Droplet delivery and nebulization system using surface acoustic wave for mass spectrometry.

We present a piezoelectric transducer for standing wave surface acoustic wave nebulization (SW-SAWN). The transducer nebulizes nonvolatile analytes present in bulk fluid into ambient air after which the aerosolized drops are sampled by mass spectrometry (MS) for detection. Furthermore, we report for the first time integration of anisotropic ratchet conveyors (ARCs) on the SAWN transducer surfaces to automate the sample preparation and droplet delivery process. The ARCs employ micro-sized hydrophilic patterns on hydrophobic Cytop coatings. Moving, positioning, merging, and mixing of droplets at a designated nebulization location are demonstrated. To create the ARCs, we adopt parylene C as a stencil mask so that the hydrophobicity of the Cytop does not degrade during the microfabrication process. MS measurements with the SAWN chip are performed under different input frequencies. The SAWN transducer can provide a controllable nebulization rate by varying the input nebulization frequency while maintaining a reasonable signal to noise ratio for MS detection.

Quantitative analysis of chondroitin sulfate disaccharides from human and rodent fixed brain tissue by electrospray ionization-tandem mass spectrometry.

Chondroitin sulfates (CS) are long, negatively charged, unbranched glycosaminoglycan (GAG) chains attached to CS-proteoglycan (CSPG) core proteins that comprise the glycan component in both loose interstitial extracellular matrices (ECMs) and in rigid, structured perineuronal net (PNN) scaffolds within the brain. As aberrant CS-PNN formations have been linked to a range of pathological states, including Alzheimer's disease (AD) and schizophrenia, the analysis of CS-GAGs in brain tissue at the disaccharide level has great potential to enhance disease diagnosis and prognosis. Two mass-spectrometry (MS)-based approaches were adapted to detect CS disaccharides from minute fixed tissue samples with low picomolar sensitivity and high reproducibility. The first approach employed a straightforward, quantitative direct infusion (DI)-tandem mass spectrometry (MS/MS) technique to determine the percentages of Δ4S- and Δ6S-CS disaccharides within the 4S/6S-CS ratio, while the second used a comprehensive liquid chromatography (LC)-MS/MS technique to determine the relative percentages of Δ0S-, Δ4S-, Δ6S-, Δ4S6S-CS and Δ2S6S-CS disaccharides, with internal validation by full chondroitin lyase activity. The quantitative accuracy of the five primary biologically relevant CS disaccharides was validated using a developmental time course series in fixed rodent brain tissue. We then analyzed the CS disaccharide composition in formalin-fixed human brain tissue, thus providing the first quantitative report of CS sulfation patterns in the human brain. The ability to comprehensively analyze the CS disaccharide composition from fixed brain tissue provides a means with which to identify alterations in the CS-GAG composition in relation to the onset and/or progression of neurological diseases.

Surface acoustic wave nebulization device with dual interdigitated transducers improves SAWN-MS performance.

We compared mass spectrometric (MS) performance of surface acoustic wave nebulization (SAWN) generated by a single interdigitated transducer (IDT) designed to produce a progressive wave (PW) to one with a dual IDT that can in theory generate standing waves (SW). Given that devices using dual IDTs had been shown to produce fewer large size droplets on average, we hypothesized they would improve MS performance by improving the efficiency of desolvation. Indeed, the SW-SAWN chip provided an improved limit of detection of 1 femtomole of peptide placed on chip making it 100× more sensitive than the PW design. However, as measured by high-speed image recording and phase Doppler particle analyzer measurements, there was only a 26% increase in the small diameter (1-10 µm) droplets produced from the new device, precluding a conclusion that the decrease in droplet size was solely responsible for the improvement in MS signal/noise. Given that the dual IDT design produced a more instantaneous plume than the PW design, the more likely contributor to improved MS signal/noise was concluded to be a higher ion flux entering the mass spectrometer for the dual IDT designs. Notably, the dual IDT device allowed production of much higher quality protein mass spectra up to about 20 kDa, compared with the single IDT device. Copyright © 2016 John Wiley & Sons, Ltd.

Use of captive spray ionization to increase throughput of the data-independent acquisition technique PAcIFIC.

RATIONALE: The Precursor Acquisition Independent From Ion Count (PAcIFIC) method is a data-independent acquisition technique capable of identifying proteins over eight orders of magnitude in a single analysis in human plasma. Widespread application of this technique in proteomic studies is hindered by its time-intensive nature. There exists a need to explore strategies to increase the throughput of the PAcIFIC method. METHODS: The PAcIFIC acquisition technique was optimized for use with an Orbitrap mass spectrometer fitted with a captive spray ionization (CSI) source. Chromatographic methods, PAcIFIC acquisition parameters, for example, channels interrogated per chromatographic gradient, time span of chromatographic gradient, and sample loading amount, were investigated to achieve a maximum number of peptide and protein identifications on a yeast proteome where protein copy number had been previously determined. RESULTS: A 24-hour CSI PAcIFIC method was developed with minimal reduction of peptide and protein identifications from the 4.2-day nano-electrospray ionization (nESI) PAcIFIC method. Analysis of a yeast cell lysate with the 4.2-day nESI PAcIFIC method resulted in 13,468 peptide and 2120 protein identifications. A 24-hour CSI PAcIFIC method resulted in 11,277 peptide and 1753 protein identifications. Increased sample loading of the CSI system allowed for an 8% increase in peptide and protein identifications. CONCLUSIONS: A dramatic decrease in the overall analysis time of the PAcIFIC method (24 h with CSI versus 100.8 h with nESI) was achieved with minimal reduction of peptide and protein identifications. Furthermore, the CSI PAcIFIC method demonstrated a high degree of sensitivity and capability of identifying proteins across a large dynamic range observed with the nESI PAcIFIC method (CSI PAcIFIC identified proteins as low as 46 molecules per cell).

Screen-printed digital microfluidics combined with surface acoustic wave nebulization for hydrogen-deuterium exchange measurements.

An inexpensive digital microfluidic (DMF) chip was fabricated by screen-printing electrodes on a sheet of polyimide. This device was manually integrated with surface acoustic wave nebulization (SAWN) MS to conduct hydrogen/deuterium exchange (HDX) of peptides. The HDX experiment was performed by DMF mixing of one aqueous droplet of angiotensin II with a second containing various concentrations of D2O. Subsequently, the degree of HDX was measured immediately by SAWN-MS. As expected for a small peptide, the isotopically resolved mass spectrum for angiotensin revealed that maximum deuterium exchange was achieved using 50% D2O. Additionally, using SAWN-MS alone, the global HDX kinetics of ubiquitin were found to be similar to published NMR data and back exchange rates for the uncooled apparatus using high inlet capillary temperatures was less than 6%.

Surface acoustic wave nebulization facilitating lipid mass spectrometric analysis.

Surface acoustic wave nebulization (SAWN) is a novel method to transfer nonvolatile analytes directly from the aqueous phase to the gas phase for mass spectrometric analysis. The lower ion energetics of SAWN and its planar nature make it appealing for analytically challenging lipid samples. This challenge is a result of their amphipathic nature, labile nature, and tendency to form aggregates, which readily precipitate clogging capillaries used for electrospray ionization (ESI). Here, we report the use of SAWN to characterize the complex glycolipid, lipid A, which serves as the membrane anchor component of lipopolysaccharide (LPS) and has a pronounced tendency to clog nano-ESI capillaries. We also show that unlike ESI SAWN is capable of ionizing labile phospholipids without fragmentation. Lastly, we compare the ease of use of SAWN to the more conventional infusion-based ESI methods and demonstrate the ability to generate higher order tandem mass spectral data of lipid A for automated structure assignment using our previously reported hierarchical tandem mass spectrometry (HiTMS) algorithm. The ease of generating SAWN-MS(n) data combined with HiTMS interpretation offers the potential for high throughput lipid A structure analysis.

Surface acoustic wave nebulization produces ions with lower internal energy than electrospray ionization.

Surface acoustic wave nebulization (SAWN) has recently been reported as a novel method to transfer non-volatile analytes directly from solution to the gas phase for mass spectrometric analysis. Here we present a comparison of the survival yield of SAWN versus electrospray ionization (ESI) produced ions. A series of substituted benzylpyridinium (BzPy) compounds were utilized to measure ion survival yield from which ion energetics were inferred. We also estimated bond dissociation energies using higher level quantum chemical calculations than previously reported for BzPy ions. Additionally, the effects on BzPy precursor ion survival of SAWN operational parameters such as inlet capillary temperature and solution flow-rate were investigated. Under all conditions tested, SAWN-generated BzPy ions displayed a higher tendency for survival and thus have lower internal energies than those formed by ESI.

A new USP Class VI-compliant substrate for manufacturing disposable microfluidic devices.

As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification. We report two production processes that are compatible with the existing methods of rapid prototyping and present characterizations of the resultant PUMA microfluidic devices.

Compartmentalization of chemically separated components into droplets.

Not merely a drop in the ocean: The integration of capillary electrophoresis (CE) with droplet generation driven by electroosmotic flow enabled the compartimentalization of molecular components separated by CE in a series of droplets (see picture; the green bars represent the separated analytes). The droplet-confined bands can be docked and studied on a chip.

Quantitative force mapping of an optical vortex trap.

This paper describes the quantitative force mapping of micron-sized particles held in an optical vortex trap. We present a simple and efficient model, which accounts for the diffraction of the strongly localized optical field of the tightly focused laser beam, the spherical aberration introduced by the dielectric glass-to-water interface, employs the multidipole approximation for force calculations, and is able to reproduce, with quantitative agreement, the experimentally measured force map.

Spin-to-orbital angular momentum conversion in a strongly focused optical beam.

As a fundamental property of light, the angular momentum of photons has been of great interest. Here, we demonstrate that optical spin-to-orbital angular momentum conversion can occur in a homogeneous and isotropic medium. This Letter presents both theoretical and experimental studies of this conversion in a tightly focused beam and shows that the orbital rotation speeds of trapped particles are altered because of this conversion as predicted by theory.

Using polarization-shaped optical vortex traps for single-cell nanosurgery.

Single-cell nanosurgery and the ability to manipulate nanometer-sized subcellular structures with optical tweezers has widespread applications in biology but so far has been limited by difficulties in maintaining the functionality of the transported subcellular organelles. This difficulty arises because of the propensity of optical tweezers to photodamage the trapped object. To address this issue, this paper describes the use of a polarization-shaped optical vortex trap, which exerts less photodamage on the trapped particle than conventional optical tweezers, for carrying out single-cell nanosurgical procedures. This method is also anticipated to find broad use in the trapping of any nanoparticles that are adversely affected by high-intensity laser light.

Vortex-trap-induced fusion of femtoliter-volume aqueous droplets.

This paper describes the use of an optical vortex trap for the transport and fusion of single femtoliter-volume aqueous droplets. Individual droplets were generated by emulsifying water in acetophenone with SPAN 80 surfactant. We demonstrate the ability of optical vortex traps to position trapped droplets precisely while excluding surrounding aqueous droplets from entering the trap, thereby preventing unwanted cross contamination by other nearby droplets. Additionally, the limitation of optical vortex traps for inducing droplet fusion is illustrated, and a remedy is provided through modulation of the spatial intensity profile of the optical vortex beam. Spatial modulation was achieved by translating the computer-generated hologram (CGH) with respect to the input Gaussian beam, thereby shifting the location of the embedded phase singularity (dark core) within the optical vortex beam. We present both simulated and experimentally measured intensity profiles of the vortex beam caused by translation of the CGH. We further describe the use of this technique to achieve controlled and facile fusion of two aqueous droplets.

Capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis.

This paper demonstrates the ability to use capillary electrophoresis (CE) separation coupled with laser-induced fluorescence for analyzing the contents of single femtoliter-volume aqueous droplets. A single droplet was formed using a T-channel (3 microm wide by 3 microm tall) connected to microinjectors, and then the droplet was fluidically moved to an immiscible boundary that isolates the CE channel (50 microm wide by 50 microm tall) from the droplet generation region. Fusion of the aqueous droplet with the immiscible boundary effectively injects the droplet content into the separation channel. In addition to injecting the contents of droplets, we found aqueous samples can be introduced directly into the separation channel by reversibly penetrating and resealing the immiscible partition. Because droplet generation in channels requires hydrophobic surfaces, we have also investigated the advantages to using all hydrophobic channels versus channel systems with patterned hydrophobic and hydrophilic regions. To fabricate devices with patterned surface chemistry, we have developed a simple strategy based on differential wetting to deposit selectively a hydrophilic polymer (poly(styrenesulfonate)) onto desired regions of the microfluidic chip. Finally, we applied our device to the separation of a simple mixture of fluorescein-labeled amino acids contained within a approximately 10-fL droplet.

Microfluidic and optical systems for the on-demand generation and manipulation of single femtoliter-volume aqueous droplets.

This paper describes a fluidic and optical platform for the generation and manipulation of single femtoliter-volume aqueous droplets. Individual droplets were generated on-demand using a microfluidic chamber that confers environmental flow stability. Optical vortex traps were implemented to manipulate and transport the generated droplets, which have a lower refractive index than the immiscible medium in which the droplets are immersed and thus cannot be trapped using conventional optical tweezers. We also demonstrated the ability to shrink and increase the refractive index of the generated droplet, thereby permitting its facile fusion with another droplet using an optical tweezer. To illustrate the versatility of this platform, we have performed both fast (<1 s) and slow (>1 h) chemical reactions in these femtoliter-volume aqueous droplets.

Monitoring cell survival after extraction of a single subcellular organelle using optical trapping and pulsed-nitrogen laser ablation.

This paper characterizes cell viability in three different cell lines--Chinese hamster ovary cells (CHO), neuroblastoma cells fused with glialoma cells (NG108-15) and murine embryonic stem cells (ES-D3)--after N2 laser disruption of the cell membrane and removal, via optical trapping, of a single subcellular organelle. Morphological changes and viability (as determined by live/dead fluorescent stains) of the cell were monitored every half hour over a 4-h period postsurgery. The ability of the cell to survive organelle extraction was found to depend both on the conditions under which surgery was performed and on the cell type. The average viability after surgery for CHO cells was approximately 80%, for NG 108 cells it was approximately 30% and for ES-D3 cells postsurgery viability was approximately 10%. From over 600 surgeries we found the survival of the cell is determined almost exclusively within the first hour postsurgery regardless of cell line. The optimal pulse energy for N2 laser ablation was approximately 0.7 microJ. The N2 pulse produced an approximately 1-3 microm hole in the cell membrane and proved to be the primary source of cell death in those cells that did not survive the procedure.

Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets.

This paper describes a method, which combines optical trapping and microfluidic-based droplet generation, for selectively and controllably encapsulating a single target cell or subcellular structure, such as a mitochondrion, into a picoliter- or femtoliter-volume aqueous droplet that is surrounded by an immiscible phase. Once the selected cell or organelle is encased within the droplet, it is stably confined in the droplet and cannot be removed. We demonstrate in droplet the rapid laser photolysis of the single cell, which essentially "freezes" the state that the cell was in at the moment of photolysis and confines the lysate within the small volume of the droplet. Using fluorescein di-beta-d-galactopyranoside, which is a fluorogenic substrate for the intracellular enzyme beta-galactosidase, we also assayed the activity of this enzyme from a single cell following the laser-induced lysis of the cell. This ability to entrap individual selected cells or subcellular organelles should open new possibilities for carrying out single-cell studies and single-organelle measurements.