Crown ether dopant to reduce ion suppression and improve detection in the liquid microjunction surface sampling probe.

RATIONALE: Sodium and potassium are required in agar media for the growth of some microorganisms (e.g., marine bacteria). However, alkali cations are a significant source of contamination for mass spectrometry causing ion suppression and adduct formation. Conventionally, salts can be removed before mass spectrometric analysis with appropriate and often lengthy sample preparation. The direct mass spectrometric sampling of bacterial colonies grown on agar media seeks to minimize or eliminate sample preparation to improve workflow. However, this may exacerbate ion suppression and contamination since these metal cations will degrade spectral quality and limit the rapid profiling of microbial metabolites. Different approaches are needed to sequester sodium and potassium ions to minimize unwanted background interferences. Herein, we use crown ethers (CEs) in combination with a liquid microjunction surface sampling probe (LMJ-SSP) to directly sample the surface of the bacterial colonies from two marine bacteria species (Pseudoalteromonas rubra DSM6842 and Pseudoalteromonas tunicata DSM 14096). CEs (e.g., 18-crown-6 or 15-crown-5) are added to the carrier solvent of the LMJ-SSP, the chemical noise is reduced, and spectra are easier to interpret. METHODS: The liquid microjunction formed at the tip of LMJ-SSP was used to directly touch bacterial colonies on agar. The carrier solvent was either methanol (100%) or methanol: H2O (50:49.9%) with or without 0.01% CEs. Information-theoretic measures are employed to investigate qualitative changes between spectra before and after adding CEs. RESULTS: Our work demonstrates the capability of CEs to reduce background interferences within the direct profiling of bacterial colonies from agar plates. The data obtained from both P. rubra DSM6842 and P. tunicata DSM 14096 show that CEs can be used to mitigate the salty background and improve compound detection. CONCLUSION: Our approach can be implemented in natural product discovery using LMJ-SSP to allow fast and accurate detection of interesting/novel compounds.

Hyperspectral Visualization-Based Mass Spectrometry Imaging by LMJ-SSP: A Novel Strategy for Rapid Natural Product Profiling in Bacteria.

Mass spectrometry imaging (MSI) has been widely used to discover natural products (NPs) from underexplored microbiological sources. However, the technique is limited by incompatibility with complicated/uneven surface topography and labor-intensive sample preparation, as well as lengthy compound profiling procedures. Here, liquid micro-junction surface sampling probe (LMJ-SSP)-based MSI is used for rapid profiling of natural products from Gram-negative marine bacteria Pseudoalteromonas on nutrient agar media without any sample preparation. A conductance-based autosampling platform with 1 mm spatial resolution and an innovative multivariant analysis-driven method was used to create one hyperspectral image for the sampling area. NP discovery requires general spatial correlation between m/z and colony location but not highly precise spatial resolution. The hyperspectral image was used to annotate different m/z by straightforward color differences without the need to directly interrogate the spectra. To demonstrate the utility of our approach, the rapid analysis of Pseudoalteromonas rubra DSM6842, Pseudoalteromonas tunicata DSM14096, Pseudoalteromonas piscicida JCM20779, and Pseudoalteromonas elyakovii ATCC700519 cultures was directly performed on Agar. Various natural products, including prodiginine and tambjamine analogues, were quickly identified from the hyperspectral image, and the dynamic extracellular environment was shown with compound heatmaps. Hyperspectral visualization-based MSI is an efficient and sensitive strategy for direct and rapid natural product profiling from different Pseudoalteromonas strains.

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.

Discontinuously Dewetting Solvent Arrays: Droplet Formation and Poly-Synchronous Surface Extraction for Mass Spectrometry Imaging Applications.

We describe a new liquid tissue stamping method called poly-synchronous surface extraction (PSSE) that utilizes an omniphobic substrate patterned with hydrophilic surface energy traps (SETs), which when wet with a solvent form a dense microdroplet array. When contacted with a tissue sample, each droplet locally extracts analytes from the tissue surface, which subsequentially can be analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-IMS) or ambient ionization-MS techniques. Optimization of the patterned surface with six different solvents was carried out to increase the droplet density, height, and reproducibility of volume deposition. Once optimized, sister slices of a strawberry (Fragaria × ananassa) were spatially extracted using the PSSE technique and the chemical distribution of selected compounds was analyzed with both MALDI-IMS and a lower resolution but faster ambient liquid microjunction surface sampling probe (LMJ-SSP) approach. Heat maps for target analytes for the PSSE approach are compared to those produced using traditional MALDI-IMS analysis. The PSSE method aligned well with direct analysis and demonstrated the potential to increase the speed of ambient MS tissue imaging techniques by decreasing the number of steps required for sample preparation.

The liquid micro junction-surface sampling probe (LMJ-SSP); a versatile ambient mass spectrometry interface.

Ambient ionization methods have become important tools in mass spectrometry. The LMJ-SSP can significantly simplify/reduce lengthy sample preparation requirements associated with mass spectrometry analysis. Samples may be introduced through direct contact, insertion and droplet injection, enabling applications from drug discovery and surface analysis to tissue profiling and metabolic mapping. This review examines the underlying principles associated with the LMJ-SSP interface and highlights modifications of the original design that have extended its capability. We summarize different application areas that have exploited the method and describe potential future directions for the adaptable ambient ionization source.

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.

Hydrophobic/hydrophilic patterned surfaces for directed evaporative preconcentration.

The deposition of micro- and nanolitre volumes is crucial in sessile droplet microfluidic systems. Several techniques exist for the fabrication of surfaces with patterned wettabilities; however, many of these fabrication techniques are time-consuming and complex. Here, we present a device that allows for deposition of multiple droplets within seconds followed by directed evaporative preconcentration. Hydrophobic-coated glass substrates are fashioned with hydrophilic surface energy traps (SETs) using picosecond laser micromachining. SETs can capture nanolitre volumed droplets of both aqueous and organic liquids through discontinuous dewetting. Modification of the machined hydrophilic shape yields a passive mechanism that preconcentrates analyte through evaporation. Studies and optimizations of SET parameters/dimensions (laser power, laser passes, ring/patch diameter) and their effect on patch wettability and degree of preconcentration are presented. As a demonstration, the optimized platform was used to improve the colourimetric detection of cadmium-containing aqueous samples. The optimized SET design demonstrated an 18-fold increase in colourimetric sensitivity compared to conventional milled SETs, suggesting the design would be well-suited for trace analysis. The evaporative preconcentration was also applied to MALDI-IMS analysis of peptides where it resulted in improved uniformity of deposited analyte and decreased analysis times. The rapid droplet deposition and directed evaporative approach can be tailored to provide different concentration factors and is compatible with a wide variety of detection schemes.

Reliable identification of prostate cancer using mass spectrometry metabolomic imaging in needle core biopsies.

Metabolomic profiling can aid in understanding crucial biological processes in cancer development and progression and can also yield diagnostic biomarkers. Desorption electrospray ionization coupled to mass spectrometry imaging (DESI-MSI) has been proposed as a potential adjunct to diagnostic surgical pathology, particularly for prostate cancer. However, due to low resolution sampling, small numbers of mass spectra, and little validation, published studies have yet to test whether this method is sufficiently robust to merit clinical translation. We used over 900 spatially resolved DESI-MSI spectra to establish an accurate, high-resolution metabolic profile of prostate cancer. We identified 25 differentially abundant metabolites, with cancer tissue showing increased fatty acids (FAs) and phospholipids, along with utilization of the Krebs cycle, and benign tissue showing increased levels of lyso-phosphatidylethanolamine (PE). Additionally, we identified, for the first time, two lyso-PEs with abundance that decreased with cancer grade and two phosphatidylcholines (PChs) with increased abundance with increasing cancer grade. Importantly, we developed and internally validated a multivariate metabolomic classifier for prostate cancer using 534 spatial regions of interest (ROIs) in the training cohort and 430 ROIs in the test cohort. With excellent statistical power, the training cohort achieved a balanced accuracy of 97% and validation on testing data set demonstrated 85% balanced accuracy. Given the validated accuracy of this classifier and the correlation of differentially abundant metabolites with established patterns of prostate cancer cell metabolism, we conclude that DESI-MSI is an effective tool for characterizing prostate cancer metabolism with the potential for clinical translation.

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.

Fabrication of axicon microlenses on capillaries and microstructured fibers by wet etching.

A facile method is presented for the fabrication of microlenses at the facet of fused silica capillaries and microstructured fibers. After submersion in hydrogen fluoride solution water is pumped slowly through the center hole of the capillary microchannel to create an etchant gradient extending from the capillary axis. The desired axicon angle is generated by adjusting the etching time and/or concentration of the etchant. Similarly, flow- assisted HF etching of a custom microstructured fiber containing nine microchannels produces nine individual microlenses simultaneously at the fiber facet, where each microaxicon lens shows a similar focusing pattern. A theoretical model of the flow-assisted etching process is used to determine the axicon angle and post angle. Also, a simple ray-based model was applied to characterize the focusing properties of the microaxicons in good agreement with experimental observations.

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.

Digital microfluidic platform for human plasma protein depletion.

Many important biomarkers for disease diagnosis are present at low concentrations in human serum. These biomarkers are masked in proteomic analysis by highly abundant proteins such as human serum albumin (HSA) and immunoglobulins (IgGs) which account for up to 80% of the total protein content of serum. Traditional depletion methods using macro-scale LC-columns for highly abundant proteins involve slow separations which impart considerable dilution to the samples. Furthermore, most techniques lack the ability to process multiple samples simultaneously. We present a method of protein depletion using superparamagnetic beads coated in anti-HSA, Protein A, and Protein G, manipulated by digital microfluidics (DMF). The depletion process was capable of up to 95% protein depletion efficiency for IgG and HSA in 10 min for four samples simultaneously, which resulted in an approximately 4-fold increase in signal-to-noise ratio in MALDI-MS analysis for a low abundance protein, hemopexin. This rapid and automated method has the potential to greatly improve the process of biomarker identification.

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.