphone-imaged microfluidic biochip for detecting nCD64 expression in under 50 min. In our assay, first unprocessed whole blood is injected into a capture chamber to immunologically capture nCD64 along a staggered array of pillars, which were previously functionalized with an antibody against CD64. Then, an image of the capture channel is taken using a smartphone-based microscope. This image is used to measure the cumulative fraction of captured cells (γ) as a function of length in the channel. During the image analysis, a statistical model is fitted to γ in order to extract the probability of capture of neutrophils per collision with a pillar (ε). The fitting shows a strong correlation with nCD64 expression measured using flow cytometry (R2 = 0.82). Finally, the applicability of the device to sepsis was demonstrated by analyzing nCD64 from 8 patients (37 blood samples analyzed) along the time they were admitted to the hospital. Results from this analysis, obtained using the smartphone-imaged microfluidic biochip were compared with flow cytometry. Again, a correlation coefficient R2 = 0.82 (slope = 0.99) was obtained demonstrating a good linear correlation between the two techniques. Deployment of this technology in ICU could significantly enhance patient care worldwide.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Neutrófilos/imunologia , Receptores de IgG/sangue , Sepse/diagnóstico , Smartphone , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Citometria de Fluxo , Humanos , Masculino , Técnicas Analíticas Microfluídicas/instrumentação , Pessoa de Meia-Idade , Testes ImediatosRESUMO
Deoxynivalenol (DON) is a toxic secondary metabolite produced by several species of Fusarium fungi, which can be predominantly found in agricultural crops such as wheat. In livestock, deoxynivalenol-contaminated grain can produce vomiting, feed refusal, weight loss, and diarrhea. This paper reports an electrochemical immunosensor for the detection of residual DON mycotoxin in food samples. The device uses electrochemical nanoprobes (CdSNP-AbDON) and antigen biofunctionalized magnetic µ-particles (DON-BSAMP) to detect the mycotoxin. CdSNP-AbDON are prepared by labelling the DON-specific antibodies with CdS nanoparticles (CdSNPs). Nanoparticle size and CdSNP-AbDON conjugation ratio are characterized using TEM images. The metal ions released by the CdSNP are reduced at the working electrode and read by anodic stripping voltammetry. DON can be detected in PBST buffer with an IC50 of 6.74 ± 0.19 µg L-1. The high detectability of the immunosensor developed allows detection of DON residues in 50-fold diluted wheat extracts. The limit of detection (LOD, IC90) accomplished in wheat is of 342.4 µg kg-1, which is below the maximum residue limit (MRL, 1750 µg kg-1 for unprocessed durum wheat, 750 µg kg-1 for cereals intended for direct human consumption) established by the EU for this mycotoxin. The working range is in the interval between 610 and 6210 µg kg-1. The performance of the immunosensor was compared with the ELISA assay. DON naturally contaminated wheat samples were analyzed with the immunosensor, showing acceptable recoveries. Graphical abstract.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Sondas Moleculares , Micotoxinas/análise , Nanopartículas , Tricotecenos/análise , Triticum/química , Limite de Detecção , Microscopia Eletrônica de Transmissão , Reprodutibilidade dos TestesRESUMO
The gold standard for diagnosing viruses such as the Hepatitis B Virus has remained largely unchanged, relying on conventional methods involving extraction, purification, and polymerase chain reaction (PCR). This approach is hindered by limited availability, as it is time-consuming and requires highly trained personnel. Moreover, it suffers from low recovery rates of the nucleic acid molecules for samples with low copy numbers. To address the challenges of complex instrumentation and low recovery rate of DNA, a drying process coupled with thermal treatment of whole blood is employed, resulting in the creation of a dried blood matrix characterized by a porous structure with a high surface-to-volume ratio where it also inactivates the amplification inhibitors present in whole blood. Drawing on insights from Brunauer-Emmett-Teller (BET)- Barrett-Joyner-Halenda (BJH) analysis, scanning electron microscopy (SEM), and fluorescence recovery after photobleaching (FRAP), detection assay is devised for HBV, as a demonstration, from whole blood with high recovery of DNA and simplified instrumentation achieving a limit of detection (LOD) of 10 IU mL-1. This assay can be completed in <1.5 h using a simple heater, can be applied to other DNA viruses, and is expected to be suitable for point-of-care, especially in low-resource settings.
RESUMO
The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.
RESUMO
An electrochemical magneto immunosensor for the detection of low concentrations of paraquat (PQ) in food samples has been developed and its performance evaluated in a complex sample such as potato extracts. The immunosensor presented uses immunoreagents specifically developed for the recognition of paraquat, a magnetic graphite-epoxy composite (m-GEC) electrode and biofunctionalized magnetic micro-particles (PQ1-BSAMP) that allow reduction of the potential interferences caused by the matrix components. The amperometric signal is provided by an enzymatic probe prepared by covalently linking an enzyme to the specific antibodies (Ab198-cc-HRP). The use of hydroquinone, as mediator, allows recording of the signal at a low potential, which also contributes to reducing the background noise potentially caused by the sample matrix. The immunocomplexes formed on top of the modified MP are easily captured by the m-GEC, which acts simultaneously as transducer. PQ can be detected at concentrations as low as 0.18 ± 0.09 µg L(-1). Combined with an efficient extraction procedure, PQ residues can be directly detected and accurately quantified in potato extracts without additional clean-up or purification steps, with a limit of detection (90% of the maximum signal) of 2.18 ± 2.08 µg kg(-1), far below the maximum residue level (20 µg kg(-1)) established by the EC. The immunosensor presented here is suitable for on-site analysis. Combined with the use of magnetic racks, multiple samples can be run simultaneously in a reasonable time.
Assuntos
Eletroquímica , Inocuidade dos Alimentos/métodos , Magnetismo , Paraquat/análise , Resíduos de Praguicidas/análise , Solanum tuberosum/química , HumanosRESUMO
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.
Assuntos
Luminescência , Nanopartículas , Humanos , Nanopartículas/química , Resinas Acrílicas , Zinco/químicaRESUMO
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.
RESUMO
Spatial mapping of heterogeneity in gene expression in cancer tissues can improve our understanding of cancers and help in the rapid detection of cancers with high accuracy and reliability. Significant advancements have been made in recent years in OMICS technologies, which possess the strong potential to be applied in the spatial mapping of biopsy tissue samples and their molecular profiling to a single-cell level. The clinical application of OMICS technologies in spatial profiling of cancer tissues is also advancing. The current review presents recent advancements and prospects of applying OMICS technologies to the spatial mapping of various analytes in cancer tissues. We benchmark the current state of the art in the field to advance existing OMICS technologies for high throughput spatial profiling. The factors taken into consideration include spatial resolution, types of biomolecules, number of different biomolecules that can be detected from the same assay, labeled versus label-free approaches, and approximate time required for each assay. Further advancements are still needed for the widespread application of OMICs technologies in performing fast and high throughput spatial mapping of cancer tissues as well as their effective use in research and clinical applications.
Assuntos
Neoplasias , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Reprodutibilidade dos TestesRESUMO
Since the beginning of the COVID-19 pandemic, several mutations of the SARS-CoV-2 virus have emerged. Current gold standard detection methods for detecting the virus and its variants are based on PCR-based diagnostics using complex laboratory protocols and time-consuming steps, such as RNA isolation and purification, and thermal cycling. These steps limit the translation of technology to the point-of-care and limit accessibility to under-resourced regions. While PCR-based assays currently offer the possibility of multiplexed gene detection, and commercial products of single gene PCR and isothermal LAMP at point-of-care are also now available, reports of isothermal assays at the point-of-care with detection of multiple genes are lacking. Here, we present a microfluidic assay and device to detect and differentiate the Alpha variant (B.1.1.7) from the SARS-CoV-2 virus early strains in saliva samples. The detection assay, which is based on isothermal RT-LAMP amplification, takes advantage of the S-gene target failure (SGTF) to differentiate the Alpha variant from the SARS-CoV-2 virus early strains using a binary detection system based on spatial separation of the primers specific to the N- and S-genes. We use additively manufactured plastic cartridges in a low-cost optical reader system to successfully detect the SARS-CoV-2 virus from saliva samples (positive amplification is detected with concentration ≥10 copies per µL) within 30 min. We demonstrate that our platform can discriminate the B.1.1.7 variant (USA/CA_CDC_5574/2020 isolate) from SARS-CoV-2 negative samples, but also from the SARS-CoV-2 USA-WA1/2020 isolate. The reliability of the developed point-of-care device was confirmed by testing 38 clinical saliva samples, including 20 samples positive for Alpha variant (sensitivity > 90%, specificity = 100%). This study highlights the current relevance of binary-based testing, as the new Omicron variant also exhibits S-gene target failure and could be tested by adapting the approach presented here.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Microfluídica , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , RNA Viral/genética , Reprodutibilidade dos Testes , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
In Fall 2020, universities saw extensive transmission of SARS-CoV-2 among their populations, threatening health of the university and surrounding communities, and viability of in-person instruction. Here we report a case study at the University of Illinois at Urbana-Champaign, where a multimodal "SHIELD: Target, Test, and Tell" program, with other non-pharmaceutical interventions, was employed to keep classrooms and laboratories open. The program included epidemiological modeling and surveillance, fast/frequent testing using a novel low-cost and scalable saliva-based RT-qPCR assay for SARS-CoV-2 that bypasses RNA extraction, called covidSHIELD, and digital tools for communication and compliance. In Fall 2020, we performed >1,000,000 covidSHIELD tests, positivity rates remained low, we had zero COVID-19-related hospitalizations or deaths amongst our university community, and mortality in the surrounding Champaign County was reduced more than 4-fold relative to expected. This case study shows that fast/frequent testing and other interventions mitigated transmission of SARS-CoV-2 at a large public university.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , COVID-19/prevenção & controle , Teste para COVID-19 , Humanos , SARS-CoV-2/genética , Sensibilidade e Especificidade , UniversidadesRESUMO
Point-of-care (POC) detection technologies that enable decentralized, rapid, sensitive, low-cost diagnostics of COVID-19 infection are urgently needed around the world. With many technologies approved for commercialization in the past 10 months, the field of COVID-19 POC diagnostics is rapidly evolving. In this Perspective, we analyze the current state of POC technologies for the diagnosis and monitoring of COVID-19 infection and discuss future challenges in COVID-19 diagnostics. As the COVID-19 pandemic becomes endemic, the advances gained during this past year will likely also be utilized for future prediction of emerging outbreaks and pandemics.