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A new method for the sampling and off-site analysis of hemoglobin variants by mass spectrometry is reported. This technique uses a nonporous polyurethane membrane as the collection device and transportation medium of a blood sample for analysis. The same membrane is then used as the MALDI-TOF MS sample support for mass spectrometric analysis. Minimal invasive sample collection is afforded by collecting less than 1 microL of blood using a common lancet device. MALDI-TOF MS is performed directly on the membrane, after washing off the interfering plasma components, followed by the addition of matrix. This reduces the time of analysis and prevents sample loss. Enzymatic digestion can be performed directly on the membrane, using in this case trypsin, allowing for further characterization of the sample. The method is much less invasive compared to drawing blood with a syringe. The sample may be transported to the laboratory by regular mail, and thus the method can serve remote locations. We demonstrate the procedure by characterizing the Hb Shepherds Bush hemoglobin variant, b74-(E18)Gly-->Asp.

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: H 2 O (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., (© 2024 The Author(s). Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Ambient mass spectrometry (MS) technologies have been applied to spatial metabolomic profiling of various samples in an attempt to both increase analysis speed and reduce the length of sample preparation. Recent studies, however, have focused on improving the spatial resolution of ambient approaches. Finer resolution requires greater analysis times and commensurate computing power for more sophisticated data analysis algorithms and larger data sets. Higher resolution provides a more detailed molecular picture of the sample; however, for some applications, this is not required. A liquid microjunction surface sampling probe (LMJ-SSP) based MS platform combined with unsupervised multivariant analysis based hyperspectral visualization is demonstrated for the metabolomic analysis of marine bacteria from the genus Pseudoalteromonas to create a rapid and robust spatial profiling workflow for microbial natural product screening. In our study, metabolomic profiles of different Pseudoalteromonas species are quickly acquired without any sample preparation and distinguished by unsupervised multivariant analysis. Our robust platform is capable of automated direct sampling of microbes cultured on agar without clogging. Hyperspectral visualization-based rapid spatial profiling provides adequate spatial metabolite information on microbial samples through red-green-blue (RGB) color annotation. Both static and temporal metabolome differences can be visualized by straightforward color differences and differentiating m / z values identified afterward. Through this approach, novel analogues and their potential biosynthetic pathways are discovered by applying results from the spatial navigation to chromatography-based metabolome annotation. In this current research, LMJ-SSP is shown to be a robust and rapid spatial profiling method. Unsupervised multivariant analysis based hyperspectral visualization is proven straightforward for facile/rapid data interpretation. The combination of direct analysis and innovative data visualization forms a powerful tool to aid the identification/interpretation of interesting compounds from conventional metabolomics analysis., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

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., (© 2023 The Authors. Small Methods published by Wiley-VCH GmbH.)

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.

DNA evidence in sexual assault cases have proven increasingly difficult to obtain and analyse due to increased condom use. With more interest in alternatives to DNA evidence, prophylactic lubricants, spermicides and residues may be interesting prospects. Current interest in the analysis of prophylactic residues focuses on the evaluation and identification of lubricants and constituents, primarily through gas chromatography or Fourier transform infrared spectroscopy. Though cost-effective methods, extensive sample preparation and destructive modes of analysis remain an area for improvement. As a result, the focus has since shifted to ambient ionization methods that offer adequate sensitivity and reduced sample preparation. The Liquid Microjunction Surface Sampling Probe (LMJSSP) is a versatile ambient ionization source that employs a probe that supports a continuously flushing droplet that extracts analytes when placed in contact with a surface. The analytes are aspirated into the mass spectrometer with a Venturi pressure. In this work we use the LMJSSP to analyse the trace transfer of condom lubricant to different types of fabric (cotton, cotton-spandex, and denim). Furthermore, we examine the sensitivity and storage conditions for the direct analysis method on different swab types (cotton, silicone, and foam). Additionally, Principal Component Analysis (PCA) and Maximally Collapsing Metric Learning (MCML) are utilized for visualization of differentiability of commercially available condom brands including Durex™ and Trojan™, and product subtypes. The results present an interesting multi-disciplinary approach of using a direct liquid extraction ambient ionization technique and machine learning to improve the overall workflow for the analysis of lubricants, swabs and fabrics. Machine learning algorithms were able to differentiate between inherent differences of Durex™ and Trojan™ condoms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Richard David Oleschuk reports equipment, drugs, or supplies was provided by AB SCIEX LLC., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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., (© 2023 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

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.

The use of 3D printing in the chemical and analytical sciences has gained a lot of momentum in recent years. Some of the earliest publications detailed 3D-printed interfaces for mass spectrometry, which is an evolving family of powerful detection techniques. Since then, the application of 3D printing for enhancing mass spectrometry has significantly diversified, with important reasons for its application including flexible integration of different parts or devices, fast customization of setups, additional functionality, portability, cost-effectiveness, and user-friendliness. Moreover, computer-aided design (CAD) and 3D printing enables the rapid and wide distribution of scientific and engineering knowledge. 3D printers allow fast prototyping with constantly increasing resolution in a broad range of materials using different fabrication principles. Moreover, 3D printing has proven its value in the development of novel technologies for multiple analytical applications such as online and offline sample preparation, ionization, ion transport, and developing interfaces for the mass spectrometer. Additionally, 3D-printed devices are often used for the protection of more fragile elements of a sample preparation system in a customized fashion, and allow the embedding of external components into an integrated system for mass spectrometric analysis. This review comprehensively addresses these developments, since their introduction in 2013. Moreover, the challenges and choices with respect to the selection of the most appropriate printing process in combination with an appropriate material for a mass spectrometric application are addressed; special attention is paid to chemical compatibility, ease of production, and cost. In this review, we critically discuss these developments and assess their impact on mass spectrometry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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-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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

Magnetic actuation provides a low-cost, simple method for droplet manipulation on a digital microfluidic platform. The impetus to move the droplets on a low friction surface can come from internal superparamagnetic particles or paramagnetic salts. Recently, the use of microbes for bio-actuation has been established, where the thrust produced by the microbes can be exploited to exert the force required for droplet movement. This study presents biologically-driven magnetic actuation of droplets on a superhydrophobic surface using magnetotactic bacteria (MTB). MTB-droplets were impelled along various trajectories such as rectangular and figure-of-eight-shaped paths. Droplets were reproducibly actuated with speeds up of to 30 mm s -1 . We demonstrated the ability to sequentially merge and mix multiple droplets by merging a 10 μL MTB droplet with two 4 μL colored droplets. The reorientation of MTB in the droplet enhanced mixing rate of the merged fluids by ∼40% compared with the control experiment where no actuation was used. Biologically-driven magnetic actuation was compared with actuation by superparamagnetic particles and paramagnetic salts, in terms of controllability and speed. MTB droplet was moved with the same average speed as other two methods and showed higher response time as the magnet acceleration increased. Lastly, MTB were used to perform a phosphatase assay using endogenous enzyme. The relative absorbance at 405 nm, indicating the production of the yellow product, increased over time and levels off after 75 min., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Metal clusters of gold and silver with highly tunable optical and electronic properties are attractive candidates for next generation medical imaging and therapy. Of these two most commonly studied metals, silver clusters often exhibit superior optical properties (i.e. stronger absorbance and higher emission quantum yield). The atomically precise synthesis of these clusters is essential before their use in biological applications can be realized. However, most cluster synthetic routes result in complex mixtures, where isolation and/or characterization can become incredibly challenging. Using photochemistry, we demonstrate a synthetic route for silver thiolate clusters resulting in the isolation of a pure eighteen-atom silver cluster capped by fourteen captopril ligands, Ag 18 (Capt) 14 . The facile control over the reduction of Ag(i) salt that this photochemical route affords can be readily applied as a general synthesis for isolating other new, atomically precise clusters.

Digital microfluidics employs water-repellant surfaces to exquisitely manipulate droplets of water for chemical analysis. However, the actuation and manipulation of organic droplets is still relatively unexplored as it is significantly more difficult to synthesize organic-repellent surfaces compared to water-repellent surfaces. Here, we present the fabrication of slippery liquid-infused porous surfaces (SLIPS) based on a porous polymer monolithic approach. The synthesized SLIPS were able to repel organic liquids such as hexane and methanol with a contact angle of 42.1 ± 0.4° and 69.0 ± 1.8°, respectively, as well as water with a contact angle of 115.8 ± 0.8°. More importantly for digital microfluidic applications, the sliding angle of liquids tested was between 4° and 6°. As a result, droplets containing magnetically susceptible material could be facilely manipulated on the SLIPS surface. A systematic actuation study was carried out to explore how actuation parameters including speed, paramagnetic particle (PMP) concentrations, and droplet volume impacted the outcomes (droplet actuation, disengagement, and PMP extraction). Two different applications were used to demonstrate the utility of actuating organic droplets on SLIPS surfaces including on-chip liquid-liquid extractions of natural products (NPs) from marine bacteria and droplet-based polymer synthesis with different polymerization conditions. Both applications employ an aqueous droplet and organic droplet interface at which either phase transfer or a chemical reaction is carried out. Two NPs (prodigiosin from Pseudoalteromonas rubra and violacein from Pseudoalteromonas luteoviolacea ) were extracted, from aqueous droplets containing the bacteria, into butanol droplets and characterized with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Nylon 6,6 was synthesized on-chip via magnetic actuation of organic droplets containing adipoyl chloride and hexamethylamine. Relative intensities of the characteristic polymer masses suggest that droplet-based microfluidic synthesis on slips can be used to probe reaction conditions. The compatibility of SLIPS with both aqueous and organic solutions opens up a wider number of droplet-based sample preparation protocols and chemical transformations.

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.

Silver and gold clusters have received a lot of recent attention for their use in biomedical imaging. However, crude solutions of clusters are often complex mixtures, leading to discrepancies in their identification and characterization; important factors in determining their utility in biological applications. In the present study, silver clusters were separated for analysis using reverse-phase high performance liquid chromatography, which has previously been implemented in the efficient separation of gold clusters. Using fluorescence excitation-emission matrix (EEM) spectroscopy, we have demonstrated that a certain family of glutathione-protected silver clusters, previously thought to be one optically distinct species, is better described as a complex mixture of at least three distinct silver cluster species, each possessing unique optical properties. Based on these findings, EEM spectroscopy can be implemented as a powerful technique for determining the purity of complex mixtures, especially when other techniques, including mass spectrometry, fail to provide adequate characterization of a given material., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this work we present the application of 3D-printing for the miniaturization and functionalization of an ion source for (portable) mass spectrometry (MS). Two versions of a 3D-printed cartridge for paper spray ionization (PSI) are demonstrated, assessed, and compared. We first focus on the use of 3D-printing to enable the integration of an embedded electrostatic lens and a manifold for internal sheath gas distribution and delivery. Cartridges with and without a sheath gas manifold and an electrostatic lens are compared with respect to analytical performance and operational flexibility. The sensitivity and limit of detection are improved in the cartridge with an electrostatic lens and sheath gas manifold compared to the cartridge without (15% and over 6.5× smaller, respectively). The use of these focusing elements also improved the average spray stability. Furthermore, the range of potentials required for PSI was lower, and the distance to the MS orifice over which spray could be obtained was larger. Importantly, both setups allowed quantification of a model drug in the ng/mL range with single-stage MS, after correction for spray instability. Finally, we believe that this work is an example of the impact that 3D-printing will have on the future of analytical device fabrication, miniaturization, and functionalization.

Droplet manipulation over open surfaces allows one to perform assays with a large degree of control and high throughput, making them appealing for applications in drug screening or (bio)analysis. However, the design, manufacturing and operation of these systems comes with high technical requirements. In this study we employ a commercial, low-friction, superhydrophobic coating, Ultra-Ever Dry ® , on a 3D-printed microfluidic device. The device features individual droplet compartments, which allow the manipulation of discrete droplets (10-50 μL) actuated by gravity alone. Simply by angling the device to normal in a 3D-printed holder and rocking in a "to and fro"-fashion, a sequence of droplets can be individually transferred to an electrochemical microelectrode detector and then to waste, while preserving the (chronological) order of samples. Multiple biological fluids (i.e. human saliva, urine and rat blood and serum) were successfully tested for compatibility with the device and actuation mechanism, demonstrating low slip angles and high contact angles. Biological matrix (protein) carryover was probed and effectively mitigated by incorporating aqueous rinse droplets as part of the analysis sequence. As a proof-of-concept, the enzyme-coupled, amperometric detection of glucose was carried out on individual rat serum droplets, enabling total analysis in ≈30 min, including calibration. The device is readily customizable, and the integration of droplet generation techniques and other sensor systems for different analytes of interest or applications can be realized in a plug and play fashion., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A method for performing droplet actuation, splitting, and dispensing using only magnetic force and physical confinement is reported. The combination of low-friction superhydrophobic surfaces and droplets containing superparamagnetic particles is demonstrated to reliably dispense droplets with a precision (≤6%) similar to standard air-displacement pipettes. The 3D printed microfluidic chips incorporate individual wells, a weir structure and differential channel depths to facilitate droplet splitting in differing ratios. Both empirical observations and numerical simulations show that the splitting is a combination of wetting and pressure differences. The method enables a parent drop to be dispensed and split into droplets ranging in size from 5-20 μL using different well volumes. Once dispensed/split the droplets can be further actuated, merged and mixed. An EDTA-based complexometric colorimetric titration for water hardness is conducted on-chip. The degree of colour change is then determined utilizing a cell phone camera and image analysis and used to calculate water hardness; this measurement was found to agree with the traditional, larger scale method. The simple, robust dispensing method is adaptable to other digital microfluidic assays.

The deposition of nanoliter and subnanoliter volumes is important in chemical and biochemical droplet-based microfluidic systems. There are several techniques that have been established for the deposition/generation of small volumes including the use of surfaces with patterned differences in wettability. Many such methods require complex and time-consuming lithographic techniques. Here, we present a facile method for the fabrication of superhydrophobic surfaces with patterned hydrophilic regions by laser micromachining. A comprehensive study of fabrication parameters (laser machining speed, laser power, and patch size) on the material, patch wettability, and droplet volume is presented. Patch sizes as small as 100 μm diameter and as large as 1500 μm diameter were investigated, and volumes as low as 400 pL were observed. As an example application of such patterned materials and the deposition of small volumes, halide salts were preconcentrated on the hydrophilic patches, and their fluorescence quenching constants were rapidly calculated using a 3D-printed device coupled to a fluorescence spectrometer.

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 (χ m ) 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 χ m from ≈4500 to 72 000 (× 10 -6 cm 3 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 MnCl 2 salt was examined, and the Stern-Volmer quenching constant was determined.

This work highlights the possibility of using microstructured fibres with predefined doped regions to produce functional microstructures at a fibre facet with differential chemical etching. A specially designed silica microstructured fibre (MSF) that possesses specific boron-doped silica regions was fabricated for the purpose of generating a radial micronozzle array. The MSF was drawn from a preform comprising pure silica capillaries surrounded by boron-doped silica rods. Different etching rates of the boron-doped and silica regions at the fiber facet produces raised nozzles where the silica capillaries were placed. Fabrication parameters were explored in relation to the fidelity and protrusion length of the nozzle. Using etching alone, the nozzle protrusion length was limited, and the inner diameter of the channels in the array is expanded. However with the addition of a protective water counter flow, nozzle protrusion is increased to 60 μm with a limited increase in hole diameter. The radial micronozzle array generated nine individual electrosprays which were characterized using spray current measurements and related to theoretical prediction. Signal enhancement for the higher charge state ions for two peptides showed a substantial signal enhancement compared to conventional emitter technology.

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.

Porous polymer monolithic columns are prepared from a variety of monomers and cross-linkers and can be customized to exhibit different selectivities for separate analyte classes. The composition of the monolith can be precisely controlled by selecting different monomers and or cross-linker ratios. In this work monoliths exhibiting both fluorous and hydrophobic character were prepared using butyl methacrylate and its fluorous analogue (monomer) and 1,3-butanediol diacrylate and its fluorous analogue (cross-linker) in different ratios. The selectivity of the monoliths was probed using capillary electrochromatography with several fluorous and alkyl benzene analytes. Hydrophobic stationary phases exhibited greater methylene selectivity ( [Formula: see text] ) while those with increasing fluorous character show enhanced pefluoromethylene selectivity ( [Formula: see text] ). The Gibbs free energy change associated with the sorption of the analytes on each stationary phase composition can be calculated from migration times (i.e. capacity factor) for the addition of an individual -CF2- or -CH2- moiety. Furthermore, the Gibbs free energy change associated with a single -CF2- or -CH2- moiety (analyte) interacting with an individual -CF2- or -CH2- (stationary phase) can also be estimated by plotting fluorous column composition against [Formula: see text] . Furthermore [Formula: see text] and [Formula: see text] can be plotted versus H2O percentage in mobile phase, and a new concept, hypothetical water percentage (HWP) is proposed to evaluate the hydrophobicity/fluorophilicity of a stationary phase., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

Electrospray ionization (ESI) has been invaluable to the mass spectrometric detection of biomolecules, due largely to the sensitivity afforded by the ionization technique. Lower flow rates, e.g. in the nanoelectrospray regime, result in smaller initial electrosprayed droplets, leading to higher ionization efficiency and greater signal. One approach to improving sensitivity without lowering flow rate is to generate multiple electrosprays (MESs) from the same sample, essentially splitting one larger flow into smaller flows in the nanoESI regime. Presented here is a series of novel MES emitters in the form of polycarbonate fibres. Based on microstructured fibre (MSF) technology whereby a set of homogeneous parallel channels are formed in a heat-drawn fibre intended to conduct light, a custom design was fabricated in which 3, 6, 9 and 12 holes were arranged in a radial pattern to prevent inhomogeneities in the electric field. The MSFs have dimensions that are compatible with current standards in nanoESI equipment, and the tip is more compatible with standard MS orifices than other larger multielectrospray emitters. By measuring the spray current provided by the various emitters under the same solvent/voltage/total flow rate conditions, a plot was obtained clearly demonstrating the expected dependence on the square root of the number of holes, i.e. the number of independent electrosprays. With this firm proof of principle using this design/format, further effort is justified in developing similar emitters in alternative materials that better prevent surface wetting and allow greater hole density, ultimately leading to greater signal enhancement., (Copyright © 2012 John Wiley & Sons, Ltd.)

Although commonly used in gas chromatography, open-tubular columns for liquid chromatography have seen their development hindered by a number of factors both theoretical and practical. Requiring small diameters, great lengths and specialized detection systems to achieve a proper chromatographic response, columns of this sort have largely been ignored despite the highly desirable column performance an optimized system would provide. Here, we introduce the use of microstructured fibers (MSFs) as a platform for the development of multiplexed open-tubular liquid chromatography (OTLC) columns. The multiple, parallel silica channels presented by the MSF act as a promising substrate for an OTLC column, as they have diameters near the ideal range for interactions (1-3 μm), minimize flow-induced backpressure through their many uniform paths, and increase the loading capacity compared to a single capillary channel of similar size. Additionally, with outer diameters comparable to regular fused silica capillaries, MSFs can easily be employed in conventional chromatographic systems, eliminating the need for specialized equipment. Finally, MSF columns of this type can be functionalized using silane coupling techniques to allow the introduction of a wide variety of stationary phase chemistries. While in this report we explore the potential and limitations of fluorine-functionalized MSFs as OTLC columns, other stationary phase materials could easily be substituted by choosing appropriate silanization reagents. Particular attention here will be paid to the physical and performance characteristics of the fabricated columns, as well as avenues for their improvement and implementation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel porous polymer monolith was prepared in situ in a fused-silica capillary using photoinitiated polymerization. Bisphenol A dimethacrylate (BPADMA) was selected as a crosslinker, copolymerized with benzyl methacrylate (BMA) in the presence of a binary porogenic solvent consisting of cyclohexanol and 1-decanol in < or =10 min. The resulting poly(BMA-co-BPADMA) monoliths exhibited good permeability and mechanical stability. Mixtures of alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) or phenolic compounds were successfully separated by CEC. A similar monolith was also prepared with ethylene dimethacrylate (EDMA) as the crosslinker instead of BPADMA to compare the separation ability of the resulting monoliths. The results indicated that poly(BMA-co-BPADMA) monoliths have better selectivity for aromatic analytes and greater chromatographic stability in higher aqueous mobile phase., (Copyright 2010 Elsevier B.V. All rights reserved.)

Novel capillary columns containing polymer-entrapped octadecylsilyl-modified silica microspheres were evaluated for chromatographic performance. The polymer forms only at the particle surface and in as little as 10 s strongly immobilizes them, obviating the need for a frit. The photoinitiated polymerization is patternable, and various entrapment styles were compared, all of which withstood pressure drops of at least 5000 psi (345 bar). The fritless nature of the columns allows a unique mass production capability whereby long packed columns are entrapped and simply cut afterward. Since the material has previously been shown to be a proficient nanoelectrospray emitter, the columns have enormous potential for use in capillary LC-MS. The evaluation suggests that for the designs that have minimal entrapped regions, the column performance is equivalent to commercial columns containing similar particles.

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., (Copyright 2009 Wiley Periodicals, Inc.)

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.

Novel multichannel electrospray emitters are presented that use silica-based microstructured fibers (MSFs) to split the flow allowing efficient desolvation during electrospray. The MSFs investigated in this study possess 30-168 individual fluidic channels (each channel being 5 microm in diameter) that form a 2D emitting array. Multiple flow paths afford stable electrospray at flow rates ranging from the microspray (e.g., 1000 nL/min) to the nanoelectrospray (e.g., 10 nL/min) regime with moderate to negligible flow-induced backpressures. The electrospray stability of highly aqueous solutions (up to 99.9% water with 0.1% acetic acid) is enhanced through modification of the emitting surface with a hydrophobic silylation reagent (chlorotrimethylsilane). Furthermore, by successfully spraying highly concentrated salt solutions, this study demonstrates that multichannel MSF emitters provide enhanced robustness to clogging, leading to increased operational throughput.

Poly(dimethylsiloxane) (PDMS) has shown considerable promise in the fabrication of microfluidic devices. Surface modification of PDMS by the grafting of perfluorinated alkanes allows the selective adsorption of fluorous-tagged peptides, demonstrating that this material may be used in fluorous affinity tag technology to enrich and separate specific proteins or peptides from complex mixtures. Here, we explore the non-specific adsorption of proteins which may interfere with this process. The desorption of cytochrome c, carbonic anhydrase, insulin and ubiquitin onto the surfaces of unmodified, oxidized and fluorinated PDMS in solutions of varying water/methanol concentration has been studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The interaction forces involving perfluorinated surfaces are probed using chemical force spectrometry. The denaturation of the proteins in solutions of high methanol concentration is followed using electrospray ionization mass spectrometry (ESI-MS) and the adsorption profiles discussed in the context of the surface hydrophobicity of each protein.

With applications that take advantage of highly selective fluorine-fluorine interactions appearing with greater frequency in the literature, the development of porous polymer monoliths from fluorous components is reported here for the purpose of chromatography of tagged analytes. With potential uses in fields as diverse as separation science and proteomics, facile fabrication of materials with fluorous specificity that can be applied in a high-throughput manner is greatly desirable. To this end, we have developed porous polymer capillary columns with varied fluorous content using a simple UV-initiated radical polymerization process and characterized them using flow-induced backpressure and scanning electron microscopy (SEM). With structural similarities assured (visually, and by backpressure variations of less than 42%), the monoliths were tested as chromatographic columns for the separation of a series of fluorous-tagged analytes under gradient conditions. It was found that columns made with fluorinated components exhibited greater selectivity for fluorous analytes than did equivalent, non-fluorinated monoliths, retaining analytes with either one or two fluorous tags for approximately 6% and 13% longer, respectively. This supports the idea of differences existing between fluorous and reverse-phase separation mechanisms, and encourages a broader range of potential applications for fluorous monoliths of this type.

A non-tapered open tubular emitter with 75 microm internal diameter (i.d.) and 360 microm external diameter (o.d.) was developed by simply grinding the exit aperture of a fused-silica capillary. The roughened emitter, with a relatively large aperture, generates stable electrospray signals (generally <5% relative standard deviation (RSD) for most conditions studied) at less than 500 nL/min flow rates, and was characterized with atomic force microscopy. The surface treatment greatly extends the operational range of an open tubular emitter to lower flow rates, compared to that of a cleaved capillary with similar dimensions. The stabilized nanoelectrospray is attributed to the increased surface roughness and modified wetting characteristics of the emitter exit resulting from grinding. Electrospray performance was evaluated, and as a result of the enhanced sensitivity from a roughened emitter, five femtomoles of leucine enkephalin were detected at a 50 nL/min flow rate with a signal to noise (S/N) ratio of 48. Furthermore, trypsin-digested bovine serum albumin (BSA) was used to demonstrate the application of the emitter in protein identification, giving a sequence coverage of 60%. These emitters are robust, and may become a facile alternative to tapered emitters at moderate nano flow rates (e.g. 50 to 500 nL/min).

The entrapment of silica-based microspheres, commonly used as stationary phases in chromatography, with an organic porous polymer based on poly(butyl acrylate-co-1,3-butanediol diacrylate) was explored. The spheres were immobilized by photopolymerization leading to entrapped beds within 75 microm i.d. fused silica capillaries, and were mechanically stable, resisting pressure drops of over 5600 psi (38.6 MPa) for only 1 cm of material. The morphology of the polymer formation around the spheres was investigated by SEM and corroborated with back pressure measurements, which indicated that the spheres were held together by encapsulating polymer. The entrapped material was extruded from the capillary in some cases to facilitate imaging. The entrapment conditions were explored, varying the polarity of the sphere surface, the solvent, and the monomers, revealing that polymer formation is based on partitioning of the monomers between the surface and solvent. The resulting polymer morphology is discussed with respect to the effects of confinement, supported by experiments with varying microsphere diameters. The columns described here have favourable properties for use in capillary chromatography and supported catalysis among other applications, and is suitable for lab-on-a-chip devices.

Poly(dimethylsiloxane) (PDMS) and similar polymers have proved to be of widespread interest for use in microfluidic and similar microanalytical devices. Surface modification of PDMS is required to extend the range of applications for devices made of this polymer, however. Here we report on the grafting of perfluorooctyltriethoxysilane via hydrolysis onto an oxidized PDMS substrate in order to form a fluorinated microchannel. Such a fluorinated device could be used for separating fluorous tagged proteins or peptides, similar to that which has been recently demonstrated in a capillary electrophoresis system or in an open tubular capillary column. The modified polymer is characterized using chemical force titrations, contact angle measurements, and X-ray photoelectron spectroscopy (XPS). We also report on a novel means of performing electroosmotic measurements on this material to determine the surface zeta potential. As might be expected, contact angle and chemical force titration measurements indicate the fluorinated surface to be highly hydrophobic. XPS indicates that fluorocarbon groups segregate to the surface of the polymer over a period of days following the initial surface modification, presumably driven by a lower surface free energy. One of the most interesting results is the zeta potential measurements, which show that significant surface charge can be maintained across a wide range of pH on this modified polymer, sufficient to promote electroosmotic flow in a microfluidic chip. Matrix-assisted time-of-flight mass spectrometry (MALDI-TOF MS) measurements show that a fluorous-tagged peptide will selectively adsorb on the fluorinated PDMS in aqueous solution, demonstrating that the fluorinated polymer could be used in devices designed for the enrichment or enhanced detection of fluorous-labeled proteins and peptides.

Poly(methylmethacrylate) (PMMA) is a versatile polymer that displays desirable properties for development of cheap and disposable microfluidic devices for sensing biomolecular interactions. Atomic force microscopy (AFM) and chemical force titrations were used to determine the efficacy of surface modifications made to accommodate protein-substrate linkage. AFM images show the effects on surface morphology of carboxylated-, amine-, hCG antigen- and anti-hCG antibody-modified PMMA substrates. Confocal microscopy was used to determine the fluorescent intensity of labeled antibody species on the PMMA substrate, confirming the success of surface antigen/antibody immobilization. Surface pK(1/2) value for carboxylic acid and amine species grafted on PMMA were determined. When carboxylic acid or amine-terminated tips were titrated against PMMA samples terminated with the hCG antigen and anti-hCG antibody, peaks appeared in the force titration curve consistent with the pI range of the antigen or antibody species. Strong adhesive forces were present at pH values above 7.0 when the antigen was present on the PMMA substrate, and these were attributed to hydrophobic interactions between the antigen and the alkane "linker" chain attaching the amine or carboxylate group to the AFM tip. Such hydrophobic interactions were not observed with the carboxylic acid or amine/antibody combinations suggesting that the surface-linked antibody was more resistant to denaturation under higher pH. The results demonstrated the feasibility of using AFM approaches for interrogating protein grafting strategies in the fabrication of PMMA-based microsystems.

An affinity porous polymer monolith is utilized as a nanoelectrospray emitter as well as an online affinity capture column for the preconcentration of glycans. Porous polymer monolith (PPM) assisted electrospray provides a facile methodology for coupling microfluidics to mass spectrometry that is sheathless and with zero dead volume. Affinity PPM was photopolymerized using glycidyl methacrylate/ethylene dimethacrylate utilizing different porogenic solvents based on aliphatic alcohols to provide PPMs with a variety of pore sizes. The use of longer alkyl chain alcohols decreased the pore size of the formed PPM as indicated by the higher flow back pressure generated. The effect of the pore size on the stability of the electrospray was tested showing higher stability of the TIC with lower pore size. A lectin, namely Concanavaline A, was immobilized on glycidyl methacrylate/ethylene dimethacrylate using the Schiff base method to provide an affinity monolith for high mannose glycans. The amount of the lectin immobilized was studied as a function of the porogenic solvent used in the polymerization. The glycopeptides of the glycoprotein Ribonuclease B was preconcentrated on the affinity PPM sprayer and detected by tandem MS.

The application of phase-shift, fiber-loop, ring-down spectroscopy (PS-FLRDS) as an on-line detector for capillary electrophoresis (CE) of biomolecules is demonstrated. CE was conducted using a custom-designed capillary/fiber interface coupled to an absorption detector, which is based on the ring-down of an optical signal in a closed fiber waveguide loop. The ring-down times were obtained by measuring the phase difference between intensity modulated light entering and exiting the fiber loop. The incorporation of a microlens to enhance transmission through the sample gap led to an improvement of the sensitivity by up to 80% compared to the square-cut fiber and a reduction in the detection limit. The performance of the PS-FLRDS absorption technique as an online detector was characterized by flow injection through a capillary. Good repeatability and linear response were obtained, and the detection limit using the lensed fiber/capillary interface system was determined to be alpha(min) = 1.6 cm(-1) for an absorption path of approximately 30 microm. PS-FLRDS coupled to CE was also applied to the analysis of human serum albumin (HSA) by using a NIR dye as a noncovalent label. The excess free dye and the dye/protein complex were resolved. The labeling coefficient was determined to be approximately 6, and good repeatability of peak areas (RSD = 8.7%) was obtained for the analysis of HSA. Furthermore, an excellent linear response (R2 > 0.99) was obtained between the peak areas and concentrations of HSA. The detection limit of labeled HSA was determined to be 1.67 microM.

Coupling of polymeric microfluidic devices to mass spectrometry is reported using porous polymer monoliths (PPM) as nanoelectrospray emitters. Lauryl acrylate-co-ethylene dimethacrylate porous polymer monolith was photopatterned for 5 mm at the end of the channel of microfluidic devices fabricated from three different polymeric substrate materials, including the following: poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and cyclic olefin copolymer (COC). Spraying directly from the end of the chip removes any dead volume associated with inserted emitters or transfer lines, and the presence of multiple pathways in the PPM prevents the clogging of the channels, which is a common problem in conventional nanospray emitters. Spraying from a microfluidic channel having a PPM emitter produced a substantial increase in TIC stability and increased sensitivity by as much as 70x compared to spraying from an open end chip with no PPM. The performance of PPM emitter in COC, PMMA, and PDMS chips was compared in terms of stability and reproducibility of the electrospray. COC chips showed the highest reproducibility in terms of chip-to-chip performance, which can be attributed to the ease and reproducibility of the PPM formation due to the favorable optical and chemical properties of COC. We have further tested the performance of the COC chips by constant infusion of poly(propylene glycol) solution at organic content ranging from 10 to 90% methanol and at flow rates ranging from 50 to 1000 nL/min, showing optimum spraying conditions (RSD < 5%) at 50-70% organic content and at flow rates from 100 to 500 nL/min. The PPM sprayer was also used for protein preconcentration and desalting prior to mass spectrometric detection, and results were comparable with a chip spraying from an electrospray tip.

An optical sensor integrated into a polymer microfluidic chip is proposed as a low cost solution to highly parallel biochemical analysis. The sensor consists of a single high-finesse optical resonator for direct analytes detection. High quality silica microspheres (diameter approximately 300 microm) are easily produced and low-loss whispering gallery modes were excited through evanescent coupling at wavelengths near 1550 nm and 544 nm. The quality factor (Q) and ring down time of these modes is sensitive to minute changes in the microresonator environment thus making it an excellent candidate for a sensor. Instead of the traditional time domain studies, we determine quality factors and ring down times as long as 53.8 +/- 0.6 ns (Q approximately 10(6)) from phase shift measurements using optical sources with sinusoidal intensity modulations of 300 kHz and below.