3D printer platform and conductance feedback loop for automated imaging of uneven surfaces by liquid microjunction-surface sampling probe mass spectrometry.

RATIONALE: Molecular imaging of samples using mass spectrometric techniques, such as matrix-assisted laser desorption ionization or desorption electrospray ionization, requires the sample surface to be even/flat and sliced into thin sections (c. 10 µm). Furthermore, sample preparation steps can alter the analyte composition of the sample. The liquid microjunction-surface sampling probe (LMJ-SSP) is a robust sampling interface that enables surface profiling with minimal sample preparation. In conjunction with a conductance feedback system, the LMJ-SSP can be used to automatically sample uneven specimens. METHODS: A sampling stage was built with a modified 3D printer where the LMJ-SSP is attached to the printing head. This setup can scan across flat and even surfaces in a predefined pattern ("static sampling mode"). Uneven samples are automatically probed in "conductance sampling mode" where an electric potential is applied and measured at the probe. When the probe contacts the electrically grounded sample, the potential at the probe drops, which is used as a feedback signal to determine the optimal position of the probe for sampling each location. RESULTS: The applicability of the probe/sensing system was demonstrated by first examining the strawberry tissue using the "static sampling mode." Second, porcine tissue samples were profiled using the "conductance sampling mode." With minimal sample preparation, an area of 11 × 15 mm was profiled in less than 2 h. From the obtained results, adipose areas could be distinguished from non-adipose parts. The versatility of the approach was further demonstrated by directly sampling the bacteria colonies on agar and resected human kidney (intratumoral hemorrhage) specimens with thicknesses ranging from 1 to 4 mm. CONCLUSION: The LMJ-SSP in conjunction with a conductive feedback system is a powerful tool that allows for fast, reproducible, and automated assessment of uneven surfaces with minimal sample preparation. This setup could be used for perioperative assessment of tissue samples, food screening, and natural product discovery, among others.

Fabrication and characterization of laser-heated, multiplexed electrospray emitter.

We present a one-step fabrication method for a new multiplexed electrospray emitter with nine parallel micronozzles. The nozzles were formed by wet chemical etching of the end of a microstructured silica fiber containing nine 10 µm flow channels. By carefully adjusting the water flow through the channels while etching, we controlled the shape of the conical micronozzles and were able to obtain conditions under which the micronozzles, together with the flow channels, formed optical micro-axicon lenses. When 1064 nm light was guided through the flow channels and focused by the micro-axicon lenses into the Taylor cones, we were able to increase the desolvation of a model analyte and thereby increased the spray current produced by the emitter. This work paves the way towards a rapidly modulated mass-spectrometry source having a greatly enhanced throughput.

Ice recrystallization inhibition activity varies with ice-binding protein type and does not correlate with thermal hysteresis.

Ice-binding proteins (IBPs) inhibit the growth of ice through surface adsorption. In some freeze-resistant fishes and insects, circulating IBPs serve as antifreeze proteins to stop ice growth by lowering the freezing point. Plants are less able to avoid freezing and some use IBPs to minimize the damage caused in the frozen state by ice recrystallization, which is the growth of large ice grains at the expense of small ones. Here we have accurately and reproducibly measured the ice recrystallization inhibition (IRI) activity of over a dozen naturally occurring IBPs from fishes, insects, plants, and microorganisms using a modified 'splat' method on serial dilutions of IBPs whose concentrations were determined by amino acid analysis. The endpoint of IRI, which was scored as the lowest protein concentration at which no recrystallization was observed, varied for the different IBPs over two orders of magnitude from 1000 nM to 5 nM. Moreover, there was no apparent correlation between their IRI levels and reported antifreeze activities. IBPs from insects and fishes had similar IRI activity, even though the insect IBPs are typically 10x more active in freezing point depression. Plant IBPs had weak antifreeze activity but were more effective at IRI. Bacterial IBPs involved in ice adhesion showed both strong freezing point depression and IRI. Two trends did emerge, including that basal plane binding IBPs correlated with stronger IRI activity and larger IBPs had higher IRI activity.

An investigation into the kinematics of magnetically driven droplets on various (super)hydrophobic surfaces and their application to an automated multi-droplet platform.

Magnetic actuation on digital microfluidic (DMF) platforms may provide a low-cost, less cumbersome alternative for droplet manipulation in comparison to other techniques such as electrowetting-on-dielectric. Precise control of droplets in magnetically driven DMF platforms is achieved using a low-friction surface, magnetically susceptible material/droplet(s), and an applied magnetic field. Superhydrophobic (SH) surfaces offer limited friction for aqueous media as defined by their high water contact angles (WCA) (>150°) and low sliding angles (<10°). The low surface friction of such coatings and materials significantly reduces the force required for droplet transport. Here, we present a study that examines several actuation parameters including the effects of particle and particle-free actuation mechanisms, porous and non-porous SH materials, surface chemistry, droplet speed/acceleration, and the presence of surface energy traps (SETs) on droplet kinematics. Automated actuation was performed using an XY linear stepper gantry, which enabled sequential droplet actuation, mixing, and undocking operations to be performed in series. The results of this study are applied to a quantitative fluorescence-based DNA assay in under 2 min. Graphical abstract ᅟ.

High-capacity ice-recrystallization endpoint assay employing superhydrophobic coatings that is equivalent to the 'splat' assay.

We have developed an ice recrystallization inhibition (IRI) assay system that allows the side-by-side comparison of up to a dozen samples treated in an identical manner. This system is ideal for determining, by serial dilution, the IRI 'endpoint' where the concentration of a sample is reached that can no longer inhibit recrystallization. Samples can be an order of magnitude smaller in volume (<1 µL) than those used for the conventional 'splat' assay. The samples are pipetted into wells cut out of a superhydrophobic coating on sapphire slides that are covered with a second slide and then snap-frozen in liquid nitrogen. Sapphire is greatly superior to glass in its ability to cool quickly without cracking. As a consequence, the samples freeze evenly as a multi-crystalline mass. The ice grain size is slightly larger than that obtained by the 'splat' assay but can be followed sufficiently well to assess IRI activity by changes in mean grain boundary size. The slides can be washed in detergent and reused with no carryover of IRI activity even from the highest protein concentrations.

"Particle-Free" Magnetic Actuation of Droplets on Superhydrophobic Surfaces Using Dissolved Paramagnetic Salts.

Magnetic actuation is a droplet manipulation mechanism in digital microfluidics (DMF), where droplets can be actuated over a (super)hydrophobic surface with a magnetic force. Superparamagnetic particles or ferromagnetic liquids are added to the droplets to provide a "handle" by which the magnet can exert a force on the droplet. In this study, we present a novel method of magnetic manipulation, where droplets instead contain paramagnetic salts with molar magnetic susceptibilities (χm) approximately ≈10 000× < that for superparamagnetic particles. Droplet actuation is facilitated by low surface friction on fluorous silica nanoparticle-based superhydrophobic coatings, where <2 µN is required for reproducible droplet actuation. Different paramagnetic salts with χm from ≈4500 to 72 000 (× 10-6 cm3 mol-1) were used to make aqueous solutions of different concentration and tested for droplet actuation and sliding angle using permanent magnets (1.8-2.1 kG). Paramagnetic salts are compared in terms of solubility, minimum required concentration, and maximum droplet velocity before disengagement. There is a strong correlation between the magnetic susceptibility of the salt solution, its concentration, and ease of actuation. As an application example, droplets containing a paramagnetic salt and doxorubicin (leukemia drug) are magnetically actuated and interrogated using laser-induced fluorescence. Signal attenuation due to the MnCl2 salt was examined, and the Stern-Volmer quenching constant was determined.

Polymer micronozzle array for multiple electrosprays produced by templated synthesis and etching of microstructured fibers.

Microstructured fibers (MSFs) having raised polymer nozzles in each channel are custom designed, fabricated, and tested for use as multiple electrospray (MES) emitters for mass spectrometry (MS). There is strong motivation to develop electrospray emitters that operate at practical flow rates but give the much greater ionization efficiency associated with lower (nano) flow rates. This can be accomplished by splitting the flow into many lower-volume electrosprays, an approach known as MES. To couple with most modern mass spectrometers, the MES emitter must have a small diameter to allow efficient ion collection into the MS. In this work, a MSF, defined as a fiber having many empty channels running along its length, was designed to have 9 channels, 9 µm each, >100 µm apart arranged in a radial pattern, all in a fiber having a compatible diameter with both front-end LC equipment and typical MS inlets. This design seeks to promote independent electrospray from each channel while maintaining electric field homogeneity. While the MSFs themselves do not support MES, the formation of polymer nozzles protruding from each channel at the tip face enables independent electrospray from each nozzle. Microscope imaging, electrospray current measurement, and ESI-MS detection of a model analyte all confirm the MES behavior of the 9-nozzle emitter, showing significant signal enhancement relative to a single-nozzle emitter at the same total flow rate. LC/MS data from a protein digest obtained at an independent laboratory demonstrates the applicability and robustness of the emitter for real scientific challenges using modern LC/MS equipment.

A fluorous porous polymer monolith photo-patterned chromatographic column for the separation of a flourous/fluorescently labeled peptide within a microchip.

A fluorous porous polymer stationary phase is photo-patterned within a glass microfluidic chip to conduct CEC. During free radical-initiated polymerization, extraneous polymer forms and contributes to excessive microfluidic channel clogging. Nitrobenzene is explored as free radical quencher to limit clogging by minimizing extraneous polymer formation and a number of initiator to quencher ratios are explored with a 0.5:1 quencher (nitrobenzene): initiator (benzoin methyl ether) molar ratio shown to be optimal. The microchip patterned with a fluorous monolith was used to carry out the electrochromatographic analysis of a mixture containing fluorescent and fluorous labeling products. The fluorous monolithic column shows fluorous selectivity for compounds labeled with perfluoromethylene tags and a custom peptide is synthesized that possesses functional groups that can be both fluorescently and fluorously labeled. MALDI MS was used to identify the labeled fragments and microchip based electrochromatography was used to analyze the resulting labeling mixture. This is the first report to our knowledge that uses fluorous porous polymer monolith within a microchip to separate analytes using fluorous-fluorous interactions.

Micro "Hyper-Channels" on Laser-Refined Cellulose Structures.

Controlled liquid transportation is widely applied in both academia and industry. However, liquid transport applications are limited by parameters such as driving forces, precision, and velocity. Herein, a simple laser-refining technology is presented to produce micro "hyper-channels". A cellulose substrate is rendered hydrophobic through silanization and refined with a laser to produce both hierarchical nanostructures and a wettability contrast simultaneously. Such a method enables faster ("hyper"-channel) aqueous liquid transportation (≈25X, 50 mm s-1 ) compared to conventional methods. Complex patterns can be readily produced at different scales with spatial resolution as low as 50 µm. This technique also controls the refining depth on the thin paper substrate. Shallow channels can be fabricated on thin paper substrates that enable fluidic channel-crossover without liquid mixing. With certain parameters, the technique creates "portals" through the substrate, allowing trans-dimensional liquid transportation between two layers of a single sheet of substrate. The fluid throughput can be increased, while also permitting fluidic channel crossover without liquid mixing. By introducing multiple portals, the controlled fluid can transfer trans-dimensionally several times, enabling further fluidic complexity. The real-life utility of the method is demonstrated by creating a trans-dimensional microfluidic device for colorimetric detection.

SpectraX: A Straightforward Tool for Principal Component Analysis-Based Spectral Analysis.

The analysis of complex spectra is an important component of direct/ambient mass spectrometry (MS) applications such as natural product screening. Unlike chromatography-based metabolomics or proteomics approaches, which rely on software and algorithms, the work of spectral screening is mostly performed manually in the initial stages of research and relies heavily on the experience of the analyst. As a result, throughput and spectral screening reliability are problematic when dealing with large amounts of data. Here, we present SpectraX, a MATLAB-based application, which can analyze MS spectra and quickly locate m/z features from them. Principal component analysis (PCA) is used to analyze the data set, and scoring plots are presented to help in understanding the clustering of data. The algorithm uses mass to charge (m/z) features to produce a list of potential natural products.

Development of fluorous porous polymer monolith (FPPM) for the capillary electrochromatographic separation of fluorous analytes based on fluorous-fluorous interaction.

This is the first report on the CEC separation of fluorous analytes on a fluorous porous polymer monolith (FPPM) stationary phase based on fluorous-fluorous interaction. Monolithic columns do not require retaining frits and can be conveniently photo-patterned within a capillary. Two groups of fluorous compounds, a N-f-Cbz-4-nitro-benzylamine (N) series and a N-f-Cbz-4-phenyl-benzylamine (P) series, each series having compounds differing only by the length of their perfluorinated tag, were employed to evaluate the ability of the fluorinated column to separate fluorous analytes using a variety of mobile phase compositions and separation conditions. Fluorous monoliths showed enhanced separation performance by providing better selectivity, higher resolution and shorter analysis time compared to a similar non-fluorous (reversed phase) monolithic column. Under optimal conditions, column efficiency as high as 234,000 plates per metre was achieved, and all four compounds of the N series were fully resolved in <5 minutes. Perfluoromethylene selectivity was used to quantitatively evaluate the interaction between the perfluorinated chain on the analytes and both the FPPM and non-FPPM columns. It was found that the non-FPPM column resolves fluorous analytes mainly based on reversed phase interaction while the FPPM column resolves them mainly based on fluorous-fluorous interaction. Results are compared to fluorous monolith columns used in a nano-liquid-chromatographic (nano-LC) separation with gradient elution. The FPPM column required less than one fifth the analysis time in CEC mode than was required in nanoLC mode, with superior separation efficiency and resolution. FPPM stationary phases provide an attractive option for the analysis of perfluorinated analytes, which is expected to be useful in areas such as proteomics for the separation of fluorously tagged proteins, and in environmental analysis where fluorinated species are of increasing concern.

Open Port Interface for Coupling Capillary Electrophoresis and Mass Spectrometry: Performance Evaluation for Capillary Isoelectric Focusing.

Capillary electrophoresis (CE) combined with mass spectrometry (MS) is a powerful analytical technique that utilizes the resolving power of CE and the mass-detection capabilities of MS. In many cases, CE is coupled to MS via a sheath-flow interface (SFI). This interface has a simple design and can be easily constructed; however, it often suffers from issues such as MS signal suppression, interference of MS and CE electrical circuits, and the inability to set an optical point of detection close to the capillary end due to the specific design of the coupling union. In this paper, we describe a novel coupling of CE and MS based upon the open port interface (OPI). The OPI differs from classical sheath flow interfaces by operating at flow rates at least 1 order of magnitude higher. In addition to the flow rate difference, the OPI provides more efficient mixing of the capillary eluates with the transport fluid and thus minimizes MS signal suppression. In this work, we compared the performance of OPI and SFI in a series of capillary isoelectric focusing (cIEF) experiments with 5 pI markers, carbonic anhydrase II and NIST antibody. The evaluation criteria for the comparison of the OPI and SFI were analytical sensitivity, reproducibility, and pI marker linearity. Given the extent of sample dilution in the OPI, we also compared the peak resolution determined using an upstream UV detector to those determined by the downstream mass spectrometer. The results suggested that the OPI configuration reduced signal suppression, with no adverse effect on peak resolution. In addition, the OPI provided better decoupling of the CE and MS potentials as well as reduced signal dependence upon the sheath liquid composition. While these results are preliminary, they suggest that the OPI is a viable approach for CE-MS coupling.

Characterization of microstructured fibre emitters: in pursuit of improved nano electrospray ionization performance.

Full-dimensional computational fluid dynamics (CFD) simulations are presented for nano electrospray ionization (ESI) with various emitter designs. Our CFD electrohydrodynamic simulations are based on the Taylor-Melcher leaky-dielectric model, and the volume of fluid technique for tracking the fast-changing liquid-gas interface. The numerical method is first validated for a conventional 20 µm inner diameter capillary emitter. The impact of ESI voltage, flow rate, emitter tapering, surface hydrophobicity, and fluid conductivity on the nano-ESI behavior are thoroughly investigated and compared with experiments. Multi-electrospray is further simulated with 2-hole and 3-hole emitters with the latter having a linear or triangular hole arrangement. The simulations predict multi-electrospray behavior in good agreement with laboratory observations.

Rapid Mass Spectrometric Calibration and Standard Addition Using Hydrophobic/Hydrophilic Patterned Surfaces and Discontinuous Dewetting.

The rapid calibration chip (RCC) is a device that uses the fast and reproducible wetting behavior of hydrophilic/hydrophobic patterned surfaces to confine a series of differently sized droplets on a substrate to obtain a calibration curve. Multiple series of droplets can be formed within seconds by dipping an RCC into a calibration solution. No pipetting, sequential droplet deposition, or advanced equipment is required. The performance and reproducibility of RCCs were evaluated with an electrospray ionization triple-quadrupole mass spectrometer equipped with a liquid microjunction-surface sampling probe (LMJ-SSP) that allows for fast sampling of surfaces. Using circular hydrophilic areas with diameters ranging from 0.25 to 2.00 mm, liquid volumes of 4.6-70.6 nL could be deposited. Furthermore, the use of a second hydrophobic/hydrophilic patterned transfer chip can be used to add internal standard solutions to each calibration spot of the RCC, allowing to transfer a liquid volume of 22.5 nL.

Fluorous monolith specificity: the effects of polymer density and secondary interactions on column performance and amenability to biological samples.

Continuing from the foundation laid by our previous work in the field, we present here an examination of the effects of monolith density and overall composition on the efficacy of performance in the realm of fluorous separations. By variation of the proportions of monomer and cross-linking agent relative to a static porogenic solvent composition, it was found that a composition of 30% polymer-forming material provides the optimal results in terms of resolution and peak shape for fluorous chromatography of a mixture of similarly labeled benzylamines. The presence of so-called "secondary interactions" that can compete with fluorous specificity in columns of this type were also examined and discussed, with similar results to those observed for commercial fluorous columns being noted. We suggest that these effects may actually be positive if they can be properly harnessed, as the ability to provide a second dimension for fluorous separations based on polarity may allow more complex analyses of labeled proteomic samples to be effectively undertaken. Finally, we present some initial results on the effectiveness of our optimized fluorous monoliths in a series of tagging and separation experiments using a custom-synthesized peptide. With successful resolution of labeled biological samples from their nonfluorous counterparts achieved, we discuss the potential expansion and further applicability of fluorous monoliths of this type in proteomic avenues, as well as their amenability to the greater analytical community.

Bundled capillary electrophoresis using microstructured fibres.

Joule heating, arising from the electric current passing through the capillary, causes many undesired effects in CE that ultimately result in band broadening. The use of narrow-bore capillaries helps to solve this problem as smaller cross-sectional area results in decreased Joule heating and the rate of heat dissipation is increased by the larger surface-to-volume ratio. Issues arising from such small capillaries, such as poor detection sensitivity, low loading capacity and high flow-induced backpressure (complicating capillary loading) can be avoided by using a bundle of small capillaries operating simultaneously that share buffer reservoirs. Microstructured fibres, originally designed as waveguides in the telecommunication industry, are essentially a bundle of parallel ∼5 µm id channels that extend the length of a fibre having otherwise similar dimensions to conventional CE capillaries. This work presents the use of microstructured fibres for CZE, taking advantage of their relatively high surface-to-volume ratio and the small individual size of each channel to effect highly efficient separations, particularly for dye-labelled peptides.

Nanoelectrospray emitters: trends and perspective.

The benefits of electrospray ionization are many, including sensitivity, robustness, simplicity and the ability to couple continuous flow methods with mass spectrometry. The technique has seen further improvement by lowering flow rates to the nanoelectrospray regime (<1,000 nL/min), where sample consumption is minimized and sensitivity increases. The move to nanoelectrospray has required a shift in the design of the electrospray source which has mostly involved the emitter itself. The emitter has seen an evolution in architecture as the shape and geometry of the device have proved pivotal in the formation of sufficiently small droplets for sensitive MS detection at these flow rates. There is a clear movement toward the development of emitters that produce multiple Taylor cones. Such multielectrospray emitters have been shown to provide enhanced sensitivity and sample utilization. This article reviews the development of nanoelectrospray emitters, including factors such as geometry and the manner of applying voltage. Designs for emitters that take advantage of multielectrospray are emphasized.

Open sessile droplet viscometer with low sample consumption.

This paper reports a portable viscometer that requires less than 10 µL of sample for a measurement. Using a two-droplet Laplace-induced pumping system on an open microfluidic substrate, the device measures the viscosity of a liquid by determining the time required for one droplet to completely pump into a second droplet. The pumping behaviour follows the Hagen-Poiseuille and Laplace relations where the flow rate, Q, is proportional to the liquid's kinematic viscosity, µ. The progress of pumping is measured by tracking the change in curvature of one of the droplets using a laser that is positioned perpendicular to the microfluidic chip and directed at the "tail" of the shrinking droplet. The angle of incidence and degree of refraction changes depending on the size of the droplet, which is tracked by a linear diode array placed beneath the microfluidic chip. Droplet reservoirs and connecting channels were defined by precise patterning of a glass substrate coated with a commercially available omniphobic coating (Ultra Ever Dry®) using laser micromachining. A 500 µm wide and 20 mm long channel with circular reservoirs (d = 1.5 mm) enabled the measurement of dynamic viscosities in the range of η = 1.0-2.87 mPa s. The materials cost for the entire viscometer (fluidics and electronics, etc.) is <15 USD.

405 nm absorption detection in nanoliter volumes.

Analytical UV absorption detection for microfluidic devices, capillary electrophoresis, and even high-performance liquid chromatography is hampered by the small detection volumes, short absorption paths, and the need to sample at a high rate with a stable background and low noise. Fiber-loop ring-down spectroscopy (FLRDS) permits absorption detection of dilute liquid samples in volumes as small as a few nanoliters, while being insensitive to light source fluctuations and permitting a millisecond temporal resolution. We demonstrate a FLRDS based detection scheme that is compatible in dimensions (<200 microm absorption path, 6.0 nL detection volume) and optical design (405 nm detection wavelength, fiber coupled) with existing separation systems. An optical/fluidic interface has been built that allows injection of laser light into the loop while also permitting delivery of the sample. The detection limit of tartrazine was determined to be 5 microM (30 fmol) corresponding to an absorption of 0.11 cm(-1). Equivalent results were obtained when detecting myoglobin, a heterocyclic pharmaceutical ingredient, and 5.17 microm diameter polystyrene beads.