Microfluidics enabled multi-omics triple-shot mass spectrometry for cell-based therapies.

In recent years, cell-based therapies have transformed medical treatment. These therapies present a multitude of challenges associated with identifying the mechanism of action, developing accurate safety and potency assays, and achieving low-cost product manufacturing at scale. The complexity of the problem can be attributed to the intricate composition of the therapeutic products: living cells with complex biochemical compositions. Identifying and measuring critical quality attributes (CQAs) that impact therapy success is crucial for both the therapy development and its manufacturing. Unfortunately, current analytical methods and tools for identifying and measuring CQAs are limited in both scope and speed. This Perspective explores the potential for microfluidic-enabled mass spectrometry (MS) systems to comprehensively characterize CQAs for cell-based therapies, focusing on secretome, intracellular metabolome, and surfaceome biomarkers. Powerful microfluidic sampling and processing platforms have been recently presented for the secretome and intracellular metabolome, which could be implemented with MS for fast, locally sampled screening of the cell culture. However, surfaceome analysis remains limited by the lack of rapid isolation and enrichment methods. Developing innovative microfluidic approaches for surface marker analysis and integrating them with secretome and metabolome measurements using a common analytical platform hold the promise of enhancing our understanding of CQAs across all "omes," potentially revolutionizing cell-based therapy development and manufacturing for improved efficacy and patient accessibility.

Early detection and metabolic pathway identification of T cell activation by in-process intracellular mass spectrometry.

BACKGROUND AIMS: In-process monitoring and control of biomanufacturing workflows remains a significant challenge in the development, production, and application of cell therapies. New process analytical technologies must be developed to identify and control the critical process parameters that govern ex vivo cell growth and differentiation to ensure consistent and predictable safety, efficacy, and potency of clinical products. METHODS: This study demonstrates a new platform for at-line intracellular analysis of T-cells. Untargeted mass spectrometry analyses via the platform are correlated to conventional methods of T-cell assessment. RESULTS: Spectral markers and metabolic pathways correlated with T-cell activation and differentiation are detected at early time points via rapid, label-free metabolic measurements from a minimal number of cells as enabled by the platform. This is achieved while reducing the analytical time and resources as compared to conventional methods of T-cell assessment. CONCLUSIONS: In addition to opportunities for fundamental insight into the dynamics of T-cell processes, this work highlights the potential of in-process monitoring and dynamic feedback control strategies via metabolic modulation to drive T-cell activation, proliferation, and differentiation throughout biomanufacturing.

Comparisons of healthy human brain temperature predicted from biophysical modeling and measured with whole brain MR thermometry.

Brain temperature is an understudied parameter relevant to brain injury and ischemia. To advance our understanding of thermal dynamics in the human brain, combined with the challenges of routine experimental measurements, a biophysical modeling framework was developed to facilitate individualized brain temperature predictions. Model-predicted brain temperatures using our fully conserved model were compared with whole brain chemical shift thermometry acquired in 30 healthy human subjects (15 male and 15 female, age range 18-36 years old). Magnetic resonance (MR) thermometry, as well as structural imaging, angiography, and venography, were acquired prospectively on a Siemens Prisma whole body 3 T MR scanner. Bland-Altman plots demonstrate agreement between model-predicted and MR-measured brain temperatures at the voxel-level. Regional variations were similar between predicted and measured temperatures (< 0.55 °C for all 10 cortical and 12 subcortical regions of interest), and subcortical white matter temperatures were higher than cortical regions. We anticipate the advancement of brain temperature as a marker of health and injury will be facilitated by a well-validated computational model which can enable predictions when experiments are not feasible.

Altering apparent optical properties with an array of semitransparent mesoscale structures.

The ability to control and optimize interactions between light and matter has much utility in engineering design. A well-researched way to achieve optical property modulation is via the use of optical metamaterials, which feature sub-wavelength scale surface structures. In this work, an alternative approach for modulating optical properties is presented using a composite surface modified with a periodic array of semitransparent hemispherical shell mesoscale structures which are larger than the incident light wavelength. A ray-tracing simulation approach is used to predict the optical behavior for an arrayed surface. At oblique angles of incidence, significant increases and decreases in apparent absorptance are achieved via the use of optically thick and thin shells, respectively. Additionally, a potential application to solar cells is described with optimal spectral behavior achieved via the use of semitransparent external structures.

Integrated photoelectrochemical energy storage cells prepared by benchtop ion soft landing.

The exceptional photochromic and redox properties of polyoxometalate anions, PW12O403-, have been exploited to develop an integrated photoelectrochemical energy storage cell for conversion and storage of solar energy. Elimination of strongly coordinating cations using benchtop ion soft landing leads to a ∼370% increase in the maximum power output of the device. Additionally, the photocathode displayed a pronounced color change from clear to blue upon irradiation, which warrants the potential application of the IPES cell in advanced smart windows and photochromic lenses.

Radiolytic redox interplay defines nanomaterial synthesis in liquids.

Irradiation of a liquid solution generates solvated electrons and radiolysis products, which can lead to material deposition or etching. The chemical environment dictates the dominant reactions. Radiolysis-induced reactions in salt solutions have substantially different results in pure water versus water-ammonia, which extends the lifetime of solvated electrons. We investigate the interplay between transport and solution chemistry via the example of solid silver formation from e-beam irradiation of silver nitrate solutions in water and water-ammonia. The addition of ammonia results in the formation of a secondary ring-shaped deposit tens of micrometers in diameter (formed over tens of seconds) around the primary point of deposition (formed over milliseconds). Simulations uncover the relative importance of oxidizing and reducing reactions and transport effects. Our explanation of this behavior involves mechanisms beyond ammonia's role in extending solvated electron lifetimes.

Sample-to-analysis platform for rapid intracellular mass spectrometry from small numbers of cells.

Real-time, advanced diagnostics of the biochemical state within cells remains a significant challenge for research and development, production, and application of cell-based therapies. The fundamental biochemical processes and mechanisms of action of such advanced therapies are still largely unknown, including the critical quality attributes that correlate to therapeutic function, performance, and potency and the critical process parameters that impact quality throughout cell therapy manufacturing. An integrated microfluidic platform has been developed for in-line analysis of a small number of cells via direct infusion nano-electrospray ionization mass spectrometry. Central to this platform is a microfabricated cell processing device that prepares cells from limited sample volumes removed directly from cell culture systems. The sample-to-analysis workflow overcomes the labor intensive, time-consuming, and destructive nature of existing mass spectrometry approaches for analysis of cells. By providing rapid, high-throughput analyses of the intracellular state, this platform enables untargeted discovery of critical quality attributes and their real-time, in-process monitoring.

Localized Sampling Enables Monitoring of Cell State via Inline Electrospray Ionization Mass Spectrometry.

Nascent advanced therapies, including regenerative medicine and cell and gene therapies, rely on the production of cells in bioreactors that are highly heterogeneous in both space and time. Unfortunately, advanced therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. This can be attributed largely to a void in existing process analytical technologies (PATs) capable of characterizing the secreted critical quality attribute (CQA) biomolecules that correlate with the final product quality. The Dynamic Sampling Platform (DSP) is a PAT for cell bioreactor monitoring that can be coupled to a suite of sensor techniques to provide real-time feedback on spatial and temporal CQA content in situ. In this study, DSP is coupled with electrospray ionization mass spectrometry and direct-from-culture sampling to obtain measures of CQA content in bulk media and the cell microenvironment throughout the entire cell culture process (≈3 weeks). Post hoc analysis of this real-time data reveals that sampling from the microenvironment enables cell state monitoring (e.g., confluence, differentiation). These results demonstrate that an effective PAT should incorporate both spatial and temporal resolution to serve as an effective input for feedback control in biomanufacturing.

Hydrodynamics of Vortical Gas Jets Coupled to Point-Like Suction.

Vortical jet flows in the Reynolds number (Re) range from 1000 to 3425 and swirl number (S) below 0.5, alone and in combination with suction through a small aperture, are experimentally investigated using optical visualization. Schlieren photography is employed to assess the vortical flow structure and establish the fundamental understanding of the source-to-sink gas-dynamic coupling, including the role played by flow rate, jet diameter, and the separation distance between the gas jet source and the suction sink. Compared to vortex-free jets, vortical jets for Re>2700 with swirl number S>0.27 experience earlier laminar-to-turbulent transition, with resulting rapid growth of the jet boundary. The ability to control growth of the jet expansion and mass and momentum dissipation into the surrounding is demonstrated via use of a coaxially aligned flow suction placed in the path of a jet. When a swirling jet is completely coupled with a flow suction, jet expansion is significantly suppressed. The suction/sink flow rate imposes a limit on the maximum input/source flow rate of gas jet to achieve complete coupling. Furthermore, there is a maximum distance over which effective coupling can occur, and for all Reynolds numbers considered this distance is shorter than the distance at which the jet structure breaks up into turbulent eddies in the absence of a sink.

Electrohydrodynamics of Gas-Assisted Electrospray Ionization Mass Spectrometry.

Gas-flow assistance is commonly used in ESI-MS for improved transport and desolvation, and fundamental understanding of the underlying phenomena is essential for improvement of aerodynamic interfaces that couple ESI sources and MS. For this purpose, an electrohydrodynamic model is developed for simulation of charged droplet dynamics under the combined effects of gas flow and electric fields with consideration of space charge interactions within the charged aerosol plume. The model is implemented in COMSOL by exploiting a formalism for establishing the droplet trajectories as a sequence of successive droplets ejected at a frequency defined by the electrospray current. The model is used to assess the effect of two distinct flow configurations and compared to the baseline care of electrospray without assist gas. The simulated flows are jet flows oriented coaxially with the ESI spray, with and without imposed vorticity (swirling). Droplet trajectory simulations of a bimodal droplet population consisting of large primary droplets and small progeny droplets reveal a unique capability for vortical assist jet flow to selectively transmit smaller droplets into the MS due to inertial separation. ESI-MS analysis of fluorinated phosphazines subjected to the different gas flow conditions supports the model predictions. The electrohydrodynamic model developed in this work provides a versatile tool to analyze and design aerodynamic ESI interfaces with rigorous incorporation of drag, inertia, and space-charge repulsion and can be used as a powerful simulation methodology for optimizing charged droplet transmission and ultimately improved analytical performance of gas-assisted ESI-MS workflows.

Dynamic mass spectrometry probe for electrospray ionization mass spectrometry monitoring of bioreactors for therapeutic cell manufacturing.

Large-scale manufacturing of therapeutic cells requires bioreactor technologies with online feedback control enabled by monitoring of secreted biomolecular critical quality attributes (CQAs). Electrospray ionization mass spectrometry (ESI-MS) is a highly sensitive label-free method to detect and identify biomolecules, but requires extensive sample preparation before analysis, making online application of ESI-MS challenging. We present a microfabricated, monolithically integrated device capable of continuous sample collection, treatment, and direct infusion for ESI-MS detection of biomolecules in high-salt solutions. The dynamic mass spectrometry probe (DMSP) uses a microfluidic mass exchanger to rapidly condition samples for online MS analysis by removing interfering salts, while concurrently introducing MS signal enhancers to the sample for sensitive biomolecular detection. Exploiting this active conditioning capability increases MS signal intensity and signal-to-noise ratio. As a result, sensitivity for low-concentration biomolecules is significantly improved, and multiple proteins can be detected from chemically complex samples. Thus, the DMSP has significant potential to serve as an enabling portion of a novel analytical tool for discovery and monitoring of CQAs relevant to therapeutic cell manufacturing.

DRILL Interface Makes Ion Soft Landing Broadly Accessible for Energy Science and Applications.

Polyoxometalates (POM) have been deposited onto carbon nanotube (CNT) electrodes using benchtop ion soft landing (SL) enabled by a vortex-confined electrohydrodynamic desolvation process. The device is based on the dry ion localization and locomotion (DRILL) mass spectrometry interface of Fedorov and co-workers. By adding electrospray emitters, heating the desolvation gas, and operating at high gas flow rates, it is possible to obtain stable ion currents up to -15 nA that are ideal for deposition. Coupled with ambient ion optics, this interface enables desolvated ions to be delivered to surfaces while excluding solvent and counterions. Electron microscopy of surfaces prepared using the device reveal discrete POM and no aggregation that degrades electrode performance. Characterization of POM-coated CNT electrodes in a supercapacitor showed an energy storage capacity similar to that achieved with SL in vacuum. For solutions that produce primarily a single ion by electrospray ionization, benchtop SL offers a simpler and less costly approach for surface modification with applications in catalysis, energy storage, and beyond.

DRILL: An Electrospray Ionization-Mass Spectrometry Interface for Improved Sensitivity via Inertial Droplet Sorting and Electrohydrodynamic Focusing in a Swirling Flow.

We describe the DRILL (dry ion localization and locomotion) device, which is an interface for electrospray ionization (ESI)-mass spectrometry (MS) that exploits a swirling flow to enable the use of inertial separation to prescribe different fates for electrosprayed droplets based on their size. This source adds a new approach to charged droplet trajectory manipulation which, when combined with hydrodynamic drag forces and electric field forces, provides a rich range of possible DRILL operational modes. Here, we experimentally demonstrate sensitivity improvement obtained via vortex-induced inertial sorting of electrosprayed droplets/ions: one possible mode of DRILL operation. In this mode, DRILL removes larger droplets while accelerating the remainder of the ESI plume, producing a high velocity stream of gas-enriched spray with small, highly charged droplets and ions and directing it toward the MS inlet. The improved signal-to-noise ratio (10-fold enhancement) in the detection of angiotensin I is demonstrated using the DRILL interface coupled to ESI-MS along with an improved limit of detection (10-fold enhancement, 100 picomole) in the detection of angiotensin II. The utility of DRILL has also been demonstrated by liquid chromatography (LC)-MS: a stable isotope labeled peptide cocktail was spiked into a complex native tissue extract and quantified by unscheduled multiple reaction monitoring on a TSQ Vantage. DRILL demonstrated improved signal strength (up to a 700-fold) for 8 out of 9 peptides and had no effects on the peak shape of the transitions.

Rapid Electron Beam Writing of Topologically Complex 3D Nanostructures Using Liquid Phase Precursor.

Advancement of focused electron beam-induced deposition (FEBID) as a versatile direct-write additive nanoscale fabrication technique has been inhibited by poor throughput, limited choice of precursors, and restrictions on possible 3D topologies. Here, we demonstrate FEBID using nanoelectrospray liquid precursor injection to grow carbon and pure metal nanostructures via direct decomposition and electrochemical reduction of the relevant precursors, achieving growth rates 10(5) times greater than those observed in standard gas-phase FEBID. Initiating growth at the free surface of a liquid pool enables fabrication of complex 3D carbon nanostructures with strong adhesion to the substrate. Deposition of silver microstructures at similar growth rates is also demonstrated as a promising avenue for future development of the technique.

Microfabricated ultrarapid desalting device for nanoelectrospray ionization mass spectrometry.

Salt removal is a prerequisite for electrospray ionization mass spectrometry (ESI-MS) analysis of biological samples. Rapid desalting and a low volume connection to an electrospray tip are required for time-resolved measurements. We have developed a microfabricated desalting device that meets both requirements, thus providing the foundational technology piece for transient ESI-MS measurements of complex biological liquid specimens. In the microfabricated device, the sample flows in a channel separated from a higher flow rate, salt-free counter solution by a monolithically integrated nanoporous alumina membrane, which can support pressure differences between the flow channels of over 600 kPa. Salt is removed by exploiting the large difference in diffusivities between salts and the typical ESI-MS target bioanalytes, e.g., peptides and proteins. We demonstrate the capability to remove 95% of salt from a sample solution in ∼1 s while retaining sufficiently high concentration of a relatively low molecular weight protein, cytochrome-c, for ESI-MS detection.

Ambient nanoelectrospray ionization with in-line microdialysis for spatially resolved transient biochemical monitoring within cell culture environments.

We have developed a new mass spectrometry (MS) based approach for continuous, spatially resolved in vitro biochemical detection and demonstrated its utility in a 3-D cell culture system. Extracellular liquid is passively extracted at a low flow rate (~10 nL/s) through a small bore silica capillary (ID 50 µm); inline microdialysis (MD) removes ions that would interfere with mass spectrometric analysis, and the sample is ionized by nanoelectrospray ionization (nano-ESI) and mass analyzed in a time-of-flight mass spectrometer. The system successfully detects low-volume, low-concentration releases of a small protein (8 µL of 5 µM cytochrome-c, molecular mass ~12 kDa) and exhibits ~1 min temporal resolution. The system also displays sensitivity to probe proximity to the sample release point. Due to the sensitivity of ESI-MS and its ability to simultaneously detect and identify multiple unanticipated biochemicals, this approach shows considerable potential as a biomarker discovery tool.

Scanning mass spectrometry probe: a scanning probe electrospray ion source for imaging mass spectrometry of submerged interfaces and transient events in solution.

The scanning mass spectrometry (SMS) probe is a new electrospray ion source. Motivated by the need for untargeted chemical imaging of dynamic events in solution, we have exploited an approach to electrospray ionization (ESI) that allows continuous sampling from a highly localized volume (approximately picoliters) in a liquid environment, softly ionizes molecules in the sample to render them amenable for mass spectrometric analysis, and sends the ions to the mass spectrometer. The key underlying concepts for our approach are (1) treating the electrospray capillary inlet as a chemical scanning probe and (2) locating the electrospray point as close as possible to the sampling point, thus providing the shortest response time possible. This approach enables chemical monitoring or imaging of submerged interfaces, providing access to details of spatial heterogeneity and temporal changes within liquid samples. It also permits direct access to liquid/ liquid interfaces for ESI-MS analysis. In this letter we report the first demonstrations of these capabilities of the SMS probe and describe some of the probe's basic characteristics.

Theory of Polymer Entrapped Enzyme Ultramicroelectrodes: Application to Glucose and Adenosine Triphosphate Detection.

We validate, by comparison with experimental data, a theoretical description of the amperometric response of microbiosensors formed via enzyme entrapment. The utility of the theory is further illustrated with two relevant examples supported by experiments: (1) quantitative detection of glucose and (2) quantitative detection of adenosine triphosphate (ATP).

Theory of Polymer Entrapped Enzyme Ultramicroelectrodes: Fundamentals.

We have developed a theoretical description of the amperometric response of ultramicroelectrode (UME) biosensors formed via enzyme entrapment. Our model allows for multiple enzymes and co-substrates, and results in a closed-form analytical expression for the steady-state current response of the disk ultramicroelectrode. It captures the effects of enzyme-entrapment domain size, species transport properties (which can be different in the polymer and surrounding electrolyte), enzyme kinetics, and axisymmetric diffusion. Assumptions inherent in the derivation are carefully explained, as are the resulting limits on the applicability of the results. The ability to theoretically predict the response of enzyme entrapped UMEs should enable improved design, operation, and data interpretation for this important class of biosensors.

Droplet growth and transition to coalescence in confined geometries.

A thermodynamic theory is developed to predict growth, rearrangement to a close-packed ensemble, and transition to a deformed or coalesced state for droplets in a confined space. For the close-packed configuration, analysis of forced interactions between confined droplets yields analytical criteria for predicting whether droplets will deform and if they will coalesce. Relevant nondimensional parameters are identified to generalize results in terms of energy barrier maps, and their use for predicting interacting droplet behavior is described.