RÉSUMÉ
Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.
Sujet(s)
Anopheles , Serpines , Animaux , Femelle , Immunité humorale , Serine endopeptidases/génétique , Serine endopeptidases/métabolisme , Protéases à sérine/génétique , Serpines/génétique , Serpines/métabolisme , Protéines d'insecte/génétique , Protéines d'insecte/métabolismeRÉSUMÉ
Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip domain serine proteases (cSPs) and/or their non-catalytic homologs (cSPHs), which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.
RÉSUMÉ
We report the design and adaptation of iron/iron oxide nanoparticle-based optical nanobiosensors for enzymes or cytokine/chemokines that are established biomarkers of lung diseases. These biomarkers comprise ADAM33, granzyme B, MMP-8, neutrophil elastase, arginase, chemokine (C-C motif) ligand 20 and interleukin-6. The synthesis of nanobiosensors for these seven biomarkers, their calibration with commercially available enzymes and cytokines/chemokines, as well as their validation using bronchoalveolar lavage (BAL) obtained from a mouse model of TLR3-mediated inflammation are discussed here. Exhaled Breath Condensate (EBC) is a minimally invasive approach for sampling airway fluid in the diagnosis and management of various lung diseases in humans (e.g., asthma, COPD and viral infections). We report the proof-of-concept of using human EBC in conjunction with nanobiosensors for diagnosis/monitoring airway inflammation. These findings suggest that, with nanosensor technology, human EBC can be utilized as a liquid biopsy to monitor inflammation/remodeling in lung disease.
Sujet(s)
Asthme , Maladies pulmonaires , Animaux , Marqueurs biologiques , Tests d'analyse de l'haleine , Inflammation/diagnostic , SourisRÉSUMÉ
Chronic neuroinflammation has long been considered to be a central factor in accelerating the progression of neurodegenerative diseases such as Alzheimer's diseases, Parkinson's disease and chronic traumatic encephalopathy. Under pathological conditions microglia produce inflammatory signaling molecules, such as nitric oxide (NO), that can damage DNA and proteins and ultimately induce neuronal apoptosis. One strategy for treating neurodegenerative diseases is to specifically target NO production through inhibition of inducible nitric oxide synthase (iNOS). However, accurately measuring changes in microglial NO production in response to potential therapeutics is challenging due to NO's short half-life and microglial heterogeneity. In this paper we report the application of a microfluidic device for the high-throughput measurement of intracellular NO in SIM-A9 microglial cells. NO production was measured in response to treatment with lipopolysaccharides (LPS) and interferon gamma (IFN-γ) with and without a potent iNOS inhibitor (1400 W dihydrochloride). Cells were labeled with a fluorogenic NO probe, 4-amino-5-methylamino-2',7'-difluorofluoescein diacetate (DAF-FM DA), and 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. Separation and quantitation of intracellular NO was achieved using microchip electrophoresis and laser induced fluorescence detection (LIF). Statistical analysis suggests that the populations fit a lognormal distribution and are better represented by their geometric mean values. Comparison of the geometric means indicated a 1.6-fold increase in NO production between untreated and stimulated cells and a decrease by a factor of approximately 0.5 comparing stimulated and inhibited cells. Additionally, we report experimental data demonstrating the improvement in the sensitivity of our integrated optical fiber-based detection system through the use of refractive index matching gel.
Sujet(s)
Microglie , Monoxyde d'azote , Microfluidique , Facteur de transcription NF-kappa B , Analyse sur cellule uniqueRÉSUMÉ
Here we describe in detail the design, fabrication and operation of our automated high-throughput single cell microchip electrophoresis device with laser induced fluorescence detection. Our device features on-board integrated peristaltic pumps that generate flow directly within the microfluidic channels. Additionally, we have incorporated an optical fiber bridge that enables simultaneous fluorescence detection at two points of interest within the device without the need for additional optical components or detectors. The second detection spot is used to detect the intact cell immediately prior to lysis giving a signal at t=0s for each single-cell electropherogram. We can also use this signal to measure the absolute migration time of the separated analytes to confidently determine the identity of each peak. Finally, we demonstrate the application of our device for the measurement of intracellular nitric oxide (NO) levels in T-lymphocytes. Changes in NO levels within cells is associated with a number of chronic diseases including neurodegenerative, cardiovascular and cancers. We show that our system is capable of measuring NO levels under the following conditions: native, lipopolysaccharide stimulation, and inhibition of inducible nitric oxide synthase. It is our hope that the information and procedures described in this chapter may enable others to use or adapt our system for other analyses at the single cell level.
Sujet(s)
Électrophorèse sur puce/instrumentation , Analyse sur cellule unique/instrumentation , Dosages enzymatiques/instrumentation , Conception d'appareillage , Humains , Cellules Jurkat , Monoxyde d'azote/analyse , Fibres optiques , Lymphocytes T/composition chimiqueRÉSUMÉ
This chapter provides step-by-step procedures for the fabrication of glass-based microfluidic devices. These procedures include device design, photomask generation, photolithography, channel etching, and high-temperature bonding.
Sujet(s)
Conception d'appareillage/méthodes , Techniques d'analyse microfluidique/instrumentation , Verre , Laboratoires sur puces , Processus photochimiques , Propriétés de surfaceRÉSUMÉ
Numerous proteases, such as matrix metalloproteinases (MMPs), cathepsins (CTS), and urokinase plasminogen activator (UpA), are dysfunctional (that is, over- or under-expressed) in solid tumors, when compared to healthy human subjects. This offers the opportunity to detect early tumors by liquid biopsies. This approach is of particular advantage for the early detection of pancreatic cancer, which is a "silent killer". We have developed fluorescence nanobiosensors for ultrasensitive (sub-femtomolar) arginase and protease detection, consisting of water-dispersible Fe/Fe3O4 core/shell nanoparticles and two tethered fluorescent dyes: TCPP (Tetrakis(4-carboxyphenyl)porphyrin) and cyanine 5.5. Upon posttranslational modification or enzymatic cleavage, the fluorescence of TCPP increases, which enables the detection of proteases at sub-femtomolar activities utilizing conventional plate readers. We have identified an enzymatic signature for the detection of pancreatic adenocarcinomas in serum, consisting of arginase, matrix metalloproteinase-1, -3, and -â¯9, cathepsin-B and -E, urokinase plasminogen activator, and neutrophil elastase, which is a potential game-changer.
Sujet(s)
Techniques de biocapteur , Carcinome du canal pancréatique/diagnostic , Dépistage précoce du cancer/méthodes , Colorants fluorescents/composition chimique , Nanoparticules/composition chimique , Tumeurs du pancréas/diagnostic , Études cas-témoins , Femelle , Humains , Biopsie liquide , MâleRÉSUMÉ
Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.
RÉSUMÉ
In this paper a single particle/cell-tracking microfluidic device that integrates an out-of-plane multimode optical fiber (OP-MMF) is reported. This OP-MMF is used to generate three excitation light-lines and three detection spots using only one excitation source and one detector. It takes advantage of an optical tunneling mode to create two excitation lines in a microfluidic channel emanating from a single fiber end. This method was used to accurately count particles/cells and perform velocity measurements and size discrimination. The velocity and size distributions of 5, 7, and 10 µm fluorescently labeled polystyrene beads were determined using the OP-MMF. Additionally, this method was used to analyze cell lysates with the third excitation line in the separation channel. The OP-MMF setup accurately detected an intact cell twice â¼2 mm prior to lysis, determined its velocity, and detected the injected cell lysate 3 mm downstream of the injection point in the separation channel. Using this setup, the velocity of cells entering the lysis intersection and the absolute migration times of fluorescently labeled analytes injected into the separation channel were determined in an automated fashion. This method enabled us to determine a lysing/injection efficiency coefficient (K) using signals from the injected lysate signal and from the intact cell before lysing. K provided a reliable measurement of the amount of cell lysate that was injected into the separation channel. The approach reported here could be used in the future to track particles, cells or droplets in a variety of existing microfluidic devices without the need for multiplexed masks, layers, bulky optical elements or complex optical designs.
Sujet(s)
Microfluidique/méthodes , Fluorescéines/composition chimique , Humains , Cellules Jurkat , Laboratoires sur puces , Microfluidique/instrumentation , Fibres optiques , Taille de particule , Polystyrènes/composition chimique , RhéologieRÉSUMÉ
A microfluidic device is reported that employs an out-of-plane optical fiber bridge to generate two excitation and two detection spots in a microfluidic channel using only one excitation source and one detector. This fiber optic bridge was integrated into a single cell analysis device to detect an intact cell just prior to lysis and the injected lysate 2, 5, 10, or 15 mm downstream of the injection point. Using this setup the absolute migration times for analytes from cells stochastically entering the lysis intersection could be determined for the first time in an automated fashion. This allowed the evaluation of several separation parameters, including analyte band velocity, migration time drift, diffusion coefficient, injection plug length, separation efficiency (N), and plate height (H), which previously could only be estimated. To demonstrate the utility of this system, a peptide substrate for protein kinase B (PKB) was designed, synthesized, and loaded into T-lymphocytes in order to measure PKB activity in individual cells. The optical fiber bridge is easy to implement, inexpensive, and flexible in terms of changing the distances between the two detection points.
Sujet(s)
Technologie des fibres optiques/instrumentation , Laboratoires sur puces , Techniques d'analyse microfluidique/instrumentation , Analyse sur cellule unique/méthodes , Humains , Cellules Jurkat/métabolisme , Fibres optiques , Peptides/analyse , Peptides/métabolisme , Phosphopeptides/analyse , Phosphorylation , Protéines proto-oncogènes c-akt/analyse , Protéines proto-oncogènes c-akt/métabolisme , Analyse sur cellule unique/instrumentationRÉSUMÉ
The ability to accurately control fluid transport in microfluidic devices is key for developing high-throughput methods for single cell analysis. Making small, reproducible changes to flow rates, however, to optimize lysis and injection using pumps external to the microfluidic device are challenging and time-consuming. To improve the throughput and increase the number of cells analyzed, we have integrated previously reported micropumps into a microfluidic device that can increase the cell analysis rate to â¼1000 cells/h and operate for over an hour continuously. In order to increase the flow rates sufficiently to handle cells at a higher throughput, three sets of pumps were multiplexed. These pumps are simple, low-cost, durable, easy to fabricate, and biocompatible. They provide precise control of the flow rate up to 9.2 nL/s. These devices were used to automatically transport, lyse, and electrophoretically separate T-Lymphocyte cells loaded with Oregon green and 6-carboxyfluorescein. Peak overlap statistics predicted the number of fully resolved single-cell electropherograms seen. In addition, there was no change in the average fluorescent dye peak areas indicating that the cells remained intact and the dyes did not leak out of the cells over the 1 h analysis time. The cell lysate peak area distribution followed that expected of an asynchronous steady-state population of immortalized cells.
Sujet(s)
Tests de criblage à haut débit/méthodes , Laboratoires sur puces , Techniques d'analyse microfluidique/instrumentation , Analyse sur cellule unique , Lymphocytes T , Acides carboxyliques , Séparation cellulaire , Électrophorèse , Fluorescéines , Colorants fluorescents , HumainsRÉSUMÉ
This work reports a synergistic approach to the concentration, detection and kinetic monitoring of pathogens through the integration of nanostructured dielectrophoresis (DEP) with nanotag-labelled Surface Enhanced Raman Spectroscopy (SERS). A nanoelectrode array made of embedded Vertically Aligned Carbon Nanofibers (VACNFs) at the bottom of a microfluidic chip was used to effectively capture and concentrate nanotag-labelled E. coli DHα5 cells into a 200 µm × 200 µm area on which a Raman laser probe was focused. The SERS nanotags were based on iron oxide-gold (IO-Au) core-shell nanoovals (NOVs) of â¼50 nm size, which were coated with a QSY21 Raman reporter and attached to E. coli through specific immunochemistry. The combination of the greatly enhanced Raman signal by the SERS nanotags and the effective DEP concentration significantly improved the detection limit and speed. The SERS signal was measured with both a confocal Raman microscope and a portable Raman probe during DEP capture, and was fully validated with fluorescence microscopy measurements under all DEP conditions. The SERS measurements were sensitive enough to detect a single bacterium. A concentration detection limit as low as 210 cfu ml(-1) using a portable Raman system was obtained with a DEP capture time of only â¼50 s. These results demonstrate the potential to develop a compact portable system for rapid and highly sensitive detection of specific pathogens. This system is reusable, requires minimum sample preparation, and is amenable to field applications.
Sujet(s)
Électrophorèse/instrumentation , Escherichia coli , Nanostructures/composition chimique , Analyse spectrale Raman , Animaux , Carbone/composition chimique , Poulets , Conception d'appareillage , Composés du fer III/composition chimique , Or/composition chimique , Immunochimie , Laboratoires sur puces , Limite de détection , Microfluidique/instrumentation , Microscopie confocale , Microscopie de fluorescence , Nanotechnologie , Propriétés de surface , Composés de l'étain/composition chimiqueRÉSUMÉ
A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.
Sujet(s)
Microfluidique/instrumentation , Monoxyde d'azote/biosynthèse , Analyse sur cellule unique , Lymphocytes T/métabolisme , Humains , Cellules Jurkat , Normes de référenceRÉSUMÉ
This work describes efficient manipulation of bacteriophage virus particles using a nanostructured DEP device. The nonuniform electric field for DEP is created by utilizing a nanoelectrode array (NEA) made of vertically aligned carbon nanofibers versus a macroscopic indium tin oxide electrode in a "points-and-lid" configuration integrated in a microfluidic channel. The capture of the virus particles has been systematically investigated versus the flow velocity, sinusoidal AC frequency, peak-to-peak voltage, and virus concentration. The DEP capture at all conditions is reversible and the captured virus particles are released immediately when the voltage is turned off. At the low virus concentration (8.9 × 10(4) pfu/mL), the DEP capture efficiency up to 60% can be obtained. The virus particles are individually captured at isolated nanoelectrode tips and accumulate linearly with time. Due to the comparable size, it is more effective to capture virus particles than larger bacterial cells with such NEA-based DEP devices. This technique can be potentially utilized as a fast sample preparation module in a microfluidic chip to capture, separate, and concentrate viruses and other biological particles in small volumes of dilute solutions in a portable detection system for field applications.
Sujet(s)
Bactériophage T4/isolement et purification , Carbone/composition chimique , Électrophorèse/instrumentation , Électrophorèse/méthodes , Nanofibres/composition chimique , Nanotechnologie/instrumentation , Nanotechnologie/méthodes , Bactériophage T4/composition chimique , Conception d'appareillage , Analyse sur microréseau/instrumentation , Analyse sur microréseau/méthodes , Microélectrodes , Taille de particuleRÉSUMÉ
This paper reports capture and detection of pathogenic bacteria based on AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) versus a macroscopic indium-tin-oxide (ITO) transparent electrode in "points-and-lid" configuration. The nano-DEP device was fabricated using photolithography processes to define an exposed active region on a randomly distributed NEA and a microfluidic channel on ITO to guide the flow of labeled Escherichia coli cells, respectively, and then bond them into a fluidic chip. A high-frequency (100 kHz) AC field was applied to generate positive DEP at the tips of exposed CNFs. Enhanced electric field gradient was achieved due to reduction in electrode size down to nanometer scale which helped to overcome the large hydrodynamic drag force experienced by E. coli cells at high flow velocities (up to 1.6 mm/s). This DEP device was able to effectively capture a significant number of E. coli cells. Significant decrease in the absolute impedance at the NEA was observed in EIS experiments. The results obtained in this study suggest the possibility of integration of a fully functional electronic device for rapid, reversible and label-free capture and detection of pathogenic bacteria.
Sujet(s)
Électrophorèse/instrumentation , Électrophorèse/méthodes , Escherichia coli/isolement et purification , Nanotechnologie/instrumentation , Carbone/composition chimique , Spectroscopie diélectrique , Escherichia coli/cytologie , Nanofibres/composition chimiqueRÉSUMÉ
Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4-10 µM with r(2) ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.
Sujet(s)
Adduits à l'ADN/isolement et purification , Électrophorèse capillaire/méthodes , Catéchol oestrogènes/isolement et purification , Techniques d'analyse microfluidique/méthodes , Adduits à l'ADN/composition chimique , Électrochimie , Électrophorèse capillaire/instrumentation , Catéchol oestrogènes/composition chimique , Humains , Techniques d'analyse microfluidique/instrumentationRÉSUMÉ
We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 microm wide with barriers as narrow as 250 microm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318-1320, 1; Martinez et al., Anal Chem 10:3699-3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606-19611, 3; Lu et al., Electrophoresis 9:1497-1500, 4; Abe et al., Anal Chem 18:6928-6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 microM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 microM) were generated. The time of device fabrication to the time of test results was about 25 min.