RESUMO
We introduce a centrifugal microfluidic disc that accepts a small volume in (â¼5 µL), performs sample cleanup on human serum samples, and delivers a small volume out, for subsequent metabolite analysis by surface assisted laser desorption/ionization (SALDI) mass spectrometry (MS) or hydrophilic interaction liquid chromatography (HILIC)-MS. The centrifugal microfluidic disc improves the MS results by removing proteins and lipids from serum. In the case of SALDI-MS, sample background electrolytes are segregated from analytes during the spotting process by the action of the SALDI-chip during drying, for further cleanup, while HILIC separates the salts in HILIC-MS. The resulting mass spectra of disc-prepared samples show a clean background and high signal-to-noise ratio for metabolite peaks. Several representative ionic metabolites from human serum samples were successfully quantified. The performances of the sample preparation disc for SALDI-MS and HILIC-MS were assessed and were comparable. Reproducibility, sample bias, and detection limits for SALDI-MS compared well to ultrafiltration sample preparation.
Assuntos
Metaboloma , Microfluídica/métodos , Centrifugação , Cromatografia Líquida de Alta Pressão , Humanos , Interações Hidrofóbicas e Hidrofílicas , Íons/química , Malatos/sangue , Nanopartículas/química , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Dióxido de Silício/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Taurina/sangueRESUMO
We present a surface assisted laser desorption ionization (SALDI) technique, coupled with fluorocarbon coating, to achieve selective segregation of ionic and/or hydrophilic analytes from background biofluid electrolytes for quantiatve mass spectrometric analysis. By controlling the contact angle of (1H,1H,2H,2H-perfluorooctyl) trichlorosilane or (1H,1H,2H,2H-perfluorooctyl) dimethylchlorosilane to a specific range (105-120°), background electrolytes can be made to segregate from hydrophilic analytes during a drying step on the surface of a highly nanoporous thin film. Nanoporous silicon films were prepared using glancing angle deposition (GLAD) thin film technology, then coated with fluorcarbon. This desalting method directly separates highly polar, ionic metabolites, such as amino acids, from salty biofluids such as aritificial cerebrospinal fluid (aCSF) and serum. Derivatization, extraction and rinsing steps are not required to separate the analytes from the bioelectrolytes. With on-chip desalting, the limit of quantitation for histidine spiked in aCSF is â¼1 µM, and calibration curves with internal standards can achieve a precision of 1-9% within a 1 to 50 µM range. Five highly polar organic acids in serum were successfully quantified, and the SALDI-MS results obtained on the desalted serum sample spots show both good reproducibility and compare well to results from NMR and liquid chromatography-mass spectrometry. Putative identification of a total of 32 metabolites was accomplished in blood using time-of-flight MS with perfluoro coated Si-GLAD SALDI, by comparison to tabulated data.
Assuntos
Aminoácidos/análise , Líquido Cefalorraquidiano/química , Fluorocarbonos/química , Nanoestruturas/química , Sais/química , Doença de Alzheimer/sangue , Doença de Alzheimer/urina , Aminoácidos/metabolismo , Líquido Cefalorraquidiano/metabolismo , Eletrólitos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Porosidade , Silício/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Propriedades de SuperfícieRESUMO
All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.
Assuntos
Mamíferos/fisiologia , Neurônios/fisiologia , Rodopsina/fisiologia , Animais , Evolução Molecular Direcionada , Proteínas Recombinantes/metabolismo , Transmissão SinápticaRESUMO
The separation behavior of 6.5-66 kDa proteins in silica particle array-based sieves formed by colloidal self-assembly in microchips is reported across a pore size range of 22-103 nm. The protein separation and resolution improves markedly with decreasing pore size. The variation of electrophoretic mobility with molecular weight of SDS-protein complexes and with particle size was evaluated using the Ogston sieving equation for a random pore gel structure, and using the modified Giddings equation developed by Wirth for uniform pore structures. The Wirth/Giddings equation provides the best fit for estimation of molecular weight of proteins, and demonstrates that even though experimental evidence shows there is some dispersion in measured pore sizes, these structures can best be described as having a uniform pore size.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Nanoestruturas/química , Análise Serial de Proteínas/métodos , Proteínas/isolamento & purificação , Tamanho da Partícula , Porosidade , Proteínas/químicaRESUMO
RATIONALE: Thin, nanoporous films fabricated using Glancing Angle Deposition (GLAD) technology are demonstrated for solid matrix laser desorption/ionization mass spectrometry (SMALDI-MS). GLAD allows facile engineering of nanoporosity, film thickness, post alignment, and material composition, as demonstrated here by the fabrication of Co-GLAD and Si-GLAD films for SMALDI, and by exploration of the SMALDI performance as a function of thickness, post density, and angle of the post relative to surface normal. METHODS: GLAD films were prepared by electron beam evaporation onto silicon substrates, using steep angles of incidence for the vacuum deposition, with computer controlled substrate rotation. LDI from the GLAD films was evaluated using an MDS-Sciex time-of-flight (TOF) MALDI mass spectrometer. RESULTS: Co-GLAD films give a limit of quantitation of 6 fmol for complex carbohydrate derivatives, and slanted-post Si-GLAD films show up to three times higher sensitivity than vertical post structures. Reproducibility of both Si and Co films is much higher than conventional MALDI methods for m/z below at least 2100 Da. Both reproducibility and detection limits are comparable to or better than other nano-structured materials. Co-GLAD films are significantly better in performance than Co powders or Co thin films on silicon substrates previously evaluated. CONCLUSIONS: The flexibility of GLAD for thin film fabrication of LDI materials is demonstrated by the range of nanoporous materials that can be grown, and the fine control over structural conformation, thickness and porosity. Copyright © 2017 John Wiley & Sons, Ltd.
RESUMO
We report on a facile method to stabilize colloidal self-assembled (CSA) nanoparticles packed in microchannels for high speed size-based separation of proteins. Silica nanoparticles, self-assembled in a network of microfluidic channels, were stabilized with a methacrylate polymer prepared in situ through photopolymerization. The entrapment conditions were investigated to minimize the effect of the polymer matrix on the structure of the packing and the separation properties of the CSA beds. SEM shows that the methacrylate matrix links the nanoparticles at specific sphere-sphere contact points, improving the stability of the CSA structure at high electric fields (up to at least 1800 V/cm), allowing fast and efficient separation. The %RSD of the protein migration times varied between 0.3 and 0.5% (n = 4, in 1 day) and <0.83% over a period of 7 days (n = 28 runs) in a single device, at high field strength. The overall %RSD of protein migration times from chip-to-chip across a single fabrication run was 4.3% (n = 3) and between fabrication runs was 11% (n = 35), with 87% fabrication yield, demonstrating reproducible packing and entrapment behavior. The optimized entrapped CSA beds demonstrated better separation performance (plate height, H â¼ 200 nm) than similarly prepared on-chip CSA beds without the polymer entrapment. Polymer-entrapped CSA beds also exhibited superior protein resolving power: the minimum resolvable molecular weight difference of proteins in the polymer-entrapped CSA bed is 0.6 kDa versus â¼9 kDa for the native silica CSA bed (i.e. without polymer entrapment).
Assuntos
Coloides/química , Nanopartículas/química , Proteínas/isolamento & purificação , Técnicas Analíticas Microfluídicas , Tamanho da Partícula , Polímeros/química , Proteínas/análise , Proteínas/química , Reprodutibilidade dos Testes , Dióxido de Silício/químicaRESUMO
We investigate the trapping mechanism of individual DNA molecules in ordered nanoporous structures generated by crystalline particle arrays. Two requisites for trapping are revealed by the dynamics of single trapped DNA, fully-stretched U/J shapes and hernia formation. The experimental results show there is a stronger possibility for hernias to lead the reorientation upon switching directions of the voltage at high field strengths, where trapping occurs. Fully stretched DNA has longer unhooking times than expected by a classic rope-on-pulley model. We propose a dielectrophoretic (DEP) force reduces the mobility of segments at the apex of the U or J, where field gradients are highest, based on simulations and observations of the trapping force dependence on field strength. A modified model for unhooking time is obtained after the DEP force is introduced. The new model explains the unhooking time data by predicting an infinite trapping time when the ratio of arm length differences (of the U or J) to molecule length Δx/L < ß, where ß is a DEP parameter that is found to strongly increase with electric field. The DNA polarizability calculated with the DEP model and experimental value of ß is of the same magnitude of reported value. The results indicate the tension at the apex of U/J shape DNA is the primary reason for DNA trapping during pulsed field separation, instead of hernias.
Assuntos
DNA/química , Nanoestruturas , Eletroforese em Gel de Campo PulsadoRESUMO
Asymmetric pulsed field electrophoresis within crystalline arrays is used to generate angular separation of DNA molecules. Four regimes of the frequency response are observed, a low frequency rise in angular separation, a plateau, a subsequent decline, and a second plateau at higher frequencies. It is shown that the frequency response for different sized DNA is governed by the relation between pulse time and the reorientation time of DNA molecules. The decline in angular separation at higher frequencies has not previously been analyzed. Real-time videos of single DNA molecules migrating under high frequency-pulsed electric field show the molecules no longer follow the head to tail switching, ratchet mechanism seen at lower frequencies. Once the pulse period is shorter than the reorientation time, the migration mechanism changes significantly. The molecule reptates along the average direction of the two electric fields, which reduces the angular separation. A freely jointed chain model of DNA is developed where the porous structure is represented with a hexagonal array of obstacles. The model qualitatively predicts the variation of DNA angular separation with respect to frequency.
Assuntos
DNA/química , Eletroforese em Gel de Campo Pulsado/métodos , Técnicas Analíticas Microfluídicas/métodos , Coloides/química , Simulação por Computador , DNA/isolamento & purificação , Eletricidade , Eletroforese em Gel de Campo Pulsado/instrumentação , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
A microfluidic device that performs "in space" sample fractionation, collection, and preconcentration for proteomics is described. Effluents from a 2.75 mm long fractionation channel, focused via sheath flow, were sequentially delivered into an array of 36-collection channels containing monolithic polymer beds for SPE. Optimum conditions for the device design, and simultaneous photolytic fabrication of 36 monolithic columns in the 36 channels, as well as for their proper performance in electrokinetic sample fractionation and collection are described. A hydrophobic butyl methacrylate-based monolithic porous polymer was copolymerized with an ionizable monomer, acryloamido-methyl-propane sulfonate, to form a polymer monolith for SPE that also sustains cathodic electroosmotic flow. The SPE bed was made deep enough to greatly reduce the linear flow rate within the bed, in order to compensate for the lower electroosmotic mobility of the cationically charged SPE bed relative to the glass walled device. Under these conditions, electrokinetic fractionation of a protein sample resulted in tightly focused sample zones delivered into each of the 36-channel polymer beds with no observed crosscontamination. Monolithic columns showed reproducible performance with preconcentration factor of 30 for 2 min loading time. The ability to fractionate, collect, and preconcentrate samples on a microfluidic platform will be especially useful for automated or continuous operation of these devices in proteomics research.
Assuntos
Eletroforese Capilar/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Extração em Fase Sólida/instrumentação , Eletroforese Capilar/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Metacrilatos/química , Polímeros/química , Proteômica/instrumentação , Proteômica/métodos , Reprodutibilidade dos Testes , Albumina Sérica/química , Extração em Fase Sólida/métodosRESUMO
We describe a microfluidic device in which integrated tryptic digestion, SPE, CE separation and electrospray ionization for MS are performed. The chip comprised of 10 × 30 µm channels for CE, and two serially connected 150 µm deep, 800 µm wide channels packed with 40 to 60 µm diameter beads, loaded with either immobilized trypsin, reversed-phase packing or both. On-chip digestion of cytochrome c using the trypsin bed showed complete consumption of the protein in 3 min, in contrast to the 2 h required for conventional solution phase tryptic digestion. SPE of 0.25 µg/mL solutions of the peptides leu-enkephalin, angiotensin II and LHRH gave concentration enhancements in the range of 4.4-12, for a ten times nominal volume ratio. A 100 nM cytochrome c sample concentrated 13.3 times on-chip gave a sequence coverage of 85.6%, with recovery values ranging from 41.2 to 106%. The same sample run without SPE showed only five fragment peaks and a sequence coverage of 41.3%. When both on-chip digestion and SPE (13.3 volume ratio concentration enhancement) were performed on 200 nM cytochrome c samples, a sequence coverage of 76.0% and recovery values of 21-105% were observed. Performing on-chip digestion alone on the same sample gave only one significant fragment peak. The above digestion/peptide concentration step was compared to on-chip protein concentration by SPE followed by on-chip digestion with solution phase trypsin. Both procedures gave similar recovery results; however, much larger trypsin autodigestion interference in the latter approach was apparent.
Assuntos
Eletroforese em Microchip/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microextração em Fase Sólida/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Tripsina/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Fatores de TempoRESUMO
We report the patternability as well as the reproducibility and stability of flow resistance of polymer monolithic beds photopatterned within microfluidic channels as a function of initial reagent composition and preparation conditions. 2-Hydroxyethyl methacrylate and ethylene dimethacrylate-based polymer monoliths were selectively photopatterned within microchannels and their flow resistance was evaluated using a photobleaching, TOF linear flow rate measurement method developed in our lab. This measurement technique was found to be significantly more informative for columns formed in microfluidic channels compared with bulk monolith characterization by mercury intrusion porosimetry. 1-Octanol was determined to provide sharp bed edge formation and relatively low flow resistance by photopatterning relative to other porogenic solvents. Compared with literature formulations which did not achieve good flow stability and reproducibility from batch to batch, using 2-hydroxyethyl methacrylate, ethylene dimethacrylate and 1-octanol as porogenic solvents, less than 4% RSD was achieved in flow stability over 7 days for monoliths prepared with 60-80% crosslinker(monomer+crosslinker) ratio. Column-to-column variation of 5% RSD was obtained in this composition range. These results demonstrate that photopatterning of uniform polymer monolithic beds, which is critical for applications in multiplexed microfluidic systems, requires careful attention to the parameters that affect reproducibility, specifically the porogenic solvent choice and the crosslinker to monomer ratio.
Assuntos
Metacrilatos/química , Microfluídica/instrumentação , PorosidadeRESUMO
We present three generations of microchip-based "in-space" sample fractionators and collectors for use in proteomics. The basic chip design consisted of a single channel for CE separation of analytes that then intersects a fractionation zone feed into multiple high aspect ratio microchannels for fractionation of separated components. Achievements of each generation are discussed in relation to important design criteria. CE-separated samples were electrokinetically driven to multiple collection channels in sequence without cross-contamination under the protection of sheath streams. A 36-channel fractionator demonstrated the efficacy of a high-throughput fractionator with no observed cross-contamination. A mixture of IgG and BSA was used to test the efficiency of the fractionator and collector. CE of the fractionated samples was performed on the same device to verify their purity. Our demonstration proved to be efficient and reproducible in obtaining non-contaminated samples over 15 sample injections. Experimental results were found to be in close agreement with PSpice simulation in terms of flow behavior, contamination control and device performance. The design presented here has a great potential to be integrated in proteomic platforms.
Assuntos
Eletroforese Capilar/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Proteômica/instrumentação , Animais , Bovinos , Fracionamento Químico/instrumentação , Desenho de Equipamento , Imunoglobulina G/isolamento & purificação , Miniaturização/instrumentação , Soroalbumina Bovina/isolamento & purificaçãoRESUMO
Glancing angle deposition (GLAD) was used to fabricate nanostructured silicon (Si) thin films with highly controlled morphology for use in laser desorption/ionization mass spectrometry (DIOS-MS). Peptides, drugs and metabolites in the mass range of 150-2500 Da were readily analyzed. The best performance was obtained with 500 nm thick films deposited at a deposition angle of 85 degrees . Low background mass spectra and attomole detection limits were observed with DIOS-MS for various peptides. Films used after three months of dry storage in ambient conditions produced mass spectra with negligible low-mass noise following a 15 min UV-ozone treatment. The performance of the Si GLAD films was as good as or better than that reported for electrochemically etched porous silicon and related materials, and was superior to matrix-assisted laser desorption/ionization (MALDI)-MS for analysis of mixtures of small molecules between 150-2500 Da in terms of background chemical noise, detection limits and spot-to-spot reproducibility. The spot-to-spot reproducibility of signal intensities (100 shots/spectrum) from 21 different Si GLAD film targets was +/-13% relative standard deviation (RSD). The single shot-to-shot reproducibility of signals on a single target was +/-19% RSD (n = 7), with no indication of sweet spots or mute spots.
RESUMO
Microchip-based bead-packed columns for electrochromatography are described for several types of stationary phases. Chromatography columns 2 mm in length were used for the separation of proteins and peptides by size- and ion-exchange modes of separation, respectively. In size-exclusion electrochromatograpgy, FITC-IgG and FITC-insulin were baseline resolved in less than 10 s, with efficiencies of up to 139,000 plates/m for FITC-insulin. In strong cation-exchange electrochromatography, a mixture of three fluorescently labeled peptides was baseline resolved in less than 40 s, with efficiencies up to 400,000 plates/m. The RSD for the analytes retention times were<3% in both size-exclusion and ion-exchange modes of separations. The use of a 1-mm-long reverse-phase column for the semiquantitative evaluation of pharmaceutical formulations in drug solubility tests illustrates the use of this microfluidic chip-based electrochromatographic approach to drug development.
Assuntos
Eletrocromatografia Capilar/instrumentação , Microfluídica/instrumentação , Resinas de Troca de Cátion , Preparações Farmacêuticas/químicaRESUMO
We introduce a single-phase flow microfluidic cell sorter with a two-point detection system capable of two-parameter screening to assist with directed evolution of a fluorescent protein based Ca2+ sensor expressed in bacterial cells. The new cell sorting system utilizes two fluorescence microscopes to obtain signals at two different points along a flow path in which a change in concentration of the analyte, Ca2+, is induced. The two detectors thus determine the magnitude of fluorescence change of the sensor following the reaction, along with the overall brightness of the sensor. A design for a 3D focusing flow was configured to enhance the spatial control of cells and signal pair-matching. The cell sorter screens the sensors at a moderate throughput, 10 cells per s and 105 cells per round, enriching top variants for the subsequent manual screening with higher accuracy. Our new µFACS greatly accelerates the directed evolution of genetically encoded Ca2+ sensors compared to the previous version with single point detection for brightness-based screening. Two rounds of directed evolution led to a variant, named Y-GECO2f, which exhibits a 26% increase in brightness and a greater than 300% larger Ca2+-dependent fluorescence change in vitro relative to the variant before evolution.
Assuntos
Cálcio/análise , Citometria de Fluxo , Técnicas Analíticas Microfluídicas , Fluorescência , Células HeLa , Humanos , Microscopia de Fluorescência , Células Tumorais CultivadasRESUMO
The fabrication of highly reliable and rugged fluidic chips designed for use in autonomous analyses for nutrient monitoring is described. The chips are based on a two-layer configuration with the fluidic channels produced in one layer using precision micromilling. The second capping layer contains through holes for sample/standard and reagent addition and waste removal post-analysis. Two optically clear polymethyl methacrylate (PMMA) windows are integrated into the opaque PMMA chip, orthogonal to a 22.5 mm-long section of the channel downstream from a serpentine reagent and sample/standard mixing region. An LED source is coupled into the channel through one of the windows, and the light intensity is monitored with a photodiode located at the distal end of the channel outside the second optically clear window. Efficient coupling of the source through the channel to the detector is achieved using custom-designed alignment units produced using 3D printing. In contrast to fluidic chips produced using solvent adhesion, the thermal-/pressure-bonded simplified method presented removes the need for surface treatment. Optimization of the thermal/pressure conditions leads to very strong adhesion between the PMMA layers, requiring forces in the region of 2000 N to separate them, which is necessary for the use in long-term deployments. Profilometry imaging shows minimal evidence of channel distortion after bonding. Finally, we show the potential of these techniques for environmental applications. The fluidic chips were integrated into prototype nutrient analyzers that display no evidence of leakage in extensive lab tests involving 2500 phosphate measurements using the yellow (vanadomolybdophosphoric acid) method. Similarly, excellent analytical performance (LOD is 0.09 µM) is reported for a 28-day field trial comprising 188 in situ autonomous phosphate measurements (564 measurements) in total including calibration.
RESUMO
We report fuel generation systems for molecular motors based on pyruvate kinase, or for the first time, mitochondria, implemented within microfluidic devices. Intact organelles acted as bio-nanopower supplies for molecular motors, using isolated mitochondria to convert chemical energy from succinate to ATP, harnessing nature's enzymatic transformation cascades directly. Motors were activated essentially equally by ATP produced by pyruvate kinase, mitochondria, or direct addition of ATP.
Assuntos
Trifosfato de Adenosina/metabolismo , Fontes Geradoras de Energia , Técnicas Analíticas Microfluídicas/métodos , Mitocôndrias/enzimologia , Nanotecnologia/métodos , Piruvato Quinase/metabolismo , Ácido Succínico/metabolismo , Trifosfato de Adenosina/química , Técnicas Analíticas Microfluídicas/instrumentação , Nanotecnologia/instrumentação , Piruvato Quinase/química , Ácido Succínico/químicaRESUMO
We have developed a series of yellow genetically encoded Ca2+ indicators for optical imaging (Y-GECOs) with inverted responses to Ca2+ and apparent dissociation constants (Kd') ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca2+ indicators, we expressed the four highest affinity variants (Kd's = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca2+ in layers 8-10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower Kd' was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.