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BACKGROUND/PURPOSE: This study aimed at screening the diagnostic potential of salivary biomarkers and pairing them to establish a prediction model with higher accuracy in diagnosing periodontitis in the Taiwanese population. METHODS: Fifty-seven participants were divided into a non-periodontitis group and a periodontitis group. Salivary biomarkers CRP, IL-6, IL-8, IL-1ß, IL-1ra, lactoferrin, MMP-8, MMP-9, PDGF-BB, TNF-α, and VEGF, were analyzed. The potential and reliability of the biomarkers for diagnosing periodontitis were analyzed dichotomously. The correlation of individual biomarkers with periodontitis was assessed using the Spearman rank correlation coefficient with logistic regression. The combinational prediction model was evaluated using the area under the receiver operating characteristic curve (AUC). RESULTS: Regarding individual biomarkers, IL-1ß and MMP-9 levels were significantly higher, and TNF-α was significantly lower in the periodontitis group. IL-1ß, MMP-8, and MMP-9 exhibited a greater odds ratio with statistical significance in the dichotomous table. The combination of three biomarkers yielded AUCs of 0.821-0.853, and the combination of IL-1ß, IL-1ra, and MMP-9 exhibited the highest AUC (0.853), with high sensitivity (73.3%) and specificity (88.9%). CONCLUSION: Regarding individual biomarkers, IL-1ß, MMP-8, and MMP-9 showed potential for identifying patients with periodontitis. The combination of IL-1ß, IL-1ra, and MMP-9 might be feasible for developing a future point-of-care device for diagnosing periodontitis.
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Biomarcadores/análise , Periodontite/diagnóstico , Saliva/química , Adulto , Idoso , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1/análise , Interleucina-1beta/análise , Modelos Logísticos , Masculino , Metaloproteinase 9 da Matriz/análise , Pessoa de Meia-Idade , Curva ROC , Reprodutibilidade dos Testes , Taiwan , Adulto JovemRESUMO
Coronary artery disease and its related complications pose great threats to human health. In this work, we aim to clinically evaluate a CMOS field-effect biomolecular sensor for cardiac biomarkers, cardiac-specific troponin-I (cTnI), N-terminal prohormone brain natriuretic peptide (NT-proBNP), and interleukin-6 (IL-6). The CMOS biosensor is implemented via a standard commercialized 0.35 µm CMOS process. To validate the sensing characteristics, in buffer conditions, the developed CMOS biosensor has identified the detection limits of IL-6, cTnI, and NT-proBNP as being 45 pM, 32 pM, and 32 pM, respectively. In clinical serum conditions, furthermore, the developed CMOS biosensor performs a good correlation with an enzyme-linked immuno-sorbent assay (ELISA) obtained from a hospital central laboratory. Based on this work, the CMOS field-effect biosensor poses good potential for accomplishing the needs of a point-of-care testing (POCT) system for heart disease diagnosis.
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Técnicas Biossensoriais , Biomarcadores , Doença da Artéria Coronariana , Humanos , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Troponina IRESUMO
Thermophoresis is a phenomenon about the migration of particles along a temperature gradient and is sensitive to the properties of particles and the surrounding medium. While a few studies have investigated its mechanisms and effects on particle motion in recent years, the applications of thermophoresis in biosensing has not been well explored. In this study, we demonstrate a thermophoresis-based method for detecting DNA. We use DNA-functionalized gold nanoparticles and fluorescent DNA probes to capture target DNA in free solution, and we demonstrate that the hybridization between the specially designed capture probes and the target DNA significantly changes the thermophoretic properties of the fluorescent probes. As a result, the target DNA can be specifically detected in serum-containing buffers based on the spatial distribution of the fluorescent probes in a laser-induced temperature gradient. The optical setup consists of only a laser and an epifluorescence microscope, and the detection does not rely on any micro- or nanofabricated devices. In addition, because the detection is based on the thermophoretic motion of molecules in free solution, no capture probes need to be immobilized on a fixed surface before detection, and no channels or pumps are needed for washing away unbound molecules. The thermophoresis-based biosensing method is found to be simple and effective for detecting DNA.
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Técnicas Biossensoriais/métodos , DNA/análise , DNA/química , Corantes Fluorescentes/química , Ouro/química , Nanopartículas Metálicas/química , Humanos , TemperaturaRESUMO
Near-field optical techniques have enabled the trapping, transport, and handling of nanoscopic materials much smaller than what can be manipulated with traditional optical tweezers. Here we extend the scope of what is possible by demonstrating angular orientation and rotational control of both biological and nonbiological nanoscale rods using photonic crystal nanotweezers. In our experiments, single microtubules (diameter 25 nm, length 8 µm) and multiwalled carbon nanotubes (outer diameter 110-170 nm, length 5 µm) are rotated by the optical torque resulting from their interaction with the evanescent field emanating from these devices. An angular trap stiffness of κ = 92.8 pN·nm/rad(2)·mW is demonstrated for the microtubules, and a torsional spring constant of 22.8 pN·nm/rad(2)·mW is measured for the nanotubes. We expect that this new capability will facilitate the development of high precision nanoassembly schemes and biophysical studies of bending strains of biomolecules.
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Microtúbulos/química , Nanotubos de Carbono/química , Nanotubos/química , Hidrodinâmica , Pinças Ópticas , FótonsRESUMO
The ability to controllably handle the smallest materials is a fundamental enabling technology for nanoscience. Conventional optical tweezers have proven useful for manipulating microscale objects but cannot exert enough force to manipulate dielectric materials smaller than about 100 nm. Recently, several near-field optical trapping techniques have been developed that can provide higher trapping stiffness, but they tend to be limited in their ability to reversibly trap and release smaller materials due to a combination of the extremely high electromagnetic fields and the resulting local temperature rise. Here, we have developed a new form of photonic crystal "nanotweezer" that can trap and release on-command Wilson disease proteins, quantum dots, and 22 nm polymer particles with a temperature rise less than ~0.3 K, which is below the point where unwanted fluid mechanical effects will prevent trapping or damage biological targets.
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Micromanipulação/instrumentação , Micromanipulação/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Pinças Ópticas , Proteínas/química , Proteínas/ultraestrutura , Sítios de Ligação , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais , Ligação ProteicaRESUMO
This paper presents a gel-based three-dimensional (3D) substrate for surface-enhanced Raman spectroscopy (SERS) mediated by photonic nanojets (PNJs) to enhance the sensitivity of SERS detection. The porous structure of the gel-based substrate allowed small molecules to diffuse into the substrate, while the placement of silica beads on the substrate surface resulted in the generation of photonic nanojets during SERS measurements. Because the gel-based SERS substrate had electromagnetic (EM) hot spots along the Z-direction for several tens of microns, the focuses of the PNJs, which were located a few microns away from the substrate surface, could excite the EM hot spots located within the substrate. Our objective was to maximize SERS signal intensity by coating the substrate with a close-packed array of silica beads to enable the generation of multiple PNJs. The bead array was formed using an optical fiber decorated with gold nanorods (AuNRs) to create a temperature gradient in a mixture containing silica beads, thereby enabling their arrangement and deposition in arbitrary locations across the substrate. In experiments, the Raman enhancement provided by multiple PNJs significantly exceeded that provided by single PNJs. The proposed PNJ-mediated SERS method reduced the limit of detection for malachite green by 100 times, compared to SERS results obtained using the same substrate without beads. The proposed enhancement scheme using a gel-based 3D SERS substrate with a close-packed array of silica beads could be utilized to achieve high-sensitivity SERS detection for a variety of molecules in a diverse range of applications.
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Heat generation and its impact on DNA transport in the vicinity of an optofluidic silicon photonic crystal resonator are studied theoretically and experimentally. The temperature rise is measured to be as high as 57 K for 10 mW of input power. The resulting optical trapping and biomolecular sensing properties of these devices are shown to be strongly affected by the combination of buoyancy driven flow and thermophoresis. Specifically, the region around the electromagnetic hot spot is depleted in biomolecules because of a high free energy barrier.
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DNA/química , Nanotecnologia/métodos , Dispositivos Ópticos , Óptica e Fotônica/métodos , Técnicas Biossensoriais/métodos , Cristalização , Temperatura Alta , Microfluídica/métodos , Silício/químicaRESUMO
Reactive oxygen species (ROS), a number of reactive molecules and free radicals derived from molecular oxygen, are generated as by-products during mitochondrial electron transport within cells. Physiologically, cells are capable of metabolizing the ROS exploiting specific mechanisms. However, if excessive ROS accumulate inside the cells, it will cause the cells apoptosis or necrosis. Hydrogen peroxide (H2O2) is one of the essential ROS often participating in chemical reactions in organisms and regulating homeostasis in the body. Therefore, rapid and sensitive detection of H2O2 is a significant task in cell biology research. Furthermore, it has been found that cells cultured in different formats can result in different cellular responses and biological activities. In order to investigate the H2O2 secretion from the cells cultured in different formats, a hydrogel-based substrate is exploited to separate relatively large molecular (e.g., proteins) for direct measurement of H2O2 secreted from living cells in complete cell culture medium containing serum. The substrate takes advantage of the localized surface plasmon resonance (LSPR) method based on enzyme immunoprecipitation. In addition, the H2O2 secreted from the cells cultured in different dimensions (suspension of single cells and three-dimensional cell spheroids) treated with identical drugs is measured and compared. The spheroid samples can be prepared with ample amount using a designed microfluidic device with precise control of size. The results show that the H2O2 secretion from the cells are great affected by their culture formats.
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We developed a method based on surface-enhanced Raman spectroscopy (SERS) and a sample pretreatment process for rapid, sensitive, reproducible, multiplexed, and low-cost detection of illegal drugs in urine. The abuse of new psychoactive substances (NPS) has become an increasingly serious problem in many countries. However, immunoassay-based screening kits for NPS are usually not available because of the lack of corresponding antibodies. SERS has a great potential for rapid detection of NPS because it can simultaneously detect multiple kinds of drugs without the use of antibodies. To achieve highly sensitive SERS detection of drugs, sodium bromide was first employed to induce the rapid formation of Ag nanoclusters by aggregating silver nanoparticles (AgNPs) in the extracted sample solution. SERS measurements were performed immediately after the sample pretreatment without incubation. The three-dimensional SERS hot spots were believed to form significantly within the nanoclusters, providing strong SERS enhancement effects. The displacement of citrate molecules on the surfaces of the AgNPs by bromide ions helped increase the adsorption of drug molecules, increasing their areal density. We demonstrated the simultaneous detection of two kinds of NPS, methcathinone and 4-methylmethcathinone, in urine at a concentration as low as 0.01 ppm.
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Living cells provide a fluctuating, out-of-equilibrium environment in which genes must coordinate cellular function. DNA looping, which is a common means of regulating transcription, is very much a stochastic process; the loops arise from the thermal motion of the DNA and other fluctuations of the cellular environment. We present single-molecule measurements of DNA loop formation and breakdown when an artificial fluctuating force, applied to mimic a fluctuating cellular environment, is imposed on the DNA. We show that loop formation is greatly enhanced in the presence of noise of only a fraction of k_{B}T, yet find that hypothetical regulatory schemes that employ mechanical tension in the DNA-as a sensitive switch to control transcription-can be surprisingly robust due to a fortuitous cancellation of noise effects.
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DNA/química , DNA/metabolismo , Modelos Biológicos , Proteínas/metabolismo , Fenômenos Biomecânicos , DNA/fisiologia , Regulação da Expressão Gênica , Movimento (Física) , Conformação de Ácido Nucleico , Processos Estocásticos , Temperatura , TermodinâmicaRESUMO
We show that minuscule entropic forces, on the order of 100 fN, can prevent the formation of DNA loops-a ubiquitous means of regulating the expression of genes. We observe a tenfold decrease in the rate of LacI-mediated DNA loop formation when a tension of 200 fN is applied to the substrate DNA, biasing the thermal fluctuations that drive loop formation and breakdown events. Conversely, once looped, the DNA-protein complex is insensitive to applied force. Our measurements are in excellent agreement with a simple polymer model of loop formation in DNA, and show that an antiparallel topology is the preferred LacI-DNA loop conformation for a generic loop-forming construct.
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DNA Bacteriano/química , DNA Bacteriano/metabolismo , Entropia , Escherichia coli/metabolismo , Repressores Lac/metabolismo , Conformação de Ácido Nucleico , Cinética , Regiões Operadoras GenéticasRESUMO
We have implemented polarization-resolved fluorescence lifetime measurement through stimulated emission based pump-probe technique, which promises much higher temporal resolution (â¼4 ps) than conventional time-correlated single-photon counting (TCSPC). The depolarization of ATTO 647N fluorescent dye is resolved through anisotropy fluorescence lifetime measurements, with variable time delay introduced between the pump and the probe beams. Importantly, the polarization anisotropy measurement and the corresponding rotational correlation time characterization of the fluorescent dye are carried out at various temperatures. We have also demonstrated the need of high temporal resolution via hetero Förster energy transfer (Hetero-FRET) through the interaction between the gold nanorods (GNRs) and the fluorescent dye ATTO 647N. Notably, our results compare highly favorably with conventional TCSPC method, which is rather limited in temporal resolution, for the above characterization. Additionally, this technique is applicable even under ambient light while being very cost-effective and robust.
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Optical tweezers have become powerful tools to manipulate biomolecular systems, but are increasingly difficult to use when the size of the molecules is <1 microm. Many important biological structures and processes, however, occur on the submicron length scale. Therefore, we developed and characterized an optical manipulation protocol that makes this length scale accessible by stretching the molecule in the axial direction of the laser beam, thus avoiding limiting artifacts from steric hindrances from the microscope coverslip and other surface effects. The molecule is held under constant mechanical tension by a combination of optical gradient forces and backscattering forces, eliminating the need for electronic feedback. We demonstrate the utility of this method through a measurement of the force-extension relationship of a 1298 bp ds-DNA molecule.
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Nanotecnologia/métodos , Pinças Ópticas , Algoritmos , Calibragem , DNA/química , ElasticidadeRESUMO
We have measured the entropic elasticity of double-stranded-DNA molecules ranging from 247 to 1298 bp in length using axial force-clamp optical tweezers. We show that entropic end effects and excluded-volume forces from surface attachments become significant for such short molecules. The effective persistence length of the shortest molecules decreases by a factor of 2 compared to the established value for long molecules, and excluded-volume forces extend the molecules to about one third of their nominal contour length. We interpret these results in the framework of an inextensible semiflexible rod model.
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DNA/química , Elasticidade , Entropia , Pinças Ópticas , Propriedades de SuperfícieRESUMO
We demonstrate three-dimensional surface-enhanced Raman spectroscopy (SERS) substrates formed by accumulating plasmonic nanostructures that are synthesized using a DNA-assisted assembly method. We densely immobilize Au nanoparticles (AuNPs) on polymer beads to form core-satellite nanostructures for detecting molecules by SERS. The experimental parameters affecting the AuNP immobilization, including salt concentration and the number ratio of the AuNPs to the polymer beads, are tested to achieve a high density of the immobilized AuNPs. To create electromagnetic hot spots for sensitive SERS sensing, we add a Ag shell to the AuNPs to reduce the interparticle distance further, and we carefully adjust the thickness of the shell to optimize the SERS effects. In addition, to obtain sensitive and reproducible SERS results, instead of using the core-satellite nanostructures dispersed in solution directly, we prepare SERS substrates consisting of closely packed nanostructures by drying nanostructure-containing droplets on hydrophobic surfaces. The densely distributed small and well-controlled nanogaps on the accumulated nanostructures function as three-dimensional SERS hot spots. Our results show that the SERS spectra obtained using the substrates are much stronger and more reproducible than that obtained using the nanostructures dispersed in solution. Sensitive detection of melamine and sodium thiocyanate (NaSCN) are achieved using the SERS substrates.
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We demonstrate an approach that utilizes DNA-functionalized gold nanorods (AuNRs) in an indirect competitive assay format to increase the spectra shift in localized surface plasmon resonance (LSPR) biosensing. We use interferon gamma (IFN-γ) as a model analyte to demonstrate the feasibility of our detection method. The LSPR chips with periodic gold nanodot arrays are fabricated using a thermal lithography process and are functionalized with IFN-γ aptamers for detection. The DNA-functionalized AuNRs and IFN-γ compete with each other to bind to the aptamers during detection, and the spectra shifts are mainly caused by the AuNRs rather than IFN-γ. When using our approach, the target molecules do not need to be captured by two capture ligands simultaneously during detection and thus do not require multiple binding sites. Both experiments and finite-difference time-domain (FDTD) simulations show that making the AuNRs as close to the chip surface as possible is very critical for increasing LSPR shifts, and the simulated results also show that the orientation of the AuNR affects the plasmon coupling between the gold nanodots on the chip surface and the nearby AuNRs. Although only the detection of IFN-γ is demonstrated in this study, we expect that the LSPR biosensing method can be applied to label-free detection of a variety of molecules as long as suitable aptamers are available.
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Aptâmeros de Nucleotídeos/química , Ouro/química , Interferon gama/análise , Nanotubos/química , Ressonância de Plasmônio de Superfície/métodos , Desenho de Equipamento , Ressonância de Plasmônio de Superfície/instrumentaçãoRESUMO
Optofluidics is a rapidly advancing field that utilizes the integration of optics and microfluidics to provide a number of novel functionalities in microsystems. In this review, we discuss how this approach can potentially be applied to address some of the greatest challenges facing both the developing and developed world, including healthcare, food shortages, malnutrition, water purification, and energy. While medical diagnostics has received most of the attention to date, here we show that some other areas can also potentially benefit from optofluidic technology. Whenever possible we briefly describe how microsystems are currently used to address these problems and then explain why and how optofluidics can provide better solutions. The focus of the article is on the applications of optofluidic techniques in low-resource settings, but we also emphasize that some of these techniques, such as those related to food production, food safety assessment, nutrition monitoring, and energy production, could be very useful in well-developed areas as well.
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In this article we review the use of near-field photonics for trapping, transport and handling of nanomaterials. While the advantages of traditional optical tweezing are well known at the microscale, direct application of these techniques to the handling of nanoscale materials has proven difficult due to unfavourable scaling of the fundamental physics. Recently a number of research groups have demonstrated how the evanescent fields surrounding photonic structures like photonic waveguides, optical resonators, and plasmonic nanoparticles can be used to greatly enhance optical forces. Here, we introduce some of the most common implementations of these techniques, focusing on those which have relevance to microfluidic or optofluidic applications. Since the field is still relatively nascent, we spend much of the article laying out the fundamental and practical advantages that near field optical manipulation offers over both traditional optical tweezing and other particle handling techniques. In addition we highlight three application areas where these techniques namely could be of interest to the lab-on-a-chip community, namely: single molecule analysis, nanoassembly, and optical chromatography.