In this work, two methods with complementary features, catalytic and molecular beacon (CAMB) and label-free fluorescent sensors using abasic site, have been combined into new label-free CAMB sensors that possess advantages of each method. The label-free method using dSpacer-containing molecular beacon makes CAMB more cost-effective and less interfering to the catalytic activity, while the CAMB allows the label-free method to use true catalytic turnovers for signal amplifications, resulting in a new label-free CAMB sensor for Pb2+ ion, with a detection limit of 3.8 nM while maintaining the same selectivity. Furthermore, by using CAMB to overcome the label-free method’s limitation of the requiring excess enzyme strand, a new label-free CAMB sensor using aptazyme is also designed to detect adenosine down to 1.4 μM, with excellent selectivity over other nucleosides.
Despite the intrinsic elemental analysis capability and lack of sample preparation requirements, laser-induced breakdown spectroscopy (LIBS) has not been extensively used for real world applications, e.g. quality assurance and process monitoring. Specifically, variability in sample, system and experimental parameters in LIBS studies present a substantive hurdle for robust classification, even when standard multivariate chemometric techniques are used for analysis. Considering pharmaceutical sample investigation as an example, we propose the use of support vector machines (SVM) as a non-linear classification method over conventional linear techniques such as soft independent modeling of class analogy (SIMCA) and partial least-squares discriminant analysis (PLS-DA) for discrimination based on LIBS measurements. Using over-the-counter pharmaceutical samples, we demonstrate that application of SVM enables statistically significant improvements in prospective classification accuracy (sensitivity), due to its ability to address variability in LIBS sample ablation and plasma self-absorption behavior. Furthermore, our results reveal that SVM provides nearly 10% improvement in correct allocation rate and a concomitant reduction in misclassification rates of 75% (cf. PLS-DA) and 80% (cf. SIMCA)-when measurements from samples not included in the training set are incorporated in the test data – highlighting its robustness. While further studies on a wider matrix of sample types performed using different LIBS systems is needed to fully characterize the capability of SVM to provide superior predictions, we anticipate that the improved sensitivity and robustness observed here will facilitate application of the proposed LIBS-SVM toolbox for screening drugs and detecting counterfeit samples as well as in related areas of forensic and biological sample analysis.
Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for biological sample extraction; however, the presence of this reagent in samples challenges LC-MS-based proteomics analyses because it can interfer with reversed-phase LC separations and electrospray ionization. This study reports a simple SDS-assisted proteomics sample preparation method facilitated by a novel peptide-level SDS removal step. In an initial demonstration, SDS was effectively (>99.9%) removed from peptide samples through ion substitution-mediated DS- precipitation using potassium chloride (KCl), and excellent peptide recovery (>95%) was observed for <20 μg peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage obtained for both mammalian tissues and bacterial samples was comparable to or better than that obtained for the same sample types prepared using standard proteomics preparation methods and analyzed using LC-MS/MS. These results suggest the SDS-assisted protocol is a practical, simple, and broadly applicable proteomics sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.
SDS removal; KDS precipitation; proteomics; sample preparation; LC-MS
We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g., in vivo cross-linking) is illustrated by Western blot detection of Cul1 complexes including known binders, Cand1 and Skp2, in HEK 293 cells, confirming good water solubility and cell-permeability.
Chemical Cross-linkers; Cross-linked Peptide; Protein Structure; Protein-Protein Interaction; Mass Spectrometry; Copper-Catalyzed Azide-Alkyne Cycloaddition; Reporter Ion
Heterogeneous conductance of individual pores on a porous membrane was studied with a four-electrode scanning ion conductance microscope (SICM). Application of a potential difference across the membrane resulted in migration of ions through nanopores, where subsequent conductance changes were measured by a nanopipet positioned above the nanopore as a change in pipet current. Current responses of single-pore membrane and individual pores within a multi-pore membrane were examined and demonstrated variations in ion current rectification (ICR) ratios due to the small differences in pore geometries.
We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD – coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA – is part of a complete workflow that includes the formation, interrogation and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2,000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions, and a tendency to produced lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tended to have lower pI values. As such NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods.
MS detection coupled with digital microfluidic (DMF) devices has only been demonstrated in an off-line manner using matrix assisted laser desorption ionization. In this work, an eductor is demonstrated which facilitated online coupling of DMF with electrospray ionization MS detection. The eductor consisted of a transfer capillary, a standard ESI needle, and a tapered gas nozzle. As a pulse of N2 was applied to the nozzle, a pressure differential was induced at the outlet of the ESI needle that pulled droplets from the DMF, past the ESI needle, and into the flow of gas exiting the nozzle, allowing detection by MS. Operating position, ionization potential, and N2 pressure were optimized, with the optimum ionization potential and N2 pressure found to be 3206 V and 80 psi, respectively. Online MS detection was demonstrated from both open and closed DMF devices using 2.5 μL and 630 nL aqueous droplets, respectively. Relative quantitation by DMF-MS was demonstrated by mixing droplets of caffeine with droplets of theophylline on an open DMF device, and comparing the peak area ratio obtained to an on-chip generated calibration curve. This eductor-based method for transferring droplets has the potential for rapid, versatile, and high throughput microfluidic analyses.
electrowetting; Venturi; droplet; atmospheric pressure ionization
IR-mediated PCR in microdevices is an established technique for rapid amplification of nucleic acids. In this report, we have expanded the applicability of IR-PCR to quantitative determination of starting copy number by integrating fluorescence detection during the amplification process. Placing the microfluidic device between an IR longpass filter and a hot mirror reduced the background to a level that enabled fluorescence measurements to be made throughout the thermal cycling process. The average fluorescence intensity during the extension step showed the expected trend of an exponential increase followed by a plateau phase in successive cycles. PUC19 template at different starting copy numbers were amplified and the threshold cycle showed an increase for decreasing amounts of starting DNA. Amplification efficiency was 80% and gel separation indicated no detectable non-specific product. A melting curve was generated using IR heating and indicated a melting temperature of 85 °C for the 304 bp amplicon which compared well to the melting temperature obtained using a conventional PCR system. This methodology will be applicable in other types of IR-mediated amplification systems, such as isothermal amplification and in highly integrated systems that combine pre- and post-PCR processes.
qPCR; IR; fluorescence; microchip
Fabrication of carbon microelectrode arrays, with up to 15 electrodes in total tips as small as 10 to 50 μm, is presented. The support structures of microelectrodes were obtained by pulling multiple quartz capillaries together to form hollow capillary arrays before carbon deposition. Carbon ring microelectrodes were deposited by pyrolysis of acetylene in the lumen of these quartz capillary arrays. Each carbon deposited array tip was filled with epoxy, followed by beveling of the tip of the array to form a deposited carbon-ring microelectrode array (CRMA). Both the number of the microelectrodes in the array and the tip size are independently tunable. These CRMAs have been characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and electrogenerated chemiluminescence. Additionally, the electrochemical properties were investigated with steady-state voltammetry. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, CRMAs containing eight microring electrodes were used for electrochemical monitoring of exocytotic events from single PC12 cells. Subcellular temporal heterogeneities in exocytosis (ie. cold spots vs. hot spots) were successfully detected with the CRMAs.
microelectrode arrays; single cell; electrochemical imaging; PC12 cells
Improvements in the mass accuracy and resolution of mass spectrometers have greatly aided mass spectrometry-based proteomics in profiling complex biological mixtures. With the use of innovative bioinformatics approaches, high mass accuracy and resolution information can be used for filtering chemical noise in mass spectral data. Using our recent algorithmic developments, we have generated the mass distributions of all theoretical tryptic peptides composed of twenty natural amino acids and with masses limited to 3.5 kDa. Peptide masses are distributed discretely, with well defined peak clusters separated by empty or sparsely populated trough regions. Accurate models for peak centers and widths can be used to filter peptide signals from chemical noise. We modeled mass defects, the difference between monoisotopic and nominal masses, peak centers and widths in the peptide mass distributions. We found that peak widths encompassing 95% of all peptide sequences are substantially smaller than previously thought. The result has implications for filtering out larger stretches of the mass axis. Mass defects of peptides exhibit an oscillatory behavior which is damped at high mass values. The periodicity of the oscillations is about 14 Da which is the most common difference between the masses of the twenty natural amino acids.
To determine the effects of amino acid modifications on our findings, we examined the mass distributions of peptides composed of the twenty natural amino acids, oxidized Met and phosphorylated Ser, Thr and Tyr. We found that extension of the amino acid set by modifications increases the 95% peak width. Mass defects decrease, reflecting the fact that the average mass defect of natural amino acids is larger than that of oxidized Met. We propose that a new model for mass defects and peak widths of peptides may improve peptide identifications by filtering chemical noise in mass spectral data.
peptide mass distributions; forbidden zones; mass defect; noise filtering; power spectrum; high mass accuracy and resolution
Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by co-incubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least six orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.
This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase ALP, and aspartate aminotransferase AST) and total serum protein. A device consists of four components: i) a top plastic sheet, ii) a filter membrane, iii) a patterned paper chip containing the reagents necessary for analysis, and iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and “spiked” standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.
Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated based on their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations.
In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the non-enzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and non-glycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision - with a limit of detection that is nearly a factor of 15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multi-component mixtures and potentially in hemolysate and whole blood lysate samples.
Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions in an entire organ or animal through registration and stacking of serial tissue sections. Here we review the current state of 3D imaging mass spectrometry as well as provide insights and perspectives on the process of generating 3D mass spectral data along with a discussion of the process necessary to generate a 3D image volume.
Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the last few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science has allowed increased numbers of proteins to be identified, especially by nano-liquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that about ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC ESI FT-ICR MS). The separation platform reduced the complexity of the protein matrix so that at 14.5 Tesla, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.
Fourier transform mass spectrometry; ion cyclotron resonance; FTMS; FT-ICR; top-down proteomics
The stoichiometry of protein phosphorylation significantly impacts protein function. The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor the regulation levels of various proteins. This study reports an integrated methodology using cerium oxide nanoparticles and isobaric tandem mass tag (TMT) labeling to assess absolute stoichiometries of protein phosphorylation. This protocol was designed to directly measure the dephosphorylation levels for a known phosphorylation site therefore allowing for quantification of phosphosites. Both the accuracy and precision of the method were verified using standard peptides and protein tryptic digests. This novel method was then applied to quantify phosphorylations on eukaryotic initiation factor 3H (eIF3H), a protein integral to overall eukaryotic protein translation initiation. To date, this is the first report of assessment of protein phosphorylation quantification on eIF3.
Iontophoresis uses electricity to deliver solutes into living tissue. Often, iontophoretic ejections from micropipettes into brain tissue are confined to millisecond pulses for highly localized delivery, but longer pulses are common. As hippocampal tissue has a ζ-potential of approximately –22 mV, we hypothesized that, in the presence of the electric field resulting from the iontophoretic current, electroosmotic flow in the tissue would carry solutes considerably farther than diffusion alone. A steady state solution to this mass transport problem predicts a spherically symmetrical solute concentration profile with the characteristic distance of the profile depending on the ζ-potential of the medium, the current density at the tip, the tip size and the solute electrophoretic mobility and diffusion coefficient. Of course, the ζ-potential of the tissue is defined by immobilized components of the extracellular matrix as well as cell-surface functional groups. As such, it cannot be changed at will. Therefore, the effect of the ζ-potential of the porous medium on ejections is examined using poly(acrylamide-co-acrylic acid) hydrogels with various magnitudes of ζ-potential, including that similar to hippocampal brain tissue. We demonstrated that nearly neutral fluorescent dextran (3 and 70 kD) solute penetration distance in the hydrogels and OHSCs depends on the magnitude of the applied current, solute properties, and, in the case of the hydrogels, the ζ-potential of the matrix. Steady state solute ejection profiles can be predicted semi-quantitatively.
Electroosmosis; Peclet number; Mass transport; Hippocampus; Microiontophoresis
The quantitative analysis of complex biological samples has emerged as a key research area in the field of proteomics. Although quantitative proteomic experiments remain challenging, these strategies have been greatly facilitated by the development of newer high-performance mass spectrometers. In this work, we have evaluated the use of the LTQ-Orbitrap, a hybrid mass spectrometer in which a linear ion trap is coupled to an Orbitrap mass analyzer, for quantitative analyses. By analyzing a range of yeast protein standards, we found that the high mass accuracy, high resolution, large ion capacity, and large dynamic range of the LTQ-Orbitrap led to a significant improvement in the number and quality of the peptide ratio measurements compared to similar analyses done on the LTQ. We also successfully quantified protein expression differences that occur in metabolically labeled rat synapses during brain development to further demonstrate the suitability of the LTQ-Orbitrap for the comparative analysis of complex tissue samples.
Mass Spectrometric Imaging (MSI) has become widely used in the analysis of a variety of biological surfaces. Biological samples are spatially, morphologically, and metabolically complex. Multimodal molecular imaging is an emerging approach that is capable of dealing with this complexity. In a multimodal approach, different imaging modalities can provide precise information about the local molecular composition of the surfaces. Images obtained by MSI can be co-registered with images obtained by other molecular imaging techniques such as microscopic images of fluorescent protein expression or histologically stained sections. In order to properly co-register images from different modalities, each tissue section must contain points of reference, which are visible in all data sets. Here, we report a newly developed co-registration technique using fiducial markers such as cresyl violet, Ponceau S, and bromophenol blue that possess a combination of optical and molecular properties that result in a clear mass spectrometric signature. We describe these fiducial markers and demonstrate an application that allows accurate co-registration and 3-dimensional reconstruction of serial histological and fluorescent microscopic images with MSI images of thin tissue sections from a breast tumor model.
Simultaneous stimulation of ex vivo pancreatic islets with dynamic oxygen and glucose is a critical technique for studying how hypoxia alters glucose-stimulated response, especially in transplant environments. Standard techniques using a hypoxic chamber cannot provide both oxygen and glucose modulations while monitoring stimulus-secretion coupling factors in real-time. Using novel microfluidic device with integrated glucose and oxygen modulations, we quantified hypoxic impairment of islet response by calcium influx, mitochondrial potentials, and insulin secretion. Glucose-induced calcium response magnitude and phase were suppressed by hypoxia, while mitochondrial hyperpolarization and insulin secretion decreased in coordination. More importantly, hypoxic response was improved by preconditioning islets to intermittent hypoxia (IH, 1min/1min 5%–21% cycling for 1 hour), translating to improved insulin secretion. Moreover, blocking mitochondrial KATP channels removed preconditioning benefits of IH, similar to mechanisms in preconditioned cardiomyocytes. Additionally, the multimodal device can be applied to a variety of dynamic oxygen-metabolic studies in other ex vivo tissues.
DNA detection is commonly used in molecular biology, pathogen analysis, genetic disorder diagnosis and forensic tests. While traditional methods for DNA detection such as polymerase chain reaction (PCR) and DNA microarrays have been well developed, they require sophisticated equipment and operations and thus it is still challenging to develop a portable and quantitative DNA detection method for the public use at home or in the field. Although many other techniques and devices have been reported to make the DNA detection simple and portable, very few of them are currently accessible to the public for quantitative DNA detection because of either the requirement of laboratory-based instrument or lack of quantitative detection.. Herein we report application of personal glucose meters (PGMs), which are widely available, low cost and simple to use, for quantitative detection of DNA, including a hepatitis B virus DNA fragment. The quantification is based on target-dependent binding of complementary DNA-invertase conjugate with the analyte DNA, thereby transforming the concentration of DNA in the sample into glucose through invertase-catalyzed hydrolysis of sucrose. Instead of amplifying DNA strands through PCR, which is vulnerable to contaminations commonly encountered for home and field usage, we demonstrate here signal amplifications based on enzymatic turnovers, making it possible to detect 40 pM DNA using PGM that can detect glucose only at mM level. The method also shows excellent selectivity toward single nucleotide mismatches.
We demonstrate an affordable low-noise SPR instrument based on extraordinary optical transmission (EOT) in metallic nanohole arrays and quantify a broad range of antibody-ligand binding kinetics with equilibrium dissociation constants ranging from 200 pM to 40 nM. This nanohole-based SPR instrument is straightforward to construct, align, and operate, since it is built around a standard microscope and a portable fiber-optic spectrometer. The measured refractive index resolution of this platform is 3.1 × 10−6 without on-chip cooling, which is among the lowest reported for SPR sensors based on EOT. This is accomplished via rapid full-spectrum acquisition in 10 milliseconds followed by frame averaging of the EOT spectra, which is made possible by the production of template-stripped gold nanohole arrays with homogeneous optical properties over centimeter-sized areas. Sequential SPR measurements are performed using a 12-channel microfluidic flow cell after optimizing surface modification protocols and antibody injection conditions to minimize mass-transport artifacts. The immobilization of a model ligand, the protective antigen of anthrax on the gold surface, is monitored in real-time with a signal-to-noise ratio of ~860. Subsequently, real-time binding kinetic curves were measured quantitatively between the antigen and a panel of small, 25 kDa single-chain antibodies at concentrations down to 1 nM. These results indicate that nanohole-based SPR instruments have potential for quantitative antibody screening and as a general-purpose platform for integrating SPR sensors with other bioanalytical tools.
Surface plasmon resonance; plasmonics; extraordinary optical transmission; nanohole arrays; template stripping; recombinant antibody; microfluidics
Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry.
We describe the development and characterization of pneumatically actuated “lifting gate” microvalves and pumps. A fluidic layer containing the gate structure and a pneumatic layer are fabricated by soft-lithography in PDMS and bonded permanently with an oxygen plasma treatment. The microvalve structures are then reversibly bonded to a featureless glass or plastic substrate to form hybrid glass-PDMS and plastic-PDMS microchannel structures. The breakthrough pressures of the microvalve increase linearly up to 65 kPa as the closing pressure increases. The pumping capability of these structures ranges from the nanoliter to microliter scale depending on the number of cycles and closing pressure employed. The micropump structures exhibit up to 86.2% pumping efficiency from flow rate measurements. The utility of these structures for integrated sample processing is demonstrated by performing an automated immunoassay. These lifting gate valve and pump structures enable facile integration of complex microfluidic control systems with a wide range of lab-on-a-chip substrates.
microvalves; micropump; soft-lithography; microfluidics; automated immunoassay; lab-on-a-chip