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Lanthanide-doped glasses and crystals are attractive for laser applications because the metastable energy levels of the trivalent lanthanide ions facilitate the establishment of population inversion and amplified stimulated emission at relatively low pump power. At the nanometre scale, lanthanide-doped upconversion nanoparticles (UCNPs) can now be made with precisely controlled phase, dimension and doping level. When excited in the near-infrared, these UCNPs emit stable, bright visible luminescence at a variety of selectable wavelengths, with single-nanoparticle sensitivity, which makes them suitable for advanced luminescence microscopy applications. Here we show that UCNPs doped with high concentrations of thulium ions (Tm3+), excited at a wavelength of 980 nanometres, can readily establish a population inversion on their intermediate metastable 3H4 level: the reduced inter-emitter distance at high Tm3+ doping concentration leads to intense cross-relaxation, inducing a photon-avalanche-like effect that rapidly populates the metastable 3H4 level, resulting in population inversion relative to the 3H6 ground level within a single nanoparticle. As a result, illumination by a laser at 808 nanometres, matching the upconversion band of the 3H4 â 3H6 transition, can trigger amplified stimulated emission to discharge the 3H4 intermediate level, so that the upconversion pathway to generate blue luminescence can be optically inhibited. We harness these properties to realize low-power super-resolution stimulated emission depletion (STED) microscopy and achieve nanometre-scale optical resolution (nanoscopy), imaging single UCNPs; the resolution is 28 nanometres, that is, 1/36th of the wavelength. These engineered nanocrystals offer saturation intensity two orders of magnitude lower than those of fluorescent probes currently employed in stimulated emission depletion microscopy, suggesting a new way of alleviating the square-root law that typically limits the resolution that can be practically achieved by such techniques.
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Water-in-oil-in-water (w/o/w) double emulsion (DE) encapsulation has been widely used as a promising platform technology for various applications in the fields of food, cosmetics, pharmacy, chemical engineering, materials science, and synthetic biology. Unfortunately, DEs formed by conventional emulsion generation approaches in most cases are highly polydisperse, making them less desirable for quantitative assays, controlled biomaterial synthesis, and entrapped ingredient release. Microfluidic devices can generate monodisperse DEs with controllable size, morphology, and production rate, but these generally require multistep fabrication processes and use of different solvents or bulky external instrumentation to pattern channel wettability. To overcome these limitations, we propose a rapid, simple, and inexpensive method to spatially pattern wettability in microfluidic devices for the continuous generation of monodisperse DEs. This is achieved by applying corona-plasma treatment to a select zone of the microchannel surface aided by a custom-designed corona resistance microchannel to strictly confine the plasma-treatment zone in a single polydimethylsiloxane (PDMS) microfluidic device. The properties of PDMS channel surfaces and key microchannel regions for DE generation are characterized under different levels of treatment. The size, shell thickness, and number of inner cores of generated DEs are shown to be highly controllable by tuning the phase flow rate ratios. Using DEs as templates, we successfully achieve a one-step generation and collection of gelatin microgels. Additionally, we demonstrate the biological capability of generated DEs by flow cytometric screening of the encapsulation and growth of yeast cells within DEs. We expect that the proposed approach will be widely used to create microfluidic devices with more complex wettability patterns.
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Dispositivos Lab-On-A-Chip , Água , Emulsões , Citometria de Fluxo , MolhabilidadeRESUMO
Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes, and phenotypes. The results showed that the average growth of single yeast cells in microdroplets experiences the same trend to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.
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Saccharomyces cerevisiae/crescimento & desenvolvimento , Engenharia MetabólicaRESUMO
BACKGROUND: Formal patient complaints are associated with increased malpractice litigation and can have adverse occupational consequences for surgeons. Our purpose was to define and categorize patient complaints within an academic pediatric orthopaedic surgery practice over a 10-year period. We further aimed to define risk factors associated with patient complaints. METHODS: We reviewed all complaints within our institution's patient advocacy service filed on behalf of a patient against 4 pediatric orthopaedic surgeons over a 10-year period. Complaints were categorized using the Patient Complaint Analysis System. A control group of all patients seen by the surgeons during the study period was created. We compared baseline demographics between the patients with a complaint and the control group and compared complaint rates between the surgeons. Any malpractice events (lawsuits and claims) associated with the surgeons were obtained. We queried our institutional MIDAS reporting system (which allows for anonymous reporting of potential patient-safety or "near-miss" events), to assess whether patients with a complaint had a reported event. RESULTS: The 4 pediatric orthopaedic surgeons saw a total of 25,747 unique patients during the study period. Forty-one patients had a formal complaint, resulting in a complaint rate of 0.15%. Complaint rates varied from 0.08% to 0.30% between surgeons. Humanness was the most frequent complaint designation category (32%) followed by Care and Treatment (19%). Of the 41 patients with a complaint, 18 (44%) underwent surgical treatment. Only 1 patient with a complaint also had an entry within our institutional patient-safety reporting system. CONCLUSIONS: The rate of patient complaints within an academic pediatric orthopaedic surgery practice over a decade was 0.15%, or ~1 complaint for every 670 new patients seen. The majority of patient complaints involved communication; a potentially modifiable area that can be targeted for improvement. While complaint rates among surgeons can vary, patient demographic factors are not associated with increased complaints. Understanding patient complaints rates and types may allow surgeons to target areas for improvement and decrease exposure to malpractice litigation. LEVEL OF EVIDENCE: Level II-prognostic.
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Lanthanide-based upconversion nanoparticles (UCNPs) generally require high power laser excitation. Here, we report wide-field upconversion microscopy at single-nanoparticle sensitivity using incoherent excitation of a 970 nm light-emitting diode (LED). We show that due to its broad emission spectrum, LED excitation is about 3 times less effective for UCNPs and generates high background compared to laser illumination. To counter this, we use time-gated luminescence detection to eliminate the residual background from the LED source, so that individual UCNPs with high sensitizer (Yb3+) doping and inert shell protection become clearly identified under LED excitation at 1.18 W cm-2, as confirmed by correlated electron microscopy images. Hydrophilic UCNPs are obtained by polysaccharide coating via a facile ligand exchange protocol to demonstrate imaging of cellular uptake using LED excitation. These results suggest a viable approach to bypassing the limitations associated with high-power lasers when applying UCNPs and upconversion microscopy to life science research.
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Efficient DNA mutation detection methods are required for diagnosis, personalized therapy development, and prognosis assessment for diseases such as cancer. To address this issue, we proposed a straightforward approach by combining active plasmonic nanostructures, surface-enhanced Raman spectroscopy (SERS), and polymerase chain reaction (PCR) with a statistical tool to identify and classify BRAF wild type (WT) and V600E mutant genes. The nanostructures provide enhanced sensitivity, while PCR offers high specificity toward target DNA. A series of positively charged plasmonic nanostructures including gold/silver nanospheres, nanoshells, nanoflowers, and nanostars were synthesized with a one-pot strategy and characterized. By changing the shape of nanostructures, we are able to vary the surface plasmon resonance from 551 to 693 nm. The gold/silver nanostar showed the highest SERS activity, which was employed for DNA mutation detection. We reproducibly analyzed as few as 100 copies of target DNA sequences using gold/silver nanostars, thus demonstrating the high sensitivity of the direct SERS detection. By means of statistical analysis (principal component analysis-linear discriminant analysis), this method was successfully applied to differentiate the WT and V600E mutant both from whole genome DNA lysed from cell line and from cell-free DNA collected from cell culture media. We further proved that this assay is capable of specifically amplifying and accurately classifying a real plasma sample. Thus, this direct SERS strategy combined with the active plasmonic nanostructures has the potential for wide applications as an alternative tool for sensitively monitoring and evaluating important clinical nucleotide biomarkers.
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DNA/genética , Nanoestruturas/química , Animais , Bovinos , Humanos , Mutação , Análise Espectral Raman , Células Tumorais CultivadasRESUMO
Upconversion nanoparticles (UCNPs) are becoming increasingly popular as biological markers as they offer photo-stable imaging in the near-infrared (NIR) biological transparency window. Imaging at NIR wavelengths benefits from low auto-fluorescence background and minimal photo-damage. However, as the diffraction limit increases with the wavelength, the imaging resolution deteriorates. To address this limitation, recently two independent approaches have been proposed for imaging UCNPs with sub-diffraction resolution, namely stimulated emission-depletion (STED) microscopy and super linear excitation-emission (uSEE) microscopy. Both methods are very sensitive to the UCNP composition and the imaging conditions, i.e. to the excitation and depletion power. Here, we demonstrate that the imaging conditions can be chosen in a way that activates both super-resolution regimes simultaneously when imaging NaYF4:Yb,Tm UCNPs. The combined uSEE-STED mode benefits from the advantages of both techniques, allowing for imaging with lateral resolution about six times better than the diffraction limit due to STED and simultaneous improvement of the axial resolution about twice over the diffraction limit due to uSEE. Conveniently, at certain imaging conditions, the uSEE-STED modality can achieve better resolution at four times lower laser power compared to STED mode, making the method appealing for biological applications. We illustrate this by imaging UCNPs functionalized by colominic acid in fixed neuronal phenotype cells.
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We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338-BHHTEGST-Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.
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Hibridização In Situ , Luminescência , Medições Luminescentes , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Cromatografia Líquida de Alta Pressão , Humanos , Hibridização In Situ/métodos , Medições Luminescentes/métodos , RNA Ribossômico 16SRESUMO
BACKGROUND: The decision to utilize portal or systemic venous drainage in pancreas transplantation is surgeon- and center-dependent. Information regarding the superior method is based on single-center reports and animal models. METHODS: UNOS data on adults receiving pancreas and kidney-pancreas transplants from 1987 to 2016 were analyzed (n = 29 078). The groups analyzed were: systemic venous pancreas graft drainage (SVD, n = 24 512) or portal venous pancreas graft drainage (PVD, n = 4566). A Cox proportional hazard model compared patient and allograft survival between groups. RESULTS: No statistically significant differences were observed for patient and allograft survival at 1, 3, 5, 10, or 15 years post-transplant at each time interval and cumulatively (patient - HR:1.041; 95% CI:0.989-1.095; allograft - HR:0.951; 95% CI:0.881-1.027). PVD reduced the risk of death by 22.0% (P = 0.017) compared to SVD for patients undergoing pancreas after kidney transplant (PAK); no statistically significant difference was found for patients undergoing other types of transplants. CONCLUSION: There is no significant clinical difference in patient or allograft survival between PVD and SVD in pancreas transplantation for the majority of patients. For the subgroup of PAK, PVD was associated with decreased mortality. For individual surgeons, center and patient scenarios should dictate which technique is performed.
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Veia Femoral/cirurgia , Circulação Hepática , Veias Mesentéricas/cirurgia , Transplante de Pâncreas/métodos , Veia Porta/cirurgia , Enxerto Vascular/métodos , Veia Cava Inferior/cirurgia , Adolescente , Adulto , Bases de Dados Factuais , Feminino , Veia Femoral/fisiopatologia , Sobrevivência de Enxerto , Humanos , Masculino , Veias Mesentéricas/fisiopatologia , Transplante de Pâncreas/efeitos adversos , Transplante de Pâncreas/mortalidade , Veia Porta/fisiopatologia , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Enxerto Vascular/efeitos adversos , Enxerto Vascular/mortalidade , Veia Cava Inferior/fisiopatologia , Adulto JovemRESUMO
Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual "grids" that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm(2) in just 5.3 min and locate individual 15 µm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 µm in X and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles.
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We describe the application of a synthetically developed tetradentate ß-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.
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Complexos de Coordenação/farmacologia , Európio/química , Imunoconjugados/farmacologia , Técnicas Imunológicas/métodos , Substâncias Luminescentes/farmacologia , Neoplasias da Próstata/diagnóstico , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Glipicanas/imunologia , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Ligantes , Luminescência , Substâncias Luminescentes/síntese química , MasculinoRESUMO
The emerging upconversion nanoparticles offer a fascinating library of ultrasensitive luminescent probes for a range of biotechnology applications from biomarker discovery to single molecule tracking, early disease diagnosis, deep tissue imaging, and drug delivery and therapies. The effective bioconjugation of inorganic nanoparticles to the molecule-specific proteins, free of agglomeration, nonspecific binding, or biomolecule deactivation, is crucial for molecular recognition of target molecules or cells. The current available protocols require multiple steps which can lead to low probe stability, specificity, and reproducibility. Here we report a simple and rapid protein bioconjugation method based on a one-step ligand exchange using the DNAs as the linker. Our method benefits from the robust DNA-protein conjugates as well as from multiple ions binding capability. Protein can be preconjugated via an amino group at the 3' end of a synthetic DNA molecule, so that the 5' end phosphoric acid group and multiple phosphate oxygen atoms in the phosphodiester bonds are exposed to replace the oleic acid ligands on the surface of upconversion nanoparticles due to their stronger chelating capability to lanthanides. We demonstrated that our method can efficiently pull out the upconversion nanoparticles from organic solvent into an aqueous phase. The upconversion nanoparticles then become hydrophilic, stable, and specific biomolecules recognition. This allows us to successfully functionalize the upconversion nanoparticles with horseradish peroxidise (HRP) for catalytic colorimetric assay and for streptavidin (SA)-biotin immunoassays.
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Peroxidase do Rábano Silvestre/química , Nanopartículas/química , Estreptavidina/química , Biotina/química , Colorimetria , DNA/química , Peroxidase do Rábano Silvestre/metabolismo , ImunoensaioRESUMO
We report a pulsed mid-infrared diamond Raman laser with output tuned from 3.38 to 3.80 µm through varying the optical parametric oscillator (OPO) pump wavelength. To our knowledge this is the longest reported wavelength from a solid-state Raman laser. We generated up to 80 µJ with good beam quality and 22% quantum conversion efficiency. Whilst the conversion process itself is efficient, approximately 40% of the generated Stokes light is lost to multiphonon absorption. By introducing a secondary pump beam at the anti-Stokes wavelength to initiate a seed at the Stokes wavelength through Raman resonant four-wave mixing, the laser threshold was reduced by approximately half, and the maximum output increased by 44% to 115 µJ.
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This article describes the use of a laser-level paralleling device for the fabrication of a unilateral auricular prosthesis. Traditional methods require use of calipers to obtain orientation of contralateral auricular anatomy, which in turn can be difficult to replicate on the patient. The purpose of this clinical report is to describe a simple means to record unilateral auricular anatomy.
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Orelha Externa , Lasers , Próteses e Implantes , Desenho de Prótese/instrumentação , Idoso , Pontos de Referência Anatômicos/anatomia & histologia , Carcinoma Basocelular/cirurgia , Neoplasias da Orelha/cirurgia , Orelha Externa/cirurgia , Humanos , Imãs , Masculino , Retenção da Prótese/instrumentaçãoRESUMO
We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of â¼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning automated microscopy (OSAM) technique to rapidly process a large population of target-only cells on microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events.
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Antígenos CD34/metabolismo , Regulação da Expressão Gênica , Microscopia/métodos , Automação , Células HEK293 , Humanos , Fatores de TempoRESUMO
Ruthenium(II) complexes have rich photophysical attributes, which enable novel design of responsive luminescence probes to selectively quantify biochemical analytes. In this work, we developed a systematic series of Ru(II)-bipyrindine complex derivatives, [Ru(bpy)(3-n)(DNP-bpy)(n)](PF(6))(2) (n = 1, 2, 3; bpy, 2,2'-bipyridine; DNP-bpy, 4-(4-(2,4-dinitrophenoxy)phenyl)-2,2'-bipyridine), as luminescent probes for highly selective and sensitive detection of thiophenol in aqueous solutions. The specific reaction between the probes and thiophenol triggers the cleavage of the electron acceptor group, 2,4-dinitrophenyl, eliminating the photoinduced electron transfer (PET) process, so that the luminescence of on-state complexes, [Ru(bpy)(3-n)(HP-bpy)(n)](2+) (n = 1, 2, 3; HP-bpy, 4-(4-hydroxyphenyl)-2,2'-bipyridine), is turned on. We found that the complex [Ru(bpy)(DNP-bpy)(2)](2+) remarkably enhanced the on-to-off contrast ratio compared to the other two (37.8 compared to 21 and 18.7). This reveals a new strategy to obtain the best Ru(II) complex luminescence probe via the most asymmetric structure. Moreover, we demonstrated the practical utility of the complex as a cell-membrane permeable probe for quantitative luminescence imaging of the dynamic intracellular process of thiophenol in living cells. The results suggest that the new probe could be a very useful tool for luminescence imaging analysis of the toxic thiophenol in intact cells.
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Desenho de Fármacos , Substâncias Luminescentes/química , Substâncias Luminescentes/síntese química , Imagem Molecular/métodos , Compostos Organometálicos/química , Compostos Organometálicos/síntese química , Rutênio/química , Sobrevivência Celular , Cor , Transporte de Elétrons , Células HeLa , Humanos , Substâncias Luminescentes/metabolismo , Compostos Organometálicos/metabolismo , Fenóis/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
We demonstrate the operation of a compact and efficient continuous wave (CW) self-Raman laser utilizing a Nd:YVO4 gain crystal and BaWO4 Raman crystal, generating yellow emission at 590 nm. We investigate the competition that occurs between Stokes lines in the Nd:YVO4 and BaWO4 crystals, and within the BaWO4 crystal itself. Through careful consideration of crystal length and orientation, we are able to suppress competition between Stokes lines, and generate pure yellow emission at 590 nm with output power of 194 mW for just 3.8 W pump power.
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Lasers de Estado Sólido , Análise Espectral Raman/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , MiniaturizaçãoRESUMO
Seafloor reflection loss and roughness measurements were taken at the Experimental Validation of Acoustic Modeling Techniques experiment in 2006. The magnitude and phase of the reflection loss was measured at frequencies from 5 to 80 kHz and grazing angles from 7° to 77°. Approximately 1500 samples were taken for each angle. The roughness was measured with a laser profiler. Geoacoustic parameters such as water and sediment sound speed and density were measured concurrently. The reflection loss data were compared with three models: A flat interface elastic model based on geoacoustic measurements; a flat interface poro-elastic model based on the Biot/Stoll model; and a rough interface model based on the measured interface roughness power spectrum. The data were most consistent with the poro-elastic model including scattering. The elastic model consistently predicted values for the reflection loss which were higher than measured. The data exhibited more variability than the model due to layering and fluctuations in the propagating medium.
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Acústica , Geologia/métodos , Som , Acústica/instrumentação , Elasticidade , Sedimentos Geológicos , Geologia/instrumentação , Lasers , Modelos Teóricos , Movimento (Física) , Oceanos e Mares , Porosidade , Espalhamento de Radiação , Processamento de Sinais Assistido por Computador , Espectrografia do Som , Fatores de Tempo , Transdutores , ÁguaRESUMO
Surface modification and functionalization is typically required to engineer upconversion nanoparticles (UCNPs) for biosensing and bioimaging applications. Nevertheless, despite various antibody conjugation methods having been applied to UCNPs, no consensus has been reached on the best choice, as the results from individual studies are largely unable to be compared due to inadequate assessment of the properties of the conjugated products. Here, we introduce a systematic approach to quantitatively evaluate the biological activity of antibody-conjugated UCNPs. We determine that the optimal antibody conjugation efficiency to our colominic acid polysaccharide-coated UCNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS) coupling is approximately 70%, corresponding to 16 antibodies per nanoparticle of 63 nm hydrodynamic diameter, with on average 12 of the 16 antibodies maintaining their affinity to the target antigens. The binding ability of the antibody-conjugated UCNPs to the antigen was well preserved, as verified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and cellular imaging. This is the first study to quantitate the active antibody binding capacity of polysaccharide coated UCNP nanoparticles, offering a practical guideline for benchmarking functionalised UCNPs in future studies.
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Nanopartículas , Anticorpos , Nanopartículas/química , PolissacarídeosRESUMO
Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4-10 cells/mL. Blinded analysis of each patient urine sample, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3-20 PCa cells per mL by TGiA, 4-13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine samples of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa.