RESUMEN
Antimicrobials (e.g., antibiotics and biocides) are invaluable chemicals used to control microbes in numerous contexts. Because of the simultaneous use of antibiotics and biocides, questions have arisen as to whether environments commonly treated with biocides (e.g., hospitals, food processing, wastewater, agriculture, etc.) could act as a reservoir for the development of antibiotic cross-resistance. Theoretically, cross-resistance could occur if the mechanism of bacterial tolerance to biocides also resulted in antibiotic resistance. On the other hand, biocides would likely present a higher evolutionary barrier to the development of resistance given the different modes of action between biocides and antibiotics and the broad-based physicochemical effects associated with most biocides. Published studies have shown that the induction of biocide tolerance in a laboratory can result in cross-resistance to some antibiotics, most commonly hypothesized to be due to efflux pump upregulation. However, testing of environmental isolates for biocide tolerance and antibiotic cross-resistance has yielded conflicting results, potentially due to the lack of standardized testing. In this review, we aim to describe the state of the science on the potential linkage between biocide tolerance and antibiotic cross-resistance. Questions still remain about whether the directed evolution of biocide tolerance and the associated antibiotic cross-resistance in a laboratory are or are not representative of real-world settings. Thus, research should continue to generate informative data to guide policies and preserve these tools' utility and availability.
RESUMEN
Seafood contamination with Vibrio bacteria is a problem for aquaculture, especially with oysters, which are often consumed raw. Current methods for diagnosing bacterial pathogens in seafood involve lab-based assays such as polymerase chain reaction or culturing, which are time consuming and must occur in a centralized location. Detection of Vibrio in a point-of-care assay would be a significant tool for food safety control measures. We report here a paper immunoassay that can detect the presence of Vibrio parahaemolyticus (Vp) in buffer and oyster hemolymph. The test uses gold nanoparticles conjugated to polyclonal anti-Vibrio antibodies in a paper-based sandwich immunoassay. A sample is added to the strip and wicked through by capillary action. If Vp is present, it results in a visible color at the test area that can be read out by eyes or a standard mobile phone camera. The assay has a limit of detection of 6.05 × 105 cfu/mL and a cost estimate of $5 per test. Receiver operating characteristic curves with validated environmental samples showed a test sensitivity of 0.96 and a specificity of 1.00. Because the assay is inexpensive and can be used on Vp directly without the requirement for culturing, or sophisticated equipment, it has the potential to be used in fieldable settings.
RESUMEN
BACKGROUND: Cells exchange information by secreting micro- and nanosized extracellular vesicles (EVs), ranging from exosomes (30-100 nm) to apoptotic bodies (ABs, 1-5 µm). There is still much to understand about fundamental EV biological, physical, and chemical properties before clinical applications can be developed. EV mechanical properties have only been measured with atomic force microscopy (AFM) with its problematic adhesion and hard substrate effects. To understand EV mechanical behavior in less extreme mechanical conditions relevant to blood flow and many soft tissue environments, a non-contact measurement technique is needed. METHODS: We measured the mechanical properties of single microscale ABs derived from human blood plasma using non-contact microfluidics. EVs were gently stretched in extensional flow, similar to a traditional tensile test, and a linear mechanical model was applied to estimate mechanical stiffnesses from the observed stretching. RESULTS: The effective shear elastic modulus of ABs in non-contact flow conditions is approximately 5.6 ± 0.5 Pa, 7 orders of magnitude lower than previously reported AFM-measured biological exosome stiffnesses and 200 times smaller than suspended cells. CONCLUSIONS: Apoptotic bodies are very soft in fluid environments and exhibit lower effective stiffnesses than suspended cells. By measuring ABs in a natural fluid environment and low-force regime without hard probes and surfaces, we achieved closer agreement with linear mechanical theory and therefore more accurate stiffness measurements. GENERAL SIGNIFICANCE: AFM manufacturers and users should consider implementing new mechanical models to interpret AFM force indentation curves so that accurate extracellular vesicle mechanical properties can be extracted.
Asunto(s)
Vesículas Extracelulares/química , Suero/citología , Fenómenos Biomecánicos , Elasticidad , Humanos , Técnicas Analíticas Microfluídicas/métodosRESUMEN
The detection of foodborne pathogens is critical for disease control and infection prevention, especially in seafood consumed raw or undercooked. Paper-based diagnostic tools are promising for rapid fieldable detection and provide a readout by eye due to the use of gold nanoparticle immunoprobes. Here we study different strategies to overcome these challenges in a real biological matrix, oyster hemolymph, for the detection of the pathogenic bacteria Vibrio parahaemolyticus (Vp). Nanoparticle surface chemistry, nitrocellulose speed and blocking, running steps, and antibody concentrations on the NP and nitrocellulose were all studied. Their effect on paper immunoassay signal intensity was quantified to determine optimal conditions, which enabled the detection of Vp directly from hemolymph below pathogenic concentrations.
Asunto(s)
Nanopartículas del Metal , Ostreidae , Vibrio parahaemolyticus , Animales , Oro , Hemolinfa , Inmunoensayo , Alimentos MarinosRESUMEN
We exploit the cross-reactivity of dengue (DENV) and Zika (ZIKV) virus polyclonal antibodies for nonstructural protein 1 (NS1) to construct a selective sensor that can detect yellow fever virus (YFV) NS1 in a manner similar to chemical olfaction. DENV and ZIKV antibodies were screened for their ability to bind to DENV, ZIKV, and YFV NS1 by enzyme linked immunosorbent assay (ELISA) and in pairs in paper immunoassays. A strategic arrangement of antibodies immobilized on paper and conjugated to different colored gold NPs was used to distinguish the three biomarkers. Machine learning of test area RGB values showed that with two spots, readout accuracies of 100% and 87% were obtained for both pure NS1 and DENV/YFV mixtures, respectively. Additional image preprocessing allowed differentiation between all four DENV serotypes with 92% accuracy. The technique was extended to hack a commercial DENV test to detect YFV and ZIKV by augmentation with DENV and ZIKV polyclonal antibodies.
Asunto(s)
Virus del Dengue , Dengue , Nanopartículas , Infección por el Virus Zika , Virus Zika , Anticuerpos Antivirales , Humanos , Proteínas no Estructurales Virales , Infección por el Virus Zika/diagnósticoRESUMEN
Myxovirus protein A (MxA) is a biomarker that can be used to distinguish between viral and bacterial infections. While MxA lateral flow assays (LFAs) have been successfully used for viral vs. bacterial differential diagnosis for children, the clinically relevant level of MxA for adults has been reported to be 100 times lower, which is too low for traditional LFAs. We present results applying the use of surface enhanced Raman spectroscopy (SERS) to detect MxA. AuAg nanoshells (AuAg NSs) were used to enhance the Raman signal of mercaptobenzoic acid (4-MBA), enabling readout by SERS. The AuAg NSs were conjugated to antibodies for the biomarker of interest, resulting in a "nanotag", that could be used in a dipstick immunoassay for detection. We first optimized the nanotag parameters using anti-human IgG/human IgG as a model antibody/antigen system, and then demonstrated detection of MxA using anti-MxA antibodies. We show that SERS readout of immunoassays for MxA can quantify MxA levels at clinically relevant levels for adult viral infection.
Asunto(s)
Anticuerpos Antivirales/química , Oro/química , Inmunoglobulina G/química , Nanopartículas del Metal/química , Proteínas de Resistencia a Mixovirus/inmunología , Nanocáscaras/química , Infecciones por Orthomyxoviridae , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/inmunología , Niño , Humanos , Inmunoensayo , Orthomyxoviridae , Infecciones por Orthomyxoviridae/diagnóstico , Infecciones por Orthomyxoviridae/inmunología , PapelRESUMEN
The effect of matrix metalloproteinases (MMPs) on preformed protein coronas around spherical gold nanoparticles (AuNPs) was studied. Protein coronas of different compositions (human serum, human serum albumin, and collagen IV) were formed around AuNPs and characterized. The protease MMP-9 had different effects on the corona depending on the corona composition, resulting in different changes to the corona hydrodynamic diameter ( DH). When incubated with PANC-1 cells, the corona showed evidence of both increases as well as decreases in DH. Varying the composition of the corona influenced the MMP-9 activity. Furthermore, the corona was influenced not only by the protease activity of the MMP-9 but also by its ability to exchange with proteins in the preformed corona. This exchange could also occur with proteins in the media. Thus, the net effect of the MMP-9 was a combination of the MMP-9 protease activity and also exchange. Time scales for the exchange varied depending on the nature that make up the protein corona (weakly vs strongly bound corona proteins). Mass spectrometry was used to probe the protein corona composition and supported the exchange and degradation model. Together, these results indicate that the mechanism of protease activity on AuNP coronas involves both rearrangement and exchange, followed by degradation.
Asunto(s)
Proteínas Sanguíneas/química , Metaloproteinasa 9 de la Matriz/metabolismo , Nanopartículas del Metal/química , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Corona de Proteínas/química , Línea Celular Tumoral , Humanos , Metaloproteinasa 9 de la Matriz/química , Neoplasias/patologíaRESUMEN
Paper-based sensors and assays have been highly attractive for numerous biological applications, including rapid diagnostics and assays for disease detection, food safety, and clinical care. In particular, the paper immunoassay has helped drive many applications in global health due to its low cost and simplicity of operation. This review is aimed at examining the fundamentals of the technology, as well as different implementations of paper-based assays and discuss novel strategies for improving their sensitivity, performance, or enabling new capabilities. These innovations can be categorized into using unique nanoparticle materials and structures for detection via different techniques, novel biological species for recognizing biomarkers, or innovative device design and/or architecture.
Asunto(s)
Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Nanopartículas/química , Papel , Biomarcadores/química , HumanosRESUMEN
We report a quantitative evaluation of the choice of reporters for multiplexed surface-enhanced Raman spectroscopy (SERS). An initial library consisted of 15 reporter molecules that included commonly used Raman dyes, thiolated reporters, and other small molecules. We used a correlation matrix to downselect Raman reporters from the library to choose five candidates: 1,2-bis(4-pyridyl)ethylene, 4-mercaptobenzoic acid, 3,5-dichlorobenzenthiol, pentachlorothiophenol, and 5,5'-dithiobis(2-nitrobenzoic acid). We evaluated the ability to distinguish the five SERS reporters in a dipstick immunoassay for the biomarker human IgG. Raman nanotags, or gold nanostars conjugated to the five reporters and anti-human IgG polyclonal antibodies were constructed. A linear discriminant analysis approach was used to evaluate the separation of the nanotag spectra in mixtures of fixed ratios.
RESUMEN
The unique size and material dependent properties of nanoparticles have made them highly attractive for biological and medical applications. However, combining nanoparticles with biomolecules and biological environments has faced many challenges. These interface issues often involve protein denaturation, steric hindrance, and orientational issues for the biomolecule, which can impair function and decrease overall performance of the nanoparticle-biomolecule conjugate. Historically, our understanding of the physical and chemical properties of nanoparticle-biomolecule conjugates as appropriate tools and experimental techniques had to be determined. We discuss here selected examples investigating the fundamental physical properties of the interface between nanoparticles and DNA and proteins and protein coronas and how they have provided insight into the properties of the biomolecule when it is interfaced to a nanoparticle.
Asunto(s)
ADN/química , Nanopartículas/química , Proteínas/química , Animales , HumanosRESUMEN
Surface enhanced Raman spectroscopy (SERS) has been attractive for enhancing the sensitivity of lateral flow immunoassays (LFA). A format that has enabled specific detection of biomarkers is to use Raman reporter molecules linked to gold nanoparticles (NPs), which are conjugated to antibodies specific for the target of interest. Many factors such as the NP and Ab properties and the method of signal readout impact the sensitivity of a SERS based immunoassay. To understand how to optimize assay sensitivity, we studied SERS readouts of multiplexed sandwich immunoassays for the zika and dengue non-structural protein 1 (NS1) biomarkers as a test case. We investigated the effect of NP shape on the SERS enhancement of the reporter molecules 1,2-bis(4-pyridyl)ethylene (BPE) and 4-mercaptobenzoic acid (MBA). We also performed SERS imaging of test lines to map the spatial distribution of signal in test lines on the nitrocellulose. Finally, we used a modified least squares analysis to differentiate reporter contributions.