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OBJECTIVE: Despite external ventricular drain (EVD) procedures being commonplace in neurosurgical practice, suboptimal placement rates remain high, and complications are not uncommon. The angle of the EVD catheter insertion and the accuracy of the drill hole placement are major factors determining successful EVD placement that are dependent on the drill bit morphology. The standard cylindrical 2-fluted twist drill bit creates a relatively deep and narrow drill hole that requires precise positioning, has limited visibility of the drill hole bottom and restricted catheter angular adjustment range, and poses the risk of inadvertent dural puncture. To overcome the standard problems associated with EVD drill bit morphology, the authors propose novel cone-shaped drill bits for EVD placement. METHODS: Conical drill bits of 30° and 45° were designed, manufactured, and tested in a simulated laboratory setting as well as in three human cadavers with intact skull, dura mater, and brain. Drill bit performance was rated by neurosurgical trainees across various domains using Likert scale-type questions. RESULTS: In the laboratory, maximum drilling temperatures adjacent to the drill hole were recorded and compared for the standard drill bit and the 30° and 45° conical drill bits and were not significantly different (p = 0.631 and p = 0.326, respectively). The maximum temperature recorded directly underneath the drilling site for the 45° drill bit was significantly higher than the temperature of the standard drill bit (p = 0.043). The differences between the standard and 30° drill bits were not significant (p = 0.783). Upon cadaver testing, the drilling times with 30° and 45° conical drill bits were significantly longer than those with the standard drill bit (p = 0.036 and p = 0.002, respectively). Likert scale scores were significantly higher for the conical 30° (median [IQR] 4.7 [3.3-5]) and 45° (4 [2-5]) drill bits than for the standard drill bit (1.7 [1-2.5], p < 0.0001), indicating significantly better performance. Conical drill bits used as a "rescue" strategy allowed for an EVD catheter angular adjustment range 6 to 9 times greater than that for the standard drill bit and resulted in a zero inadvertent dural puncture rate. CONCLUSIONS: The 30° conical drill bit can be safely used on its own or as a rescue tool to potentially achieve improved confidence, visualization, targeting, and precision of EVD placement while essentially eliminating the possibility of unintentional dural puncture with minimal increase in the total procedure time.
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Encéfalo , Catéteres , Humanos , Temperatura , Cráneo , Cadáver , Drenaje/métodos , VentriculostomíaRESUMEN
Quantitative nucleic acid amplification testing (NAAT) is a key enabling technology for infectious disease management, especially in instances where viral load informs therapeutic decisions. Inadequate access to quantitative NAATs remains a challenge to the successful deployment of antiretroviral therapy (ART) regimens for patients with chronic hepatitis B virus (CHB) in low resourced settings (LRS). Current field-deployable NAATs are generally qualitative (yes/no) rather than quantitative in nature, making them ill-suited for viral load monitoring programs for CHB patients. Here, we report the development of a proof-of-concept molecular diagnostic test, the semiquantitative ligation and amplification (SQLA) assay, which achieves semiquantitative detection of input target DNA at two independently tunable detection thresholds with a simple visual readout. The SQLA assay utilizes a duplex competitive thermophilic helicase-dependent amplification (tHDA) chemistry and can be performed in under 1 h.
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Virus de la Hepatitis B , Hepatitis B Crónica , Inmunoensayo , Técnicas de Amplificación de Ácido Nucleico , Ácidos Nucleicos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/virología , Humanos , Técnicas de Diagnóstico Molecular , Ácidos Nucleicos/análisis , Ácidos Nucleicos/genéticaRESUMEN
In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of â¼1 parasite/µL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.
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ADN Protozoario/genética , Malaria Falciparum/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Plasmodium falciparum/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , ADN Protozoario/aislamiento & purificación , Humanos , Límite de Detección , Plasmodium falciparum/aislamiento & purificación , Reproducibilidad de los ResultadosRESUMEN
Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices.
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Actinobacteria/metabolismo , Técnicas Biosensibles , Progesterona/metabolismo , Secuencia de Bases , Transferencia Resonante de Energía de Fluorescencia , Pruebas en el Punto de Atención , Reproducibilidad de los Resultados , Factores de Transcripción/metabolismoRESUMEN
Estimated to be the most common non-viral sexually transmitted infection globally, Trichomonas vaginalis (TV) can lead to pelvic inflammatory disease, pregnancy complications, and increased risk of acquiring and transmitting HIV. Once diagnosed, TV infection can be treated with oral antibiotics; however, infected individuals are often asymptomatic and do not seek treatment. The WHO and others have identified a need for point-of-care tests to expand access to TV testing and screening; ideal test characteristics include high sensitivity and specificity and the ability to use urine as a sample type, rather than invasively collected swab samples. Here, we report on a proof-of-concept prototype for rapid, electrostatic enrichment of DNA from urine samples and demonstrate the use of large volumes of urine to increase sensitivity of downstream nucleic acid amplification testing. We developed an internally controlled thermophilic helicase-dependent amplification (tHDA) assay with lateral flow immunoassay readout and demonstrate that this tHDA assay can be performed directly on our DNA capture filter. We validated our method using clinical urine samples with qPCR-quantified TV loads. Using 62 clinical urine samples and a simple sample processing device, our tHDA assay displayed 96.6% sensitivity and 100% specificity. Our analytical limit of detection was found to be approximately 7 genomic equivalents of TV DNA per mL of sample when 1 mL of sample was tested, comparable to existing isothermal tests for TV. Using large-volume simulated samples (40 mL of buffered urine with spiked-in TV DNA), we also demonstrated that sensitivity could be improved 28-fold to 0.25 genomic equivalents of TV DNA per mL, with a sample processing time of only 2 minutes.
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Developing ultrasensitive methods capable of detecting submicroscopic parasitemia-a challenge that persists in low transmission areas, asymptomatic carriers, and patients showing recrudescence-is vital to achieving malaria eradication. Nucleic acid amplification techniques offer improved analytical sensitivity but are limited by the number of copies of the amplification targets. Herein, we perform a novel genome mining approach to identify a pair of identical multirepeat sequences (IMRSs) that constitute 170 and 123 copies in the Plasmodium falciparum genome and explore their potential as primers for PCR. Real-time quantitative PCR analyses have shown the ability of P. falciparum IMRSs to amplify as low as 2.54 fg of P. falciparum genomic DNA (approximately 0.1 parasite), with a striking 100-fold increase in detection limit when compared with P. falciparum 18S rRNA (251.4 fg; approximately 10 parasites). Validation with clinical samples from malaria-endemic regions has shown 6.70 ± 1.66 cycle better detection threshold in terms of Ct value for P. falciparum IMRSs, with approximately 100% sensitivity and specificity. Plasmodium falciparum IMRS assays are also capable of detecting submicroscopic infections in asymptomatic samples. To summarize, this approach of initiating amplification at multiple loci across the genome and generating more products with increased analytical sensitivity is different from classic approaches amplifying multicopy genes or tandem repeats. This can serve as a platform technology to develop advanced diagnostics for various pathogens.
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ADN Protozoario/análisis , Genoma de Protozoos , Malaria Falciparum/diagnóstico , Parasitemia/diagnóstico , Plasmodium falciparum/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Biología Computacional/métodos , ADN Protozoario/sangre , ADN Protozoario/genética , Minería de Datos/métodos , Genes Protozoarios , Humanos , Malaria Falciparum/parasitología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Parasitemia/parasitología , Plasmodium falciparum/aislamiento & purificaciónRESUMEN
Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.
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Dispositivos Laboratorio en un Chip , Neisseria gonorrhoeae/aislamiento & purificación , Papel , Pruebas en el Punto de Atención , ADN Bacteriano/genética , Humanos , Neisseria gonorrhoeae/genéticaRESUMEN
Invasion of the extracellular matrix is a critical step in the colonization of metastatic tumors. The invasion process is thought to be driven by both chemokine signaling and interactions between invading cancer cells and physical components of the metastatic niche, including endothelial cells that line capillary walls and serve as a barrier to both diffusion and invasion of the underlying tissue. Transwell chambers, a tool for generating artificial chemokine gradients to induce cell migration, have facilitated recent work to investigate the chemokine contributions to matrix invasion. These chambers, however, are poorly designed for imaging, which limits their use in investigating the physical cell-cell and cell-matrix interactions driving matrix invasion. Microfluidic devices offer a promising model in which the invasion process can be imaged. Many current designs, however, have limited surface areas and possess intricate geometries that preclude the use of standard staining protocols to visualize cells and matrix proteins. In this work, we present a novel microfluidic platform for imaging cell-cell and cell-matrix interactions driving metastatic cancer cell matrix invasion. Our model is applied to investigate how endothelial cell-secreted matrix proteins and the physical endothelial monolayer itself interact with invading metastatic breast cancer cells to facilitate invasion of an underlying type I collagen gel. The results show that matrix invasion of metastatic breast cancer cells is significantly enhanced in the presence of live endothelial cells. Probing this interaction further, our platform revealed that, while the fibronectin-rich matrix deposited by endothelial cells was not sufficient to drive invasion alone, metastatic breast cancer cells were able to exploit components of energetically inactivated endothelial cells to gain entry into the underlying matrix. These findings reveal novel cell-cell interactions driving a key step in the colonization of metastatic tumors and have important implications for designing drugs targeted at preventing cancer metastasis.
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Neoplasias de la Mama/patología , Uniones Célula-Matriz/metabolismo , Microfluídica/métodos , Animales , Línea Celular Tumoral , Uniones Célula-Matriz/efectos de los fármacos , Colágeno Tipo I/farmacología , Matriz Extracelular/metabolismo , Femenino , Fibronectinas/metabolismo , Geles , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Ratas , Azida Sódica/farmacologíaRESUMEN
To advance the development of point-of-care technology (POCT), the National Institute of Biomedical Imaging and Bioengineering established the POCT Research Network (POCTRN), comprised of Centers that emphasize multidisciplinary partnerships and close facilitation to move technologies from an early stage of development into clinical testing and patient use. This paper describes the POCTRN and the three currently funded Centers as examples of academic-based organizations that support collaborations across disciplines, institutions, and geographic regions to successfully drive innovative solutions from concept to patient care.
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Chemotaxis is typically studied in vitro using commercially available products such as the Transwell® in which cells migrate through a porous membrane in response to one or more clearly defined chemotactic stimuli. Despite its widespread use, the Transwell assay suffers from being largely an endpoint assay, with built-in errors due to inconsistent pore size and human sampling. In this study, we report a microfluidic chemotactic chip that provides real-time monitoring, consistent paths for cell migration, and easy on-chip staining for quantifying migration. To compare its performance with that of a traditional Transwell chamber, we investigate the chemotactic response of MDA-MB-231 1833 metastatic breast cancer cells to epidermal growth factor (EGF). The results show that while both platforms were able to detect a chemotactic response, we observed a dose-dependent response of breast cancer cells towards EGF with low non-specific migration using the microfluidic platform, whereas we observed a dose-independent response of breast cancer cells towards EGF with high levels of non-specific migration using the commercially available Transwell.The microfluidic platform also allowed EGF-dependent chemotactic responses to be observed 24h, a substantially longer window than seen with the Transwell. Thus the performance of our microfluidic platform revealed phenomena that were not detected in the Transwell under the conditions tested.
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Quimiotaxis , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/fisiología , Humanos , Dispositivos Laboratorio en un ChipRESUMEN
How metastatic cancer lesions survive and grow in secondary locations is not fully understood. There is a growing appreciation for the importance of tumor components, i.e. microenvironmental cells, in this process. Here, we used a simple microfabricated dual cell culture platform with a 500 µm gap to assess interactions between two different metastatic melanoma cell lines (1205Lu isolated from a lung lesion established through a mouse xenograft; and WM852 derived from a stage III metastatic lesion of skin) and microenvironmental cells derived from either skin (fibroblasts), lung (epithelial cells) or liver (hepatocytes). We observed differential bi-directional migration between microenvironmental cells and melanoma, depending on the melanoma cell line. Lung epithelial cells and skin fibroblasts, but not hepatocytes, stimulated higher 1205Lu migration than without microenvironmental cells; in the opposite direction, 1205Lu cells induced hepatocytes to migrate, but had no effect on skin fibroblasts and slightly inhibited lung epithelial cells. In contrast, none of the microenvironments had a significant effect on WM852; in this case, skin fibroblasts and hepatocytes--but not lung epithelial cells--exhibited directed migration toward WM852. These observations reveal significant effects a given microenvironmental cell line has on the two different melanoma lines, as well as how melanoma effects different microenvironmental cell lines. Our simple platform thus has potential to provide complex insights into different strategies used by cancerous cells to survive in and colonize metastatic sites.
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Comunicación Celular , Técnicas de Cocultivo/instrumentación , Melanoma/fisiopatología , Melanoma/secundario , Ingeniería de Tejidos/métodos , Microambiente Tumoral/fisiología , Animales , Línea Celular Tumoral/clasificación , Diseño de Equipo , Análisis de Falla de Equipo , Dispositivos Laboratorio en un Chip , Melanoma/patología , Ratones , Impresión Tridimensional , Vísceras/patologíaRESUMEN
There is a critical need to formulate stable micron-sized oil droplets as hydrophobic drug carriers for efficient drug encapsulation, long-term storage, and sustained drug release. Microfluidic methods were developed to maximize the stability of micron-sized, oil-in-water (o/w) emulsions for potential use in drug delivery, using doxorubicin-loaded triacetin oil as a model hydrophobic drug formulation. Initial experiments examined multiple flow conditions for the dispersed (oil) and continuous (liposome aqueous) phases in a microfluidic device to establish the parameters that influenced droplet size. These data were fit to a mathematical model from the literature and indicate that the droplet sizes formed are controlled by the ratio of flow rates and the height of the device channel, rather than the orifice size. Next, we investigated effects of o/w emulsion production methods on the stability of the droplets. The stability of o/w emulsion produced by microfluidic flow-focusing techniques was found to be much greater (5 h vs 1 h) than for emulsions produced by mechanical agitation (vortexing). The increased droplet stability was attributed to the uniform size and lipid distribution of droplets generated by flow-focusing. In contrast, vortexed populations consisted of a wide size distribution that resulted in a higher prevalence of Ostwald ripening. Finally, the effects of shell polymerization on stability were investigated by comparing oil droplets encapsulated by a photopolymerizable diacetylene lipid shell to those with a nonpolymerizable lipid shell. Shell polymerization was found to significantly enhance stability against dissolution for flow-focused oil droplets but did not significantly affect the stability of vortexed droplets. Overall, results of these experiments show that flow-focusing is a promising technique for generating tunable, stable, monodisperse oil droplet emulsions, with potential applications for controlled delivery of hydrophobic drug formulations.
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Materiales Biocompatibles Revestidos , Portadores de Fármacos , Aceites , Fosfolípidos/química , Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , PolimerizacionRESUMEN
We report the first demonstration of using heat on a paper device to rapidly concentrate a clinically relevant analyte of interest from a biological fluid. Our technology relies on the application of localized heat to a paper strip to evaporate off hundreds of microliters of liquid to concentrate the target analyte. This method can be used to enrich for a target analyte that is present at low concentrations within a biological fluid to enhance the sensitivity of downstream detection methods. We demonstrate our method by concentrating the tuberculosis-specific glycolipid, lipoarabinomannan (LAM), a promising urinary biomarker for the detection and diagnosis of tuberculosis. We show that the heat does not compromise the subsequent immunodetectability of LAM, and in 20 min, the tuberculosis biomarker was concentrated by nearly 20-fold in simulated urine. Our method requires only 500 mW of power, and sample flow is self-driven via capillary action. As such, our technology can be readily integrated into portable, battery-powered, instrument-free diagnostic devices intended for use in low-resource settings.
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Líquidos Corporales/química , Técnicas de Química Analítica/métodos , Papel , Tuberculosis/orina , Urinálisis/métodos , Biomarcadores/sangre , Biomarcadores/orina , Calor , Humanos , Lipopolisacáridos/sangre , Lipopolisacáridos/orina , Reproducibilidad de los ResultadosRESUMEN
Monitoring cytokine release by cells allows the investigation of cellular response to specific external stimuli, such as pathogens or candidate drugs. Unlike conventional colorimetric techniques, label-free detection of cytokines enables studying cellular secretions in real time by eliminating additional wash and labeling steps after the binding step. However, label-free techniques that are based on measuring mass accumulation on a sensor surface are challenging for measuring small cytokines binding to much larger capture agents (usually antibodies) because the relative signal change is small. This problem is exacerbated when the capturing antibodies desorb from the surface, a phenomenon that almost inevitably occurs in immunoassays but is rarely accounted for. Here, we demonstrate a quantitative dynamic detection of interleukine-6 (IL-6), a pro-inflammatory cytokine, using an interferometric reflectance imaging sensor (IRIS). We improved the accuracy of the quantitative analysis of this relatively small protein (21 kDa) by characterizing the antibody desorption rate and compensating for the antibody loss during the binding experiment. By correcting for protein desorption, we achieved an analytical limit of detection at 19 ng/mL IL-6 concentration. We enhanced the sensitivity by 7-fold by using detection antibodies that recognize a different epitope of the cytokine. We demonstrate that these detection antibodies, which we call "mass tags", can be used concurrently with the target analyte to eliminate an additional wash and binding step. Finally, we report successful label-free detection of IL-6 in cell culture medium (with 10% serum) with comparable signal to that obtained in PBS. This work is the first to report quantitative dynamic label-free detection of small protein in a complex biological fluid using IRIS.
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Técnicas Biosensibles , Interleucina-6/análisis , Células Cultivadas , Humanos , Proteínas Recombinantes/análisisRESUMEN
High throughput analyses in biochemical assays are gaining popularity in the post-genomic era. Multiple label-free detection methods are especially of interest, as they allow quantitative monitoring of biomolecular interactions. It is assumed that the sensor surface is stable to the surrounding medium while the biochemical processes are taking place. Using the Interferometric Reflectance Imaging Sensor (IRIS), we found that buffers commonly used in biochemical reactions can remove silicon dioxide, a material frequently used as the solid support in the microarray industry. Here, we report 53 pm to 731 pm etching of the surface silicon oxide over a 12-h period for several different buffers, including various concentrations of SSC, SSPE, PBS, TRIS, MES, sodium phosphate, and potassium phosphate buffers, and found that PBS and MES buffers are much more benign than the others. We observe a linear dependence of the etch depth over time, and we find the etch rate of silicon dioxide in different buffers that ranges from 2.73±0.76 pm/h in 1M NaCl to 43.54±2.95 pm/h in 6×SSC. The protective effects by chemical modifications of the surface are explored. We demonstrate unaccounted glass etching leading to erroneous results with label-free detection of DNA microarrays, and offer remedies to increase the accuracy of quantitative analysis.
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Técnicas Biosensibles , Tampones (Química) , Dióxido de Silicio , Técnicas Biosensibles/métodos , Técnicas Biosensibles/normas , Interferometría/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
Monodisperse gas microbubbles, encapsulated with a shell of photopolymerizable diacetylene lipids and phospholipids, were produced by microfluidic flow focusing, for use as ultrasound contrast agents. The stability of the polymerized shell microbubbles against both aggregation and gas dissolution under physiological conditions was studied. Polyethylene glycol (PEG) 5000, which was attached to the diacetylene lipids, was predicted by molecular theory to provide more steric hindrance against aggregation than PEG 2000, and this was confirmed experimentally. The polymerized shell microbubbles were found to have higher shell-resistance than nonpolymerizable shell microbubbles and commercially available microbubbles (Vevo MicroMarker). The acoustic stability under 7.5 MHz ultrasound insonation was significantly greater than that for the two comparison microbubbles. The acoustic stability was tunable by varying the amount of diacetylene lipid. Thus, our polymerized shell microbubbles are a promising platform for ultrasound contrast agents.
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Acetileno/química , Medios de Contraste/química , Microburbujas , Polietilenglicoles/química , PolimerizacionRESUMEN
The ability to increase the concentration of target analytes in a fixed sample volume can potentially lower the limit of detection for many biosensing techniques, and thus is key in sample preparation for infectious disease diagnosis. Concentration by evaporation is an effective method to achieve target enrichment. However, concentrating human samples, including blood and plasma, by evaporation-based methods is made challenging by high concentrations of proteins and electrolytes. Dehydration of the proteins causes the sample to turn into a gel, hindering further analysis. At the same time, decreasing the volume increases the overall concentration of electrolytes, causing bacterial or viral particle lysis, and making them more difficult to detect in affinity-based biosensors. Thus, we fabricated a microfluidic chip that incorporates both dialysis and concentration in a single design. The chip dialyzes the proteins from the plasma, while maintaining an appropriate concentration of electrolytes and concentrating the sample targets. The process to concentrate plasma or serum samples by a factor of 10 takes less than 30 minutes. As a proof-of-concept, we demonstrated the chip using a defective Human Immunodeficiency Virus (HIV). To distinguish patients on antiretroviral therapy who are failing therapy from those who are not, a diagnostic must be able to detect HIV in plasma down to at least 1000 particles per milliliter. For a number of technical reasons, it is difficult to get on-chip PCR reactions to reach this level of sensitivity, so concentration of HIV from lower viral load samples has the potential to improve the sensitivity of many types of molecular point-of-care viral load tests.
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Análisis Químico de la Sangre/instrumentación , Proteínas Sanguíneas/análisis , Recolección de Muestras de Sangre/métodos , Análisis de Inyección de Flujo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Sistemas de Atención de Punto , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In this paper, we describe a technique for producing populations of ultrasound contrast agents (UCA) with a narrow size distribution. Using acoustic techniques, we measure the frequency-dependent attenuation coefficient for suspensions of ultrasound contrast agents with varying size distributions, ranging from narrow to wide. Our results demonstrate that as the size distribution becomes more uniform, attenuation occurs within a narrow frequency range. Additionally, as the mean size decreases, the peak in the attenuation coefficient occurs at a higher frequency.
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Medios de Contraste , Progresión de la Enfermedad , Diseño de Equipo , Humanos , Microburbujas , Microfluídica/métodos , Soluciones , Ultrasonido , Ultrasonografía/instrumentación , Ultrasonografía/métodosRESUMEN
Direct monitoring of primary molecular-binding interactions without the need for secondary reactants would markedly simplify and expand applications of high-throughput label-free detection methods. A simple interferometric technique is presented that monitors the optical phase difference resulting from accumulated biomolecular mass. As an example, 50 spots for each of four proteins consisting of BSA, human serum albumin, rabbit IgG, and protein G were dynamically monitored as they captured corresponding antibodies. Dynamic measurements were made at 26 pg/mm(2) SD per spot and with a detectable concentration of 19 ng/ml. The presented method is particularly relevant for protein microarray analysis because it is label-free, simple, sensitive, and easily scales to high-throughput.
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Análisis por Matrices de Proteínas/métodos , Coloración y Etiquetado/métodos , Animales , Reacciones Antígeno-Anticuerpo , Técnicas Biosensibles , Bovinos , Humanos , Técnicas de Dilución del Indicador , Cinética , ConejosRESUMEN
A significant challenge in tissue engineering is the creation of tissues with stratified morphology or embedded microstructures. This study investigated methods to fabricate composite gels from separately deposited alginate layers and examined the effects of processing methods on the mechanics of adhesion. Laminated alginate gels were created through a three step process which included: treatment of the interfaces with citrate; annealing of the gels to allow for molecular rearrangement of the alginate chains; and exposure to a CaCl(2) to crosslink the alginate sheets. Process variables included volume and concentration of applied citrate, annealing time, incubation time in CaCl(2), and CaCl(2) concentration. Laminated sheets were tested in lap-shear geometry to characterize failure phenomena and mechanical properties. The site of failure within the gel depended on the integrity of the interface, with weaker gels delaminating and gels with mechanical properties similar to that of bulk gels failing randomly throughout the thickness. Citrate volume, citrate concentration, CaCl(2) incubation time, and CaCl(2) concentration altered the mechanical properties of the laminated alginate sheets, while annealing time had little effect on all measured parameters. This study demonstrates the integration of separately fabricated alginate layers to create mechanically or chemically anisotropic or heterogeneous structures.