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High-throughput screening technologies are widely used for elucidating biological activities. These typically require trade-offs in assay specificity and sensitivity to achieve higher throughput. Microfluidic approaches enable rapid manipulation of small volumes and have found a wide range of applications in biotechnology providing improved control of reaction conditions, faster assays, and reduced reagent consumption. The integration of mass spectrometry with microfluidics has the potential to create high-throughput, sensitivity, and specificity assays. This review introduces the widely-used mass spectrometry ionization techniques that have been successfully integrated with microfluidics approaches such as continuous-flow system, microchip electrophoresis, droplet microfluidics, digital microfluidics, centrifugal microfluidics, and paper microfluidics. In addition, we discuss recent applications of microfluidics integrated with mass spectrometry in single-cell analysis, compound screening, and the study of microorganisms. Lastly, we provide future outlooks towards online coupling, improving the sensitivity and integration of multi-omics into a single platform., The integration of mass spectrometry with microfluidics has the potential to create high-throughput, sensitivity, and specificity assays.

The ability to manipulate droplets on a substrate using electric signals1—known as digital microfluidics—is used in optical2,3, biomedical4,5, thermal6 and electronic7 applications and has led to commercially available liquid lenses8 and diagnostics kits9,10. Such electrical actuation is mainly achieved by electrowetting, with droplets attracted towards and spreading on a conductive substrate in response to an applied voltage. To ensure strong and practical actuation, the substrate is covered with a dielectric layer and a hydrophobic topcoat for electrowetting-on-dielectric (EWOD)11-13; this increases the actuation voltage (to about 100 volts) and can compromise reliability owing to dielectric breakdown14, electric charging15 and biofouling16. Here we demonstrate droplet manipulation that uses electrical signals to induce the liquid to dewet, rather than wet, a hydrophilic conductive substrate without the need for added layers. In this electrodewetting mechanism, which is phenomenologically opposite to electrowetting, the liquid–substrate interaction is not controlled directly by electric field but instead by field-induced attachment and detachment of ionic surfactants to the substrate. We show that this actuation mechanism can perform all the basic fluidic operations of digital microfluidics using water on doped silicon wafers in air, with only ±2.5 volts of driving voltage, a few microamperes of current and about 0.015 times the critical micelle concentration of an ionic surfactant. The system can also handle common buffers and organic solvents, promising a simple and reliable microfluidic platform for a broad range of applications. A method of droplet manipulation is described that uses electrical signals to induce the liquid to dewet, rather than wet, a hydrophilic conductive surface.

For decades, there has been immense progress in miniaturizing analytical methods based on electrophoresis to improve sensitivity, and to reduce sample volumes, separation times, and/or equipment cost and space requirements, in applications ranging from analysis of biological samples, to environmental analysis to forensics. In the field of radiochemistry, where radiation-shielded laboratory space is limited, there has been great interest in harnessing the compactness, high efficiency, and speed of microfluidics to synthesize short-lived radiolabeled compounds. We recently proposed that analysis of these compounds could also benefit from miniaturization, and have been investigating capillary electrophoresis (CE) and hybrid microchip electrophoresis (hybrid-MCE) as alternatives to the typically-used high-performance liquid chromatography (HPLC). We previously showed separation of the positron-emission tomography (PET) imaging tracer 3′-deoxy-3′-fluorothymidine (FLT) from its impurities in a hybrid-MCE device with UV detection, with similar separation performance to HPLC, but with improved speed and lower sample volumes. In this paper, we have developed an integrated radiation detector to enable measurement of the emitted radiation from radiolabeled compounds. Though conventional radiation detectors have been incorporated into CE systems in the past, these approaches cannot be readily integrated into a compact hybrid-MCE device. We instead employed a solid-state avalanche photodiode (APD)-based detector for real-time, high-sensitivity beta particle detection. The integrated system can reliably separate [(18)F]FLT from its impurities and perform chemical identification via co-injection with non-radioactive reference standard. This system can quantitate samples with radioactivity concentrations as low as 114 MBq/mL (3.1 mCi/mL), which is sufficient for analysis of radiochemical purity of radiopharmaceuticals.

New radiolabeled probes for positron-emission tomography (PET) are providing an ever-increasing ability to answer diverse research and clinical questions and to facilitate the discovery, development, and clinical use of drugs in patient care. Despite the high equipment and facility costs to produce PET probes, many radiopharmacies and radiochemistry laboratories use a dedicated radiosynthesizer to produce each probe, even if the equipment is idle much of the time, to avoid the challenges of reconfiguring the system fluidics to switch from one probe to another. To meet growing demand, more cost-efficient approaches are being developed, such as radiosynthesizers based on disposable "cassettes," that do not require reconfiguration to switch among probes. However, most cassette-based systems make sacrifices in synthesis complexity or tolerated reaction conditions, and some do not support custom programming, thereby limiting their generality. In contrast, the design of the ELIXYS FLEX/CHEM cassette-based synthesizer supports higher temperatures and pressures than other systems while also facilitating flexible synthesis development. In this paper, the syntheses of 24 known PET probes are adapted to this system to explore the possibility of using a single radiosynthesizer and hot cell for production of a diverse array of compounds with wide-ranging synthesis requirements, alongside synthesis development efforts. Most probes were produced with yields and synthesis times comparable to literature reports, and because hardware modification was unnecessary, it was convenient to frequently switch among probes based on demand. Although our facility supplies probes for preclinical imaging, the same workflow would be applicable in a clinical setting.

PURPOSE: Metabolic imaging using [(18)F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B-cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1–2 h post-injection and a biological half-life of 2–5 h, is compatible with short-lived positron emitters such as fluorine-18 ((18)F, t(1/2) 110 min), enabling same-day imaging. METHODS: GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB), or thiol-reactive N-[2-(4-[(18)F]-fluorobenzamido)ethyl]maleimide ([(18)F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B-cell compartment in human CD20 transgenic mice (hCD20TM). [(18)F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [(18)F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution. RESULTS: The GAcDb was successfully (18)F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([(18)F]FB-GAcDb and [(18)F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 hour post injection enabling same-day imaging. [(18)F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [(18)F]FDG PET but with added specificity for the therapeutic target. CONCLUSIONS: [(18)F]FB-GAcDb and [(18)F]FBEM-GAcDb could monitor normal B cells and B-cell malignancies non-invasively and quantitatively in vivo. In contrast to [(18)F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.

The ability to manipulate droplets on a substrate using electric signals

Miniaturized synthesis of positron emission tomography (PET) tracers is poised to offer numerous advantages including reduced tracer production costs and increased availability of diverse tracers. While many steps of the tracer production process have been miniaturized, there has been relatively little development of microscale systems for the quality control (QC) testing process that is required by regulatory agencies to ensure purity, identity, and biological safety of the radiotracer before use in human subjects. Every batch must be tested, and in contrast with ordinary pharmaceuticals, the whole set of tests of radiopharmaceuticals must be completed within a short-period of time to minimize losses due to radioactive decay. By replacing conventional techniques with microscale analytical ones, it may be possible to significantly reduce instrument cost, conserve lab space, shorten analysis times, and streamline this aspect of PET tracer production. We focus in this work on miniaturizing the subset of QC tests for chemical identity and purity. These tests generally require high-resolution chromatographic separation prior to detection to enable the approach to be applied to many different tracers (and their impurities), and have not yet, to the best of our knowledge, been tackled in microfluidic systems. Toward this end, we previously explored the feasibility of using the technique of capillary electrophoresis (CE) as a replacement for the "gold standard" approach of using high-performance liquid chromatography (HPLC) since CE offers similar separating power, flexibility, and sensitivity, but can readily be implemented in a microchip format. Using a conventional CE system, we previously demonstrated the successful separation of non-radioactive version of a clinical PET tracer, 3'-deoxy-3'-fluorothymidine (FLT), from its known by-products, and the separation of the PET tracer 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)-cytosine (D-FAC) from its α-isomer, with sensitivity nearly as good as HPLC. Building on this feasibility study, in this paper, we describe the first effort to miniaturize the chemical identity and purity tests by using microchip electrophoresis (MCE). The fully automated proof-of-concept system comprises a chip for sample injection, a separation capillary, and an optical detection chip. Using the same model compound (FLT and its known by-products), we demonstrate that samples can be injected, separated, and detected, and show the potential to match the performance of HPLC. Addition of a radiation detector in the future would enable analysis of radiochemical identity and purity in the same device. We envision that eventually this MCE method could be combined with other miniaturized QC tests into a compact integrated system for automated routine QC testing of radiopharmaceuticals in the future. Graphical abstract Miniaturized quality control (QC) testing of batches of radiopharmaceuticals via microfluidic analysis. The proof-of-concept hybrid microchip electrophoresis (MCE) device demonstrated the feasibility of achieving comparable performance to conventional analytical instruments (HPLC or CE) for chemical purity testing.

A novel injector for microchip electrophoresis (MCE) has been designed and evaluated that achieves very high repeatability of injection volume suitable for quantitative analysis. It eliminates the injection biases in electrokinetic injection and the dependence on pressure and sample properties in hydrodynamic injection. The microfluidic injector, made of poly(dimethylsiloxane) (PDMS), operates similarly to an HPLC injection valve. It contains a channel segment (chamber) with a well-defined volume that serves as an "injection loop". Using on-chip microvalves, the chamber can be connected to the sample source during the "loading" step, and to the CE separation channel during the "injection" step. Once the valves are opened in the second state, electrophoretic potential is applied to separate the sample. For evaluation and demonstration purposes, the microinjector was connected to a 75μm ID capillary and UV absorbance detector. For single compounds, a relative standard deviation (RSD) of peak area as low as 1.04% (n=11) was obtained, and for compound mixtures, RSD as low as 0.40% (n=4) was observed. Using the same microchip, the performance of this new injection technique was compared to hydrodynamic injection and found to have improved repeatability and less dependence on sample viscosity. Furthermore, a non-radioactive version of the positron-emission tomography (PET) imaging probe, FLT, was successfully separated from its known 3 structurally-similar byproducts with baseline resolution, demonstrating the potential for rapid, quantitative analysis of impurities to ensure the safety of batches of short-lived radiotracers. Both the separation efficiency and injection repeatability were found to be substantially higher when using the novel volumetric injection approach compared to electrokinetic injection (performed in the same chip). This novel microinjector provides a straightforward way to improve the performance of hydrodynamic injection and enables extremely repeatable sample volume injection in MCE. It could be used in any MCE application where volume repeatability is needed, including the quantitation of impurities in pharmaceutical or radiopharmaceutical samples.