Electric Field-Modulated Electrospray Ionization Mass Spectrometry for Quantity Calibration and Mass Tracking.

Analyses conducted by electrospray ionization (ESI) mass spectrometry (MS) typically entail performing a number of preparatory steps, which include quantity calibration and mass calibration. Quantity calibration can be affected by signal noise, while mass calibration can be affected by instrumental drift if analyses are performed over an extended period of time. Here, we present two methods for achieving these calibrations using modulation of electrospray plume by alternating electric fields and demodulating the resulting MS ion currents. For this purpose, we use an ESI source fitted with three ring electrodes between the electrospray emitter and the mass spectrometer's inlet. One of these electrodes is supplied with a sine electric signal. Optionally, a nanoESI emitter is also placed between the ring electrodes and the mass spectrometer's orifice to supply calibrant ions. The ion currents, recorded with this setup, present wave-like features. In the first variant, using a triple quadrupole mass analyzer, the ion currents are subjected to data treatment by fast Fourier transform (FFT), and the resulting FFT magnitudes are correlated with analyte concentrations to produce a calibration plot. In the second variant, using a quadrupole time-of-flight mass analyzer, the mass spectra recorded at the analyte ion current maxima are mass-checked using the m/z value of the internal standard (injected via nanoESI emitter), which appears predominantly in the time intervals corresponding to the analyte ion current minima. The setup has been characterized using simulation software and optimized. Overall, the method enables the preparation of quantity calibration plots and monitoring (minor) m/z drifts during prolonged analyses.

Spontaneous Recycling of Electrosprayed Sample by Retrograde Motion of Microdroplets.

Here, we discuss an interesting phenomenon occurring spontaneously near the sample liquid meniscus at the tip of the electrospray emitter. While most ejected droplets move from the emitter tip toward the counter electrode, some of the droplets decelerate and move backward to the liquid meniscus. When they hit the surface of the liquid meniscus, they either merge with the bulk liquid or get recharged during intermittent contact with the liquid meniscus and immediately reaccelerate toward the counter electrode. In some cases, while in contact with the meniscus they spontaneously form a secondary Taylor cone and emit progeny droplets. This observation suggests that the amount of electric charge transferred to such a droplet is sufficient to surpass the Rayleigh limit. Similar effects were previously observed for water as well as for NaCl-water and ethanol-water mixtures. However, here we observed it for electrolyte solutions commonly used in electrospray ionization mass spectrometry: methanol-water solutions with the addition of ammonium acetate, formic acid, or ammonium hydroxide. The reported phenomenon reveals the ongoing recycling of sample liquid in electrosprays. Such recycling can contribute to enhancement of sample utilization efficiency in electrospray ionization.

Automation and Computerization of (Bio)sensing Systems.

Sensing systems necessitate automation to reduce human effort, increase reproducibility, and enable remote sensing. In this perspective, we highlight different types of sensing systems with elements of automation, which are based on flow injection and sequential injection analysis, microfluidics, robotics, and other prototypes addressing specific real-world problems. Finally, we discuss the role of computer technology in sensing systems. Automated flow injection and sequential injection techniques offer precise and efficient sample handling and dependable outcomes. They enable continuous analysis of numerous samples, boosting throughput, and saving time and resources. They enhance safety by minimizing contact with hazardous chemicals. Microfluidic systems are enhanced by automation to enable precise control of parameters and increase of analysis speed. Robotic sampling and sample preparation platforms excel in precise execution of intricate, repetitive tasks such as sample handling, dilution, and transfer. These platforms enhance efficiency by multitasking, use minimal sample volumes, and they seamlessly integrate with analytical instruments. Other sensor prototypes utilize mechanical devices and computer technology to address real-world issues, offering efficient, accurate, and economical real-time solutions for analyte identification and quantification in remote areas. Computer technology is crucial in modern sensing systems, enabling data acquisition, signal processing, real-time analysis, and data storage. Machine learning and artificial intelligence enhance predictions from the sensor data, supporting the Internet of Things with efficient data management.

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).

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.

Mass Specthoscope: A Hand-held Extendable Probe for Localized Noninvasive Sampling of Skin Volatome for Online Analysis.

Human skin emits a unique set of volatile organic compounds (VOCs). These VOCs can be probed in order to obtain physiological information about the individuals. However, extracting the VOCs that emanate from human skin for analysis is troublesome and time-consuming. Therefore, we have developed "Mass Specthoscope"─a convenient tool for rapid sampling and detecting VOCs emitted by human skin. The hand-held probe with a pressurized tip and wireless button enables sampling VOCs from surfaces and their transfer to the atmospheric pressure chemical ionization source of quadrupole time-of-flight mass spectrometer. The system was characterized using chemical standards (acetone, benzaldehyde, sulcatone, α-pinene, and decanal). The limits of detection are in the range from 2.25 × 10-5 to 3.79 × 10-5 mol m-2. The system was initially tested by detecting VOCs emanating from porcine skin spiked with VOCs as well as unspiked fresh and spoiled ham. In the main test, the skin of nine healthy participants was probed with the Mass Specthoscope. The sampling regions included the armpit, forearm, and forehead. Numerous skin-related VOC signals were detected. In the final test, one participant ingested a fenugreek drink, and the participant's skin surface was probed using the Mass Specthoscope hourly during the 8 h period. The result revealed a gradual release of fenugreek-related VOCs from the skin. We believe that this analytical approach has the potential to be used in metabolomic studies and following further identification of disease biomarkers─also in noninvasive diagnostics.

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.

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.

Hydrogel-enzyme micropatch array format for chemical mapping: A proof of concept.

Conventional sensing methods report on concentrations of analytes in a single point of sampled medium or provide an average value. However, distributions of substances on surfaces of sampled objects often exhibit intricate inhomogeneities. In order to obtain snapshots of the chemical distributions on surfaces, we have developed enzyme-loaded hydrogel arrays (5 × 5 and 10 × 10). The acrylic 10 × 10 array base contains 100 holes, which are filled with agarose hydrogel containing assay enzymes and substrates. Such arrays can be exposed to the analyzed surfaces to collect minute amounts of analytes. Following a brief incubation, they are subsequently visualized in a custom-built array reader device. The reader incorporates a light-emitting diode-based light source, miniature camera, and Raspberry Pi single-board computer. Two Python programs capture and analyze the images of the array to extract pixel saturation values corresponding to individual hydrogel micropatches. The method has been thoroughly optimized for mapping of glucose and lactic acid. The optimized parameters were: contact time, agarose concentration, substrate concentration, enzyme concentration ratio, and enzyme concentration. The array biosensor was further tested by mapping glucose distribution in fruit/vegetable cross-sections (apple, guava, and cucumber) and lactic acid distribution in cheese. We think that this new hydrogel-based chemical mapping method can find applications in studies related to food science, plant physiology, clinical chemistry, and forensics; wherever the distributions of analytes on the tested surfaces need to be assessed.

An economical setup for atmospheric pressure chemical ionization drift tube ion-mobility mass spectrometry.

Ion-mobility (IM) separations-performed in conjunction with mass spectrometry (MS)-increase selectivity of MS analyses. However, IM-MS instruments are costly, and many laboratories are only equipped with standard MS instruments without an IM separation stage. Therefore, it is appealing to upgrade the existing mass spectrometers with low-cost IM separation devices. Such devices can be constructed using widely available materials such as printed-circuit boards (PCBs). We demonstrate coupling of an economical PCB-based IM spectrometer (disclosed previously) with a commercial triple quadrupole (QQQ) mass spectrometer. The presented PCB-IM-QQQ-MS system incorporates an atmospheric pressure chemical ionization (APCI) source, drift tube comprising desolvation and drift regions, ion gates, and transfer line to the mass spectrometer. The ion gating is accomplished with the aid of two floated pulsers. The separated ions are divided into packets, which are sequentially introduced to the mass spectrometer. Volatile organic compounds (VOCs) are transferred with the aid of nitrogen gas flow from the sample chamber to the APCI source. The operation of the system has been demonstrated using standard compounds. The limits of detection for 2,4-lutidine, (-)-nicotine, and pyridine are 2.02 × 10-7 M, 1.54 × 10-9 mol, and 4.79 × 10-10 mol, respectively. The system was also used to monitor VOCs emitted from the porcine skin after exposure to nicotine patches, and VOCs released from meat undergoing the spoilage process. We believe this simple APCI-PCB-IM-QQQ-MS platform can be reproduced by others to augment the capabilities of the existing MS instrumentation.

Rapid skin biomarker discovery using hydrogel-phase sampling followed by semi-automated liquid-phase re-extraction high-resolution mass spectrometry.

A facile and rapid skin metabolomics protocol is proposed. The liquid microjunction-surface sampling probe system has been partly automated, and used in conjunction with hydrogel probes for skin metabolite analysis. A control device was built to precisely control the segmented solvent flow and analyte re-extraction into the liquid microjunction. This mode provides rapid online re-extraction of the analytes from hydrogel probes. Humectant was added to the hydrogel, and moist heat treatment was used to make the hydrogel probes rugged for sampling in the clinical setting. The developed method was validated for the analysis of choline - a putative biomarker of psoriasis. A linear relationship over six calibration levels from 3.18 × 10-5 to 3.18 × 10-4 mol m-2 has been obtained. The limit of detection was 6.6 × 10-6 mol m-2, while the recoveries range from 92 to 109%. The within-run and between-run precision were evaluated and the coefficients of variation range from 1.84 to 7.13%. Furthermore, the developed method has been used to screen patients (n = 10) and healthy participants (control group; n = 10) for psoriasis-related skin metabolites. Metabolomic profiling of the skin excretion-related signals identified potential biomarkers of psoriasis: choline, pipecolic acid, ornithine, urocanic acid, and methionine.

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.

Kinetic Profiling of Homogeneous and Heterogeneous Biocatalysts in Continuous Flow by Online Mass Spectrometry.

Enzyme kinetics is normally assessed by performing individual kinetic measurements using batch-type reactors (test tubes, microtiter plates), in which enzymes are mixed with different substrates. Some drawbacks of conventional methods are the large amounts of experimental materials, long analysis times, and limitations of spectrophotometry. Therefore, we have developed a method for facile determination of enzyme kinetics using online flow-based mass spectrometry. A concentration ramp of substrate or product was created by dynamically adjusting flow rates of pumps delivering stock solution of substrate and diluent. Precise kinetic measurements were performed by reaction product quantification and initial rate calculation. In the presence of ascending substrate concentrations, the rate of a target enzyme (penicillinase)-catalyzed hydrolysis was varied. By measuring the reaction product continuously, Michaelis constants (KM) could be calculated. The enzyme kinetic measurements for hydrolysis of penicillins were conducted based on this simple, rapid, and low sample consumption online flow device. In the homogeneous reaction, the KM values for amoxicillin, ampicillin, penicillin G, and penicillin V were 254.9 ± 14.5, 29.2 ± 0.3, 2.6 ± 0.1, and 5.4 ± 0.1 µM, respectively. In the heterogeneous reaction, the KM values for amoxicillin, ampicillin, penicillin G, and penicillin V were 408.9 ± 75.1, 114.4 ± 8.0, 21.8 ± 0.7, and 83.3 ± 4.8 µM, respectively. Apart from enzyme assay, the showcased method for the generation of temporal concentration ramps can be utilized to perform rapid quantity calibrations for mass spectrometric analyses.

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.

Rapid Acid/Base Switching in Flow Injection Analysis and Isocratic Elution Liquid Chromatography with Mass Spectrometric Detection for Improved Sensitivity.

Ion signals in electrospray ionization (ESI) mass spectrometry (MS) are affected by addition of acid or base. Acids or bases are typically added to samples to enhance detection of analytes in positive- or negative-ion mode, respectively. To carry out simultaneous monitoring of analytes with different ionogenic moieties by ESI-MS, a rapid acid/base switching system was developed. The system was further coupled with flow injection analysis (FIA) and liquid chromatography (LC) MS. The two variants enable detection of separated analytes immediately after alternating addition of acid and base. The methods were tested using a set of phospholipids (PLs) as analytes. The rapid acid/base switching enhanced signals of some of the PL analytes in both ion modes of MS. Both FIA-MS and LC-MS with acid/base switching show signal enhancements (∼1.3-23.2 times) of some analyte signals when compared with analysis conducted without acid/base switching. The proposed methods are suitable for simultaneous analysis of cationic and anionic analytes. The FIA-MS and LC-MS methods with acid/base switching were also applied in analysis of lipid extract from real samples (sausage and porcine liver). However, the FIA-MS results were affected by ionization competition and isobaric interference due to the complexity of the sample matrix and diversity of PL species. In contrast, the LC-MS mode provides adequate selectivity to observe signal enhancement for specific analyte ions. Overall, alternating addition of acid and base immediately before the ESI source can improve analytical performance without the need to carry out separate analyses targeting different types of analytes.

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.

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.

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.