Moderate Signal Enhancement in Electrospray Ionization Mass Spectrometry by Focusing Electrospray Plume with a Dielectric Layer around the Mass Spectrometer's Orifice.

Electrospray ionization (ESI) is among the commonly used atmospheric pressure ionization techniques in mass spectrometry (MS). One of the drawbacks of ESI is the formation of divergent plumes composed of polydisperse microdroplets, which lead to low transmission efficiency. Here, we propose a new method to potentially improve the transmission efficiency of ESI, which does not require additional electrical components and complex interface modification. A dielectric plate-made of ceramic-was used in place of a regular metallic sampling cone. Due to the charge accumulation on the dielectric surface, the dielectric layer around the MS orifice distorts the electric field, focusing the charged electrospray cloud towards the MS inlet. The concept was first verified using charge measurement on the dielectric material surface and computational simulation; then, online experiments were carried out to demonstrate the potential of this method in MS applications. In the online experiment, signal enhancements were observed for dielectric plates with different geometries, distances of the electrospray needle axis from the MS inlet, and various compounds. For example, in the case of acetaminophen (15 µM), the signal enhancement was up to 1.82 times (plate B) using the default distance of the electrospray needle axis from the MS inlet (d = 1.5 mm) and 12.18 times (plate C) using a longer distance (d = 7 mm).

Are Most Micrometer Droplets (>10 µm) Wasted in Electrospray Ionization? An Insight from Real-Time High-Speed Imaging.

Electrospray ionization (ESI) is one of the main techniques used in mass spectrometry (MS) of nonvolatile compounds. ESI is a disordered process, in which a large number of polydisperse droplets are projected from a fluctuating Taylor cone and jet protruding ESI emitter. Here, we disclose a system for sectioning electrospray plumes to discrete packets with millisecond and submillisecond lifetime, which are introduced to the MS orifice, one at a time. A high-speed camera was triggered at 10,000 frames per second to capture consecutive images of the electrospray packets transmitted to the mass spectrometer. We further correlated the high-speed images of electrospray packets with MS signals of a test analyte (acetaminophen). Following computational treatment of the images, we determined the number of droplet observations (<300), average diameter of droplets (∼10-20 µm), and average volume of droplets (few tens of picoliters) in the individual electrospray packets. The result shows that most micrometer droplets (>10 µm) do not have any significant contribution to the MS signals. This finding is in agreement with the prior conjecture that most of the MS signals are mainly attributed to nanodroplets. Based on this finding, one can deduce that only a small number of the initial microdroplets effectively carry analyte molecules that undergo ionization. We discuss that, in future, one may propose a way to "recharge" the emitted initial micrometer droplets to increase the efficiency of conventional ESI setups.

Scanning pH-metry for Observing Reversibility in Protein Folding.

One of the main factors affecting protein structure in solution is pH. Traditionally, to study pH-dependent conformational changes in proteins, the concentration of the H+ ions is adjusted manually, complicating real-time analyses, hampering dynamic pH regulation, and consequently leading to a limited number of tested pH levels. Here, we present a programmable device, a scanning pH-meter, that can automatically generate different types of pH ramps and waveforms in a solution. A feedback loop algorithm calculates the required flow rates of the acid/base titrants, allowing one, for example, to generate periodic pH sine waveforms to study the reversibility of protein folding by fluorescence spectroscopy. Interestingly, for some proteins, the fluorescence intensity profiles recorded in such a periodically oscillating pH environment display hysteretic behavior indicating an asymmetry in the sequence of the protein unfolding/refolding events, which can most likely be attributed to their distinct kinetics. Another useful application of the scanning pH-meter concerns coupling it with an electrospray ionization mass spectrometer to observe pH-induced structural changes in proteins as revealed by their varying charge-state distributions. We anticipate a broad range of applications of the scanning pH-meter developed here, including protein folding studies, determination of the optimum pH for achieving maximum fluorescence intensity, and characterization of fluorescent dyes and other synthetic materials.

Elevating Chemistry Research with a Modern Electronics Toolkit.

With the rapid development of high technology, chemical science is not as it used to be a century ago. Many chemists acquire and utilize skills that are well beyond the traditional definition of chemistry. The digital age has transformed chemistry laboratories. One aspect of this transformation is the progressing implementation of electronics and computer science in chemistry research. In the past decade, numerous chemistry-oriented studies have benefited from the implementation of electronic modules, including microcontroller boards (MCBs), single-board computers (SBCs), professional grade control and data acquisition systems, as well as field-programmable gate arrays (FPGAs). In particular, MCBs and SBCs provide good value for money. The application areas for electronic modules in chemistry research include construction of simple detection systems based on spectrophotometry and spectrofluorometry principles, customizing laboratory devices for automation of common laboratory practices, control of reaction systems (batch- and flow-based), extraction systems, chromatographic and electrophoretic systems, microfluidic systems (classical and nonclassical), custom-built polymerase chain reaction devices, gas-phase analyte detection systems, chemical robots and drones, construction of FPGA-based imaging systems, and the Internet-of-Chemical-Things. The technology is easy to handle, and many chemists have managed to train themselves in its implementation. The only major obstacle in its implementation is probably one's imagination.

Catalytic Oxygenation-Mediated Extraction as a Facile and Green Way to Analyze Volatile Solutes.

Sparging-based methods have long been used to liberate volatile organic compounds (VOCs) from liquid sample matrices prior to analysis. In these methods, a carrier gas is delivered from an external source. Here, we demonstrate "catalytic oxygenation-mediated extraction" (COME), which relies on biocatalytic production of oxygen occurring directly in the sample matrix. The newly formed oxygen (micro)bubbles extract the dissolved VOCs. The gaseous extract is immediately transferred to a separation or detection system for analysis. To start COME, dilute hydrogen peroxide is injected into the sample supplemented with catalase enzyme. The entire procedure is performed automatically-after pressing a "start" button, making a clapping sound, or triggering from a smartphone. The pump, valves, and detection system are controlled by a microcontroller board. For quality control and safety purposes, the reaction chamber is monitored by a camera linked to a single-board computer, which follows the enzymatic reaction progress by analyzing images of foam in real time. The data are instantly uploaded to the internet cloud for retrieval. The COME apparatus has been coupled on-line with the gas chromatography electron ionization mass spectrometry (MS) system, atmospheric pressure chemical ionization (APCI) MS system, and APCI ion-mobility spectrometry system. The three hyphenated variants have been tested in analyses of complex matrices (e.g., fruit-based drinks, whiskey, urine, and stored wastewater). In addition to the use of catalase, COME variants using crude potato pulp or manganese(IV) dioxide have been demonstrated. The technique is inexpensive, fast, reliable, and green: it uses low-toxicity chemicals and emits oxygen.

Sample Flow Rate Scan in Electrospray Ionization Mass Spectrometry Reveals Alterations in Protein Charge State Distribution.

Sample flow rate is one of the parameters that influence the sensitivity of electrospray ionization (ESI) mass spectrometry. By varying the sample flow rate, initial droplets of different sizes can be generated. Protein molecules in small droplets may form gas-phase ions earlier than the ones in large droplets. Here, we have systematically studied the influence of sample flow rate on the ESI charge state distributions (CSDs) of model proteins. A dedicated programmable sample flow rate scanner was used to infuse protein samples at different flow rates into a mass spectrometer. The synergistic influence of sample flow rate and various electrolytes (ammonium acetate, ammonium bicarbonate, ammonium formate, and piperidine) was studied. Significant alterations to the CSDs with increasing flow rate were observed. For example, in the presence of ammonium acetate, at low flow rates, lower charge states of proteins showed high intensities, while at high flow rates, ions related to higher charge states of proteins dominated the spectra. On the other hand, in the presence of piperidine, a significant reduction in the ion currents of all charge states was observed during the flow rate scan. Our observations suggest that at low flow rates the protein molecules follow a charged residue model of ionization mechanism, and at high flow rates-due to structural changes in protein molecules in large ESI droplets-the charged residue and chain ejection models can possibly coexist. We propose the use of sample flow rate scan as a way to reveal the influence of flow rate on the CSDs of the studied proteins.

Temporal Analysis of Conformers in the Course of pH Scan Directed by Urea-Urease Reaction-A "Protein Clock".

We demonstrate an analytical approach to monitor the effect of pH on protein tertiary structure. An autocatalytic enzyme reaction is used to stimulate refolding of proteins during real-time analysis. The method takes advantage of a nonlinear pH ramp generated by the urea-urease clock reaction. In this study, alterations to the structures of model proteins were monitored by mass spectrometry (charge pattern shift) and fluorometry (tryptophan fluorescence quenching). The pH measurements were conducted at different points of the sample flow line by different methods to minimize artifacts. Interestingly, different protein ions (corresponding to native and unfolded proteins) show distinct temporal mass spectral profiles, which reveal gradual refolding and concomitant deprotonation of higher charge state ions in the course of the clock reaction. Every multiply charged ion of a protein is characterized with its own "clock time". This approach does not require major modification of standard instrumentation. It enables determination of "high sensitivity" pH intervals for small and large molecules within a single experiment. Thus, it can be useful for characterizing the protein folding in response to pH change.

Chemical clocks, oscillations, and other temporal effects in analytical chemistry: oddity or viable approach?

Most analytical methods are based on "analogue" inputs from sensors of light, electric potentials, or currents. The signals obtained by such sensors are processed using certain calibration functions to determine concentrations of the target analytes. The signal readouts are normally done after an optimised and fixed time period, during which an assay mixture is incubated. This minireview covers another-and somewhat unusual-analytical strategy, which relies on the measurement of time interval between the occurrences of two distinguishable states in the assay reaction. These states manifest themselves via abrupt changes in the properties of the assay mixture (e.g. change of colour, appearance or disappearance of luminescence, change in pH, variations in optical activity or mechanical properties). In some cases, a correlation between the time of appearance/disappearance of a given property and the analyte concentration can be also observed. An example of an assay based on time measurement is an oscillating reaction, in which the period of oscillations is linked to the concentration of the target analyte. A number of chemo-chronometric assays, relying on the existing (bio)transformations or artificially designed reactions, were disclosed in the past few years. They are very attractive from the fundamental point of view but-so far-only few of them have be validated and used to address real-world problems. Then, can chemo-chronometric assays become a practical tool for chemical analysis? Is there a need for further development of such assays? We are aiming to answer these questions.

In-Oleo Microgasometry of Nanoliter-Scale Gas Volumes with Image-Based Detection.

Gasometric assays involve measurements of the amounts of gases that are released during physical or chemical processes. The available instrumentation for gasometric analysis is generally difficult to use and requires large sample volumes. In some cases, toxic materials (mercury) are involved in the analysis process. Here, we propose a microscale gasometric assay using silicone oil as matrix. Microliter-volume (∼2.5 µL) aqueous droplets, containing sample and reagent and/or catalyst, are introduced to the oil matrix and merged. Nanoliters of gaseous products are released to the surrounding oil matrix forming tiny spherical bubbles. Due to the huge differences between refractive indices of the released gas and the surrounding liquids (aqueous assay solution, oil), the gas bubbles are clearly visible from the top, when the assay reservoir is illuminated from the bottom with light-emitting diodes. The released gas bubbles are documented by recording videos of the assay reservoir. Individual frames within these videos are then analyzed by a graphical software to obtain diameters of every gas bubble at each time point. Following a fixed period of time (typically, 5-90 s) after the sample/reagent droplet merger, the volume of the released gas scales with the amount of the substrate (analyte) present in the sample droplet. For example, hydrogen peroxide can be decomposed to oxygen by 0.44 U catalase enzyme and semiquantified in the range up to ∼1.0 µmol. Glutathione can be detected in a two-step procedure ((1) oxidation of glutathione by hydrogen peroxide; (2) decomposition of the hydrogen peroxide residue by catalase).

High-Speed Schlieren Imaging of Vapor Formation in Electrospray Plume.

Previous mechanistic descriptions of electrosprays mostly focused on the dynamics of Taylor cones, initial droplets, and progeny droplets. However, vapor formation during droplet desolvation in an electrospray plume has not been discussed to a great extent. Here, we implement a double-pass on-axis schlieren high-speed imaging system to observe generation and propagation of vapors in an offline electrospray source under different conditions. Switching between turbulent and laminar vapor flow was observed for all of the scanned conditions, which may be attributed to randomly occurring disturbances in the sample flow inside the electrospray emitter. Calculation of mean vapor flow velocity and analysis of vapor flow patterns were performed using in-house developed image processing programs. Experiments performed at different electrospray voltages (0-6 kV), solvent flow rates (100-600 µL min-1), and methanol concentrations (50-100%), indicate only a weak dependency between electrospray voltage and mean vapor velocity, implying that the vapor is mostly neutral; thus, the vapor is not accelerated by electric field. On the other hand, electrospraying solutions of analytes (with mass 151 Da or 12 kDa) did not remarkably increase the overall vapor flow velocity. The source of vapor's velocity is attributed to the inertia of the electrospray droplets. Although there are some differences between a modern electrospray ionization (ESI) setup and the setup used in our experiment (e.g., using a higher flow rate and larger emitter), we believe the findings of our study can be projected to a modern ESI setup.

Observation of Protein Unfolding during pH Ramp Evoked by Lipase-Catalyzed Ester Hydrolysis.

Studies of protein folding often involve offline experimental methods such as titrating protein samples with denaturants or equilibrating them in the presence of denaturants. Here, we demonstrate an online analytical approach in which the protein structure is perturbed by a pH ramp evoked by immobilized lipase-catalyzed ester hydrolysis. Changes in the tertiary structure of the protein in response to a pH ramp (from approximately 6.3 to 2.8) are monitored using electrospray ionization mass spectrometry and spectrofluorometry. Interestingly, we discovered a side reaction of ammonium and formate leading to the production of cyanide that occurred during the ionization process. We also found that only certain protein analytes were bound to the formed cyanide species. Nevertheless, this problem was readily overcome by carefully selecting a specific ester substrate. Overall, the alterations in the charge-state distribution and fluorescence intensity─caused by the lipase-induced pH ramp─reveal conformational transitions in different proteins. In line with previous reports, the acid-induced denaturation of holo-myoglobin occurs through a two-step mechanism, which is supported by identification of protein-unfolding intermediates and the loss of noncovalent protein ligand (heme). The results─obtained using the developed catalytic method─are also consistent with the results of equilibrium-based experiments, while sample preparation steps are substantially reduced. The proposed approach simplifies the identification of the pH range that has the greatest impact on the protein structure. Thus, it has the potential to be a useful tool for studying protein conformational transitions in the course of pH changes.

Vending-Machine-Style Skin Excretion Sensing.

Skin metabolites show huge potential for use in clinical diagnostics. However, skin sampling and analysis workflows are tedious and time-consuming. Here, we demonstrate a vending-machine-style skin excretion sensing platform based on hydrogel-assisted sampling of skin metabolites. In this sensing platform, a sampling probe with hydrogel is held by a robotic arm. The robotic arm manoeuvres the probe to press it onto the forearm of a human subject. Due to the highly hydrophilic nature of the hydrogel, water-soluble metabolites─released by skin─are collected into the hydrogel, leaving behind the nonpolar metabolites. The probe is then inserted into a custom-made open port sampling interface coupled to an electrospray ion source of a high-resolution quadrupole-time-of-flight mass spectrometer. Metabolites in the hydrogel are immediately extracted by a solvent liquid junction in the interface and analyzed using the mass spectrometer. The ion current of the target analyte is displayed on a customized graphical user interface, which can also be used to control the key components of the analytical platform. The automated sampling and analysis workflow starts after the user inserts coins or presents an insurance card, presses a button, and extends an arm on the sampling area. The platform relies on low-cost mechanical and electronic modules (a robotic arm, a single-board computer, and two microcontroller boards). The limits of detection for standard analytes─arginine, citrulline, and histidine─embedded in agarose gel beds were 148, 205, and 199 nM, respectively. Various low-molecular-weight metabolites from human skin have been identified with the high-resolution mass spectrometer.

Portable fizzy extraction ion-mobility spectrometry system.

Fizzy extraction (FE) is a technique that utilizes effervescence phenomenon to extract volatile organic compounds (VOCs) from liquid matrices for subsequent analysis. To induce effervescence, a liquid sample is first pressurized (at âˆ¼ 150 kPa) with an extractant gas (here, nitrogen), and then rapidly depressurized. In this work, we combine an in-house-built FE system with a commercial ion-mobility spectrometry (IMS) module in order to develop a portable analytical platform for in-situ analysis of VOCs in liquid samples. The size and shape of the FE-IMS platform are similar to those of a typical airline catering trolley. Its operation is enabled by several electronic and electromechanical components (a single-board computer, two microcontroller boards, a relay board, six DC-DC converters, a pressure regulator, a solenoid valve, and a pinch valve). The platform can carry out the extraction procedure as well as acquire, process, and transmit the data to a cloud-storage service. A custom-designed graphical user interface allows the user to select one of the available operation modes: full spectrum mode, ion current profile mode, and cleaning mode. The interface also allows one to follow the analysis progress, display the final result, and upload it to the Internet cloud. The platform has been characterized using three standards: ethyl acetate, ethyl propanoate, and butanone; and their limits of detection are 4.51 × 10-8 M, 2.74 × 10-8 M, and 1.26 × 10-7 M, respectively. Furthermore, its ability to analyze real samples (alcoholic and non-alcoholic beverages) has been demonstrated.

Tuning Electrospray Ionization with Low-Frequency Sound.

Electrospray ionization (ESI) mass spectrometry (MS) is one of the key techniques used in biomolecular analysis nowadays. It relies on formation of polydisperse microdroplets, which undergo desolvation and liberate ions to the gas phase. Here we demonstrate low-frequency-sound-modulated ESI for analysis of biomolecules. By using a low-frequency (50-350 Hz) sound, it is possible to deflect electrospray microdroplets toward the mass spectrometer's orifice. Microdroplets of different sizes are deflected to a different extent leading to a partial size segregation. This effect leads to either an increase or decrease of MS signal intensity as well as signal-to-noise ratio. It also affects the selectivity of the ESI-MS analysis. The observations are rationalized by taking into account different pathways of ion formation and the likelihood of deflecting microdroplets of certain size. The online ESI-MS observations are supported with offline shadowgraphs obtained at varied sound frequencies, signal amplitudes, and phase shifts.

Telechemistry 2.0: Remote monitoring of fluorescent chemical reactions.

Implementation of the Internet-of-Things in chemistry research has the potential to improve research methodologies. Here, we describe a cloud-integrated real-time laboratory monitoring system for: (i) monitoring reactions involving fluorescent chemical species, and (ii) monitoring laboratory environment for safety purpose. A probe-type fluorescence detection system has been constructed to monitor reactions that involve fluorescent molecules. This device incorporates an in-house-built 3D-printed probe, two optical fibers, a light-emitting diode, a photoresistor, and a microcontroller board (MCB). The MCB relays experimental data to a single-board computer (SBC), which then uploads the data to a cloud-based platform (ThingSpeak) for data storage and visualization. The SBC is also connected to auxiliary sensors to measure relative alcohol vapor concentration, temperature, and humidity at different locations in the laboratory. The device has been validated and tested for its performance by monitoring a fluorescent chemical reaction (synthesis of fluorescent gold nanoclusters) for a period of 12 h.

BioChemPen for a Rapid Analysis of Compounds Supported on Solid Surfaces.

We present BioChemPen, a portable wireless biosensor device for rapid analysis of substances adsorbed on solid surfaces. The device takes advantage of (bio)luminescent reactions taking place in a hydrogel matrix. In a typical embodiment, the active element of this device is a hydrogel disk (chemotransducer) containing enzyme(s), electrolyte solution, and all of the necessary substrates. When the hydrogel is exposed to a solid sample surface containing the target analyte, light is produced. A photoresistor (phototransducer), placed in close proximity to the hydrogel disk, detects the light. The operation of the BioChemPen is enabled by a MicroPython PyBoard microcontroller board and other low-cost electronic modules. The obtained results are immediately uploaded to the Internet cloud. In one application, we demonstrate an analysis of hypochlorite-containing cleaning agents present on the surfaces of daily use objects by an assay based on hydrogel embedded with luminol and hydrogen peroxide. In another application, we use hydrogel embedded with luciferin, luciferase, and pyruvate kinase to detect adenosine triphosphate (ATP), and adenosine diphosphate (ADP), and link the ATP content with meat freshness. Lastly, we demonstrate the detection of organophosphate pesticides present on vegetables with the hydrogel containing acetylcholinesterase, choline oxidase, and horseradish peroxidase. The limits of detection for sodium hypochlorite, ATP, ADP, and chlorpyrifos-methyl (a pesticide) were 7.95 × 10-11, 2.73 × 10-13, 2.35 × 10-12, and 2.59 × 10-10 mol mm-2, respectively.

Flat Disc-Shaped Sampling Probe and Online Re-extraction Apparatus for Mass Spectrometric Analysis of Skin Metabolites: A Proof of Concept.

Sweat analysis provides an alternative and noninvasive way of clinical diagnostics. However, sampling and transferring sweat-derived samples to analytical instruments is challenging. In this report, we demonstrate a method utilizing a flat disc-shaped sampling probe, and a compatible re-extraction apparatus coupled online with extractive electrospray ionization (EESI) mass spectrometry (MS). The probe enables sampling of metabolites from a skin area of ∼2.2 cm2. The subsequent online re-extraction and analysis by EESI-MS further mitigates matrix effects caused by sweat components, thus eliminating sample preparation steps. The total analysis time is only 6 min. We have optimized the key parameters of the system, including flow rate of the nebulizing gas in ESI, pressure of the nebulizing gas in pneumatic sample nebulizer, flow rate of the solvent in ESI, and composition of extractant. The standard solutions (0.1 mL) were supplemented with 0.04 M sodium chloride to mimic the matrix effect normally observed in sweat samples. The method has been characterized with four chemical standards (positive-ion mode of histidine, leucine, urocanic acid; negative-ion mode of lactic acid). The limits of detection range from 1.09 to 95.9 nmol. We have further demonstrated the suitability of the method for analysis of sweat. An attempt was made to identify some of the recorded signals by product-ion scan and accurate/exact mass matching.

Automation of mass spectrometric detection of analytes and related workflows: A review.

The developments in mass spectrometry (MS) in the past few decades reveal the power and versatility of this technology. MS methods are utilized in routine analyses as well as research activities involving a broad range of analytes (elements and molecules) and countless matrices. However, manual MS analysis is gradually becoming a thing of the past. In this article, the available MS automation strategies are critically evaluated. Automation of analytical workflows culminating with MS detection encompasses involvement of automated operations in any of the steps related to sample handling/treatment before MS detection, sample introduction, MS data acquisition, and MS data processing. Automated MS workflows help to overcome the intrinsic limitations of MS methodology regarding reproducibility, throughput, and the expertise required to operate MS instruments. Such workflows often comprise automated off-line and on-line steps such as sampling, extraction, derivatization, and separation. The most common instrumental tools include autosamplers, multi-axis robots, flow injection systems, and lab-on-a-chip. Prototyping customized automated MS systems is a way to introduce non-standard automated features to MS workflows. The review highlights the enabling role of automated MS procedures in various sectors of academic research and industry. Examples include applications of automated MS workflows in bioscience, environmental studies, and exploration of the outer space.

Facilitating chemical and biochemical experiments with electronic microcontrollers and single-board computers.

Since the advent of modern science, researchers have had to rely on their technical skills or the support of specialized workshops to construct analytical instruments. The notion of the 'fourth industrial revolution' promotes construction of customized systems by individuals using widely available, inexpensive electronic modules. This protocol shows how chemists and biochemists can utilize a broad range of microcontroller boards (MCBs) and single-board computers (SBCs) to improve experimental designs and address scientific questions. We provide seven example procedures for laboratory routines that can be expedited by implementing this technology: (i) injection of microliter-volume liquid plugs into microscale capillaries for low-volume assays; (ii) transfer of liquid extract to a mass spectrometer; (iii) liquid-gas extraction of volatile organic compounds (called 'fizzy extraction'), followed by mass spectrometric detection; (iv) monitoring of experimental conditions over the Internet cloud in real time; (v) transfer of analytes to a mass spectrometer via a liquid microjunction interface, data acquisition, and data deposition into the Internet cloud; (vi) feedback control of a biochemical reaction; and (vii) optimization of sample flow rate in direct-infusion mass spectrometry. The protocol constitutes a primer for chemists and biochemists who would like to take advantage of MCBs and SBCs in daily experimentation. It is assumed that the readers have not attended any courses related to electronics or programming. Using the instructions provided in this protocol and the cited material, readers should be able to assemble simple systems to facilitate various procedures performed in chemical and biochemical laboratories in 1-2 d.