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On-the-Fly Mass Spectrometry in Digital Microfluidics Enabled by a Microspray Hole: Toward Multidimensional Reaction Monitoring in Automated Synthesis Platforms
Abstract
We report an approach for the online coupling of digital microfluidics (DMF) with mass spectrometry (MS) using a chip-integrated microspray hole (μSH). The technique uses an adapted electrostatic spray ionization (ESTASI) method to spray a portion of a sample droplet through a microhole in the cover plate, allowing its chemical content to be analyzed by MS. This eliminates the need for chip disassembly or the introduction of capillary emitters for MS analysis, as required by state-of-the-art. For the first time, this allows the essential advantage of a DMF device─free droplet movement─to be retained during MS analysis. The broad applicability of the developed seamless coupling of DMF and mass spectrometry was successfully applied to the study of various on-chip organic syntheses as well as protein and peptide analysis. In the case of a Hantzsch synthesis, we were able to show that the method is very well suited for monitoring even rapid chemical reactions that are completed in a few seconds. In addition, the strength of the low resource consumption in such on-chip microsyntheses was demonstrated by the example of enzymatic brominations, for which only a minute amount of a special haloperoxidase is required in the droplet. The unique selling point of this approach is that the analyzed droplet remains completely movable after the MS measurement and is available for subsequent on-DMF chip processes. This is illustrated here for the example of MS analysis of the starting materials in the corresponding droplets before they are combined to investigate the reaction progress by DMF-MS further. This technology enables the ongoing and almost unlimited tracking of multistep chemical processes in a DMF chip and offers exciting prospects for transforming digital microfluidics into automated synthesis platforms.
... A recent online DMF-MS system was developed by coupling DMF with MS through an on-chip microspray hole (DMF-lSH-MS) [125], as shown in Fig. 10(d). This unique electrostatic spray ionization can ionize a small portion of the droplet through the microhole, and the corresponding mass spectrum reveals the chemical contents. ...
... A recent online DMF-MS system was developed by coupling DMF with MS through an on-chip microspray hole (DMF-lSH-MS) [125], as shown in Fig. 10(d). This unique electrostatic spray ionization can ionize a small portion of the droplet through the microhole, and the corresponding mass spectrum reveals the chemical contents. ...
... DMF can be used to readily prepare a sample for off-chip detection, but fully integrated on-board detection provides significant advantages, especially for point-of-need measurements. To date, several integrated sensing modes have been demonstrated: nuclear magnetic resonance spectroscopy, 4,5 electrochemistry, [6][7][8] mass spectrometry, 9,10 chemiluminescence, 11 fluorescence, [12][13][14] surface plasmon resonance spectroscopy, 15 surface-enhanced Raman spectroscopy, 16 and colorimetry. 17,18 Passive radio-frequency (RF) sensors present an attractive transduction technique for DMF-integrated measurements that quantify changes in dielectric or magnetic properties of a liquid under test (LUT). ...
This paper demonstrates the integration of complementary split-ring resonators (CSSRs) with digital microfluidics (DMF) sample manipulation for passive, on-chip radio-frequency (RF) sensing. Integration is achieved by having the DMF and...
... We recently investigated biotransformations in a digital microfluidic (DMF) approach. 66 Building on our previous work with various integrated microfluidic devices for studying immobilised organocatalysts, [67][68][69] we have now transitioned these biotransformations into a microflow platform, leading to improved automation, reactor control, and expanded applications in biocatalytic processes. We used the vanadium-dependent haloperoxidase (CiVHPO) from the fungus Curvularia inaequalis as a model enzyme for evaluating a biocatalytic bromination reaction. ...
This work presents a novel microfluidic screening setup with real-time analytics for investigating reactions with immobilised biocatalysts. The setup combines microreactor technology, multi-reactor integration, and online (chip-)LC/MS analysis in a...
... 9 It has the characteristics of a high level of automation, simple internal structure and strong scalability, which has very high potential in the fields of medical diagnosis, chemical analysis and life science. In the past decade, many DMF-based platforms have been developed for automated detection, such as magnetic bead-based immunoassays, [10][11][12] cell culture, [13][14][15][16] protein analysis 17,18 and nucleic acid amplification. [19][20][21][22] Compared with the shortcomings of multi-step sample processing and cumbersome pretreatment processes in proteomics experiments, the ability of DMF to process multiple reagents at the same time makes it very suitable for this type of research. ...
The occurrence, development and prediction of various biological processes and diseases are inseparable from the protein-protein interaction (PPI), so it is extremely meaningful to perfect PPI networks. However, shortcomings of traditional detection methods, such as protein degradation, long detection time, complex operation, poor automation and high cost, restrict the rapid development of PPI networks. Here, a low-temperature digital microfluidic (LTDMF) system-based PPI detection box (LTDMF-PPI-Box) was developed to achieve rapid, lossless and efficient PPI detection. It consists of a PMMA shell, LTDMF-PPI and an integrated temperature control system. LTDMF reduces the PPI detection time from tens of hours to 1.5 hours by programmatically controlling the movement of droplets. Moreover, an integrated thermoelectric cooler (TEC) ensures an operating temperature of 4 °C, resulting in a protein protection up to 90%. The interaction between RILP protein and Rab26 protein which has a close connection to insulin secretion was demonstrated as a prototype to illustrate the feasibility of the LTDMF-PPI-Box. LTDMF with automation characteristics is capable of meeting the requirement of high-throughput screening of interacting proteins; therefore, the LTDMF-PPI-Box is expected to accelerate the establishment of the PPI network in the future.
... In addition to SPR, various other types of biosensors also exhibit different complementary advantages in coupling with mass spectrometry (Figure 4). For example, microfluidic chips are widely used in conjunction with nano ESI to provide excellent performance in reaction monitoring, sensitivity enhancement, biomolecule extraction, etc. [15,[97][98][99][100]. Surface acoustic wave (SAW) devices are used for sample delivery and assisted ionization to enable a real-time, high-throughput quantitative and qualitative analysis of heavy metals and various biomolecules in human serum [101,102]. ...
Sensitive and accurate detection of biomolecules by multiplexed methods is important for disease diagnosis, drug research, and biochemical analysis. Mass spectrometry has the advantages of high sensitivity, high throughput, and high resolution, making it ideal for biomolecular sensing. As a result of the development of atmospheric pressure mass spectrometry, researchers have been able to use a variety of means to identify target biomolecules and recognize the converted signals by mass spectrometry. In this review, three main approaches and tools are summarized for mass spectrometry sensing and biopsy techniques, including array biosensing, probe/pen-based mass spectrometry, and other biosensor–mass spectrometry coupling techniques. Portability and practicality of relevant mass spectrometry sensing methods are reviewed, together with possible future directions to promote the advancement of mass spectrometry for target identification of biomolecules and rapid detection of real biological samples.
... Digital microfluidics, which relies on the movement of drops, is, alternatively, relatively more immune to this problem [9][10][11][12]. One of the exciting areas of application of digital microfluidics is in the creation of micro-reactors [13][14][15]. The field has also been extended by the use of liquid marbles instead of drops. ...
Digital microfluidics, which relies on the movement of drops, is relatively immune to clogging problems, making it suited for micro-reactor applications. Here, graphene oxide paper of 100 μm thickness, fabricated by blade coating sedimented dispersions onto roughened substrates, followed by drying and mechanical exfoliation, was found to be relatively free of cracks and curling. It also exhibited high wettability and elasto-capillary characteristics. Possessing low enough stiffness, it could rapidly and totally self-wrap water drops of 20 μL volume placed 2 mm from its edge when oriented between 0 and 60° to the horizontal. This complete wrapping behavior allowed drops to be translated via movement of the paper over long distances without dislodgement notwithstanding accelerations and decelerations. An amount of 2 drops that were wrapped with separate papers, when collided with each other at speeds up to 0.64 m/s, were found to eschew coalescence. This portends the development of robust digital microfluidic approaches for micro-reactors.
... Currently, it is unknown whether the labile nature of LsVBPO1 is due to the purification process of the recombinant system. Further efforts to overcome its labile features, as well as new attempts, such as quasi-real-time bromination reaction monitoring using online coupling of digital microfluidics and mass spectrometry (Das et al. 2022), might shed light on the enzymatic nature of LsVBPO1. ...
The gene encoding vanadium-dependent bromoperoxidase (V-BPO) was cloned for the first time from the red alga Laurencia saitoi, which produces pharmaceutically promising brominated diterpenoids and triterpenoids. The molecular weight of V-BPO from L. saitoi (LsVBPO1) was the highest (77.0 kDa) among previously reported V-BPOs from Laurencia with a peptide insertion Asn194-Ser221 containing short Gln repeats. It shares approximately 60% amino acid sequence identity with V-BPOs from L. nipponica (LnVBPO1 and LnVBPO2) and L. okamurae (LoVBPO1a and LoVBPO2a). Heterologously expressed LsVBPO1 in Escherichia coli was partially purified and exhibited low but significant bromination activity of 38 U mg⁻¹ protein using monochlorodimedone. The pH optimum was 8.0, which was more alkaline than that for LnVBPOs and LoVBPO2a (pH 7.0). The Km for H2O2 was 0.04 mM, comparable to LnVBPO1 (0.026 mM), LnVBPO2 (0.025 mM), and LoVBPO2a (0.014 mM). LsVPBO1 retained its bromination activity until 45 °C for 20 min. When incubated at 55 °C for 20 min, catalytic activity decreased rapidly, as shown for LnVBPO1 and LoVBPO2a (retained at 45 °C, decreased at 55 °C) and LnVBPO2 (retained at 55 °C, decreased at 65 °C). Unlike other V-BPOs from Laurencia (LnVBPO1, LnVBPO2, and LoVBPO2a), dialysis and concentration during purification process were rapidly inactivated LsVBPO1, suggesting its structural instability.
In this minireview, digital microfluidics (DMF) is presented as an innovative option for automated organic synthesis at the microscale. Using DMF, valuable compounds, were successfully synthesized at the microliter scale. Additionally, organic reactions within the DMF devices were monitored using microcoil NMR and electrostatic spray ionization, allowing for kinetic analysis of rapid reactions. As DMF technology continues to advance and be integrated with synthetic organic chemistry, further miniaturization, automation, and acceleration of organic synthesis processes are anticipated.
The human body harbors diverse microbial communities essential for maintaining health and influencing disease processes. Droplet microfluidics, a precise and high-throughput platform for manipulating microscale droplets, has become vital in advancing microbiome research. This review introduces the foundational principles of droplet microfluidics, its operational capabilities, and wide-ranging applications. We emphasize its role in enhancing single-cell sequencing technologies, particularly genome and RNA sequencing, transforming our understanding of microbial diversity, gene expression, and community dynamics. We explore its critical function in isolating and cultivating traditionally unculturable microbes and investigating microbial activity and interactions, facilitating deeper insight into community behavior and metabolic functions. Lastly, we highlight its broader applications in microbial analysis and its potential to revolutionize human health research by driving innovations in diagnostics, therapeutic development, and personalized medicine. This review provides a comprehensive overview of droplet microfluidics' impact on microbiome research, underscoring its potential to transform our understanding of microbial dynamics and their relevance to health and disease.
Inspired by natural cryptic halogenation in C,C-bond formation, this study developed a synthetic approach combining biocatalytic bromination with transition-metal-catalyzed cross-coupling. Using the cyanobacterial AmVHPO, a robust and sustainable bromination-arylation cascade was created. Genetic modifications allowed enzyme immobilization, enhancing the compatibility between biocatalysis and chemocatalysis. This mild, efficient method for synthesizing biaryl compounds provides a foundation for future biochemo cascade reactions harnessing halogenation as a traceless directing tool.
Digital microfluidics enables the electrical actuation of microliter‐sized droplets on a planar surface, facilitating precise control and digitization of chemical processes. It is typically operated in a closed chip format, preventing evaporation and contamination which also complicates chemical analysis of the trapped droplets. Thus, subsequent analytics are usually carried out offline or with simple optical methods. A new method including a chip‐integrated microspray hole now allows on‐the‐fly mass spectrometry analysis on a digital microfluidics chip.
Low‐input proteomics, also referred to as micro‐ or nanoproteomics, has become increasingly popular as it allows one to elucidate molecular processes in rare biological materials. A major prerequisite for the analytics of minute protein amounts, e.g., derived from low cell numbers, down to single cells, is the availability of efficient sample preparation methods. Digital microfluidics (DMF), a technology allowing the handling and manipulation of low liquid volumes, has recently been shown to be a powerful and versatile tool to address the challenges in low‐input proteomics. Here, an overview is provided on recent advances in proteomics sample preparation using DMF. In particular, the capability of DMF to isolate proteomes from cells and small model organisms, and to perform all necessary chemical sample preparation steps, such as protein denaturation and proteolytic digestion on‐chip, are highlighted. Additionally, major prerequisites to making these steps compatible with follow‐up analytical methods such as liquid chromatography‐mass spectrometry will be discussed.
We report a new chip-integrated recyclable SERS substrate, achieved by photochemical deposition of silver nanoparticles onto titanium dioxide thin film. Facilitated by the photocatalytic activity of titanium dioxide the SERS...
The development of in situ techniques to quantitatively characterize the heterogeneous reactions is essential for understanding physicochemical processes in aqueous phase. In this work, a new approach coupling in situ UV–vis spectroscopy with a two‐step algorithm strategy is developed to quantitatively monitor heterogeneous reactions in a compact closed‐loop incorporation. The algorithm involves the inverse adding‐doubling method for light scattering correction and the multivariate curve resolution‐alternating least squares (MCR‐ALS) method for spectral deconvolution. Innovatively, theoretical spectral simulations are employed to connect MCR‐ALS solutions with chemical molecular structural evolution without prior information for reference spectra. As a model case study, the aqueous adsorption kinetics of bisphenol A onto polyamide microparticles are successfully quantified in a one‐step UV–vis spectroscopic measurement. The practical applicability of this approach is confirmed by rapidly screening a superior adsorbent from commercial materials for antibiotic wastewater adsorption treatment. The demonstrated capabilities are expected to extend beyond monitoring adsorption systems to other heterogeneous reactions, significantly advancing UV–vis spectroscopic techniques toward practical integration into automated experimental platforms for probing aqueous chemical processes and beyond.
Accurate dosing for various liquids, especially for highly viscous liquids, is fundamental in wide‐ranging from molecular crosslinking to material processing. Despite droppers or pipettes being widely used as pipetting devices, they are powerless for quantificationally splitting and dosing highly viscous liquids (>100 mPa s) like polymer liquids due to the intertwined macromolecular chains and strong cohesion energy. Here, a highly transparent photopyroelectric slippery (PS) platform is provided to achieve noncontact self‐splitting for liquids with viscosity as high as 15 000 mPa s, just with the assistance of sunlight and a cooling source to provide a local temperature difference (ΔT). Moreover, to guarantee the accuracy for pipetting liquids (>80%), the ultrathin MXene film (within a thickness of 20 nm) is self‐assembled as the photo‐thermal layers, overcoming the trade‐off between transparency and photothermal property. Compared with traditional pipetting strategies (≈1.3% accuracy for pipetting polymer liquids), this accurate microfluidic chip shows great potential in adhesive systems (bonding strength, twice than using the droppers or pipettes).
The closed-open digital microfluidic (DMF) system offers a versatile and powerful platform for various applications by combining the advantages of both closed and open structures. The current closed-open DMF system...
The worldwide pandemic covid-19 has changed people’s life and diagnostic landscape. The nucleic acid amplification test (NAT) as the gold standard for SARS-CoV-2 detection has been applied in containing its...
Ambient mass spectrometry imaging (MSI) is a powerful technique that allows for the simultaneous mapping of hundreds of molecules in biological samples under atmospheric conditions, requiring minimal sample preparation. We have developed nanospray desorption electrospray ionization (nano-DESI), a liquid extraction-based ambient ionization technique, which has proven to be sensitive and capable of achieving high spatial resolution. We have previously described an integrated microfluidic probe, which simplifies the nano-DESI setup, but is quite difficult to fabricate. Herein, we introduce a facile and scalable strategy for fabricating microfluidic devices for nano-DESI MSI applications. Our approach involves the use of selective laser-assisted etching (SLE) of fused silica to create a monolithic microfluidic probe (SLE-MFP). Unlike the traditional photolithography-based fabrication, SLE eliminates the need for the wafer bonding process and allows for automated, scalable fabrication of the probe. The chamfered design of the sampling port and ESI emitter significantly reduces the amount of polishing required to fine-tune the probe thereby streamlining and simplifying the fabrication process. We have also examined the performance of a V-shaped probe, in which only the sampling port is fabricated using SLE technology. The V-shaped design of the probe is easy to fabricate and provides an opportunity to independently optimize the size and shape of the electrospray emitter. We have evaluated the performance of SLE-MFP by imaging mouse tissue sections. Our results demonstrate that SLE technology enables the fabrication of robust monolithic microfluidic probes for MSI experiments. This development expands the capabilities of nano-DESI MSI and makes the technique more accessible to the broader scientific community.
Monoclonal antibodies are prone to form protein particles through aggregation, fragmentation, and oxidation under varying stress conditions during the manufacturing, shipping, and storage of parenteral drug products. According to pharmacopeia requirements, sub-visible particle levels need to be controlled throughout the shelf life of the product. Therefore, in addition to determining particle counts, it is crucial to accurately characterize particles in drug product to understand the stress condition of exposure and to implement appropriate mitigation actions for a specific formulation. In this study, we developed a new method for intelligent characterization of protein particles using micro-Raman spectroscopy on a digital microfluidic chip (DMF). Several microliters of protein particle solutions induced by stress degradation were loaded onto a DMF chip to generate multiple droplets for Raman spectroscopy testing. By training multiple machine learning classification models on the obtained Raman spectra of protein particles, eight types of protein particles were successfully characterized and predicted with high classification accuracy (93%-100%). The advantages of the novel particle characterization method proposed in this study include a closed system to prevent particle contamination, one-stop testing of morphological and chemical structure information, low sample volume consumption, reusable particle droplets, and simplified data analysis with high classification accuracy. It provides great potential to determine the probable root cause of the particle source or stress conditions by a single testing, so that an accurate particle control strategy can be developed and ultimately extend the product shelf-life.
The identification of tumor-specific antigens (TSAs) is critical for developing effective cancer immunotherapies. Mass spectrometry (MS)-based immunopeptidomics has emerged as a powerful tool for identifying TSAs as physical molecules. However, current immunopeptidomics platforms face challenges in measuring low-abundance TSAs in a precise, sensitive, and reproducible manner from small needle-tissue biopsies (<1 mg). Inspired by recent advances in single-cell proteomics, microfluidics technology offers a promising solution to these limitations by providing improved isolation of human leukocyte antigen (HLA)-associated peptides with higher sensitivity. In this context, we highlight the challenges in sample preparation and the rationale for developing microfluidics technology in immunopeptidomics. Additionally, we provide an overview of promising microfluidic methods, including microchip pillar arrays, valved-based systems, droplet microfluidics, and digital microfluidics, and discuss the latest research on their application in MS-based immunopeptidomics and single-cell proteomics.
Multibehavioral droplet manipulation in a precise and programmed manner is crucial for stoichiometry, biological virus detection, and intelligent lab-on-a-chip. Apart from fundamental navigation, merging, splitting, and dispensing of the droplets are required for being combined in a microfluidic chip as well. Yet, existing active manipulations including strategies from light to magnetism are arduous to use to split liquids on superwetting surfaces without mass loss and contamination, because of the high cohesion and Coanda effect. Here, we demonstrate a charge shielding mechanism (CSM) for platforms to integrate with a series of functions. In response to attachment of shielding layers from the bottom, the instantaneous and repeatable change of local potential on our platform achieves the desired loss-free manipulation of droplets, with a wide-ranging surface tension from 25.7 mN m-1 to 87.6 mN m-1, functioning as a noncontact air knife to cleave, guide, rotate, and collect reactive monomers on demand. With further refinement of the surface circuit, the droplets, just as the electron, can be programmed to be transported directionally at extremely high speeds of 100 mm s-1. This new generation of microfluidics is expected to be applied in the field of bioanalysis, chemical synthesis, and diagnostic kit.
Performance losses during the scaling-up of bioprocesses from the laboratory to the production scale are common obstacles caused by the formation of concentration gradients in bioreactors. To overcome these obstacles, so-called scale-down bioreactors are used to analyze selected large-scale conditions and are one of the most important predictive tools for the successful transfer of bioprocesses from the lab to the industrial scale. In this regard, cellular behavior is usually measured as an averaged value, neglecting possible cell-to-cell heterogeneity within the culture. In contrast, microfluidic single-cell cultivation (MSCC) systems offer the possibility of understanding cellular processes on a single-cell level. To date, most MSCC systems have a limited choice of cultivation parameters that are not representative of bioprocess-relevant environmental conditions. Herein, we critically review recent advances in MSCC that allow the cultivation and analysis of cells under dynamic (bioprocess-relevant) environmental conditions. Finally, we discuss what technological advances and efforts are needed to bridge the gap between current MSCC systems and the use of these systems as single-cell scale-down devices.
Highly sensitive and reproducible analysis of samples containing low amounts of protein is restricted by sample loss and the introduction of contaminants during processing. Here, we report an All-in-One digital microfluidic (DMF) pipeline for proteomic sample reduction, alkylation, digestion, isotopic labeling and analysis. The system features end-to-end automation, with integrated thermal control for digestion, optimized droplet additives for sample manipulation and analysis, and an automated interface to liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Dimethyl labeling was integrated into the pipeline to allow for relative quantification of the trace samples at the nanogram level, and the new pipeline was applied to evaluating cancer cell lines and cancer tissue samples. Several known proteins (including HSP90AB1, HSPB1, LDHA, ENO1, PGK1, KRT18, and AKR1C2) and pathways were observed between model breast cancer cell lines related to hormone response, cell metabolism, and cell morphology. Furthermore, differentially quantified proteins (such as PGS2, UGDH, ASPN, LUM, COEA1, and PRELP) were found in comparisons of healthy and cancer breast tissues, suggesting potential utility of the All-in-One pipeline for the emerging application of proteomic cancer sub-typing. In sum, the All-in-One pipeline represents a powerful new tool for automated proteome processing and analysis, with the potential to be useful for evaluating mass-limited samples for a wide range of applications.
The concept of digital microfluidics (DMF) enables highly flexible and precise droplet manipulation at a picoliter scale, making DMF a promising approach to realize integrated, miniaturized "lab-on-a-chip" (LOC) systems for research and clinical purposes. Owing to its simplicity and effectiveness, electrowetting-on-dielectric (EWOD) is one of the most commonly studied and applied effects to implement DMF. However, complex biomedical assays usually require more sophisticated sample handling and detection capabilities than basic EWOD manipulation. Alternatively, combined systems integrating EWOD actuators and other fluidic handling techniques are essential for bringing DMF into practical use. In this paper, we briefly review the main approaches for the integration/combination of EWOD with other microfluidic manipulation methods or additional external fields for specified biomedical applications. The form of integration ranges from independently operating sub-systems to fully coupled hybrid actuators. The corresponding biomedical applications of these works are also summarized to illustrate the significance of these innovative combination attempts.
“Green Synthesis” has been broadly applied in drug synthesis and nanomaterial preparation. The integration of automated micro‐synthesis and high‐throughput screening systems is a key way to achieve the goal of green manufacturing. Here, a one‐click green synthesis platform is designed that integrates droplets arrays‐based micro‐synthesis for Raman enhancement performance high‐throughput screening of MNPs@Cu‐MOF (AuNPs@, AgNPs@, PtNPs@Cu‐MOF) under room temperature and normal pressure. The fully integrated ultrasound units can achieve rapid multi‐component mixing in the droplet array systems, and significantly accelerate materials growth as well as enhance encapsulation of noble metal nanoparticles into Cu‐MOF structures. Based on such integrated systems, structures with high Raman enhancement performance can be high‐throughput evaluation and easily screened out. The results indicated that the AgNPs@Cu‐MOF exhibited the best Raman enhancement performance, which is ≈8 times higher than pure AgNPs. Such a green and integrated platform provides a way for automated multi‐type composites fabrication, which can be expanded to multiple fields in the future via combined robotic platforms and artificial intelligence technology.
Exploring unknown matter inside an ultrasmall volume object, such as unknown subcellular matter in a single cell, requires an analytical technique that offers identification of these unknown matter in terms of molecular structures and properties. Mass spectrometry is considered one of the best choices for such analysis of unknown matter because mass spectrometry can be utilized to identify unknown matter according to its mass-to-charge ratio. However, the use of mass spectrometry for exploring unknown matter in such a small world has been greatly impeded due to the lack of tools to conduct the sampling of complex and heterogeneous analytes with ultrasmall volumes below the picoliter order, which is the volume order of a mammalian cell. We believe that nanofluidics would be an ideal tool to resolve the critical issue owing to its ability to sample such fluid samples with ultrasmall volumes ranging from the picoliter to zeptoliter order. Thus, the integration of such nanofluidic ability into mass spectrometers would open up future avenues for the potential of mass spectrometry to explore unknown subcellular matter at a nano scale. In this perspective, we first discuss published findings in the exploration of the applicability of microfluidics/nanofluidics to mass spectrometry, then address critical issues toward nanofluidics-based mass spectrometry, and finally depict a personal outlook on the future of this field to resolve challenges on global and universal scales.
We report a novel approach for surface-enhanced Raman spectroscopy (SERS) detection in digital microfluidics (DMF). This is made possible by a microspray hole (μSH) that uses an electrostatic spray (ESTAS) for sample transfer from inside the chip to an external SERS substrate. To realize this, a new ESTAS-compatible stationary SERS substrate was developed and characterized for sensitive and reproducible SERS measurements. In a proof-of-concept study, we successfully applied the approach to detect various analyte molecules using the DMF chip and achieved micro-molar detection limits. Moreover, this technique was exemplarily employed to study an organic reaction occurring in the DMF device, providing vibrational spectroscopic data.
The use of flow reactors in biocatalysis has increased significantly in recent years. Chemists have begun to design flow systems that even allow new biocatalytic reactions to take place. This concept article will focus on the design of flow systems that have allowed enzymes to go beyond their limits in batch. The case is made for moving towards fully continuous systems. With flow chemistry increasingly seen as an enabling technology for automated synthesis, and with advancements in AI‐assisted enzyme design, there is a real possibility to fully automate the development and implementation of a continuous biocatalytic processes. This will lead to significantly improved enzyme processes for synthesis.
The implementation of continuous flow technology is critical towards enhancing the application of photochemical reactions for industrial process development. However, there are significant time and resource constraints associated with translating discovery scale vial-based batch reactions to continuous flow scale-up conditions. Herein we report the development of a droplet microfluidic platform, which enables high-throughput reaction discovery in flow to generate pharmaceutically relevant compound libraries. This platform allows for enhanced material efficiency, as reactions can be performed on picomole scale. Furthermore, high-throughput data collection via on-line ESI mass spectrometry facilitates the rapid analysis of individual, nanoliter-sized reaction droplets at acquisition rates of 0.3 samples/s. We envision this high-throughput screening platform to expand upon the robust capabilities and impact of photochemical reactions in drug discovery and development.
We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.
Experimental procedures for chemical synthesis are commonly reported in prose in patents or in the scientific literature. The extraction of the details necessary to reproduce and validate a synthesis in a chemical laboratory is often a tedious task requiring extensive human intervention. We present a method to convert unstructured experimental procedures written in English to structured synthetic steps (action sequences) reflecting all the operations needed to successfully conduct the corresponding chemical reactions. To achieve this, we design a set of synthesis actions with predefined properties and a deep-learning sequence to sequence model based on the transformer architecture to convert experimental procedures to action sequences. The model is pretrained on vast amounts of data generated automatically with a custom rule-based natural language processing approach and refined on manually annotated samples. Predictions on our test set result in a perfect (100%) match of the action sequence for 60.8% of sentences, a 90% match for 71.3% of sentences, and a 75% match for 82.4% of sentences.
Microfluidic methods for studying cell invasion can be subdivided into those in which cells invade into free space and those in which cells invade into hydrogels. The former techniques allow straightforward extraction of subpopulations of cells for RNA sequencing, while the latter preserve key aspects of cell interactions with the extracellular matrix (ECM). Here, we introduce “cell invasion in digital microfluidic microgel systems” (CIMMS), which bridges the gap between them, allowing the stratification of cells on the basis of their invasiveness into hydrogels for RNA sequencing. In initial studies with a breast cancer model, 244 genes were found to be differentially expressed between invading and noninvading cells, including genes correlating with ECM-remodeling, chemokine/cytokine receptors, and G protein transducers. These results suggest that CIMMS will be a valuable tool for probing metastasis as well as the many physiological processes that rely on invasion, such as tissue development, repair, and protection.
Technologies such as batteries, biomaterials and heterogeneous catalysts have functions that are defined by mixtures of molecular and mesoscale components. As yet, this multi-length-scale complexity cannot be fully captured by atomistic simulations, and the design of such materials from first principles is still rare1–5. Likewise, experimental complexity scales exponentially with the number of variables, restricting most searches to narrow areas of materials space. Robots can assist in experimental searches6–14 but their widespread adoption in materials research is challenging because of the diversity of sample types, operations, instruments and measurements required. Here we use a mobile robot to search for improved photocatalysts for hydrogen production from water15. The robot operated autonomously over eight days, performing 688 experiments within a ten-variable experimental space, driven by a batched Bayesian search algorithm16–18. This autonomous search identified photocatalyst mixtures that were six times more active than the initial formulations, selecting beneficial components and deselecting negative ones. Our strategy uses a dexterous19,20 free-roaming robot21–24, automating the researcher rather than the instruments. This modular approach could be deployed in conventional laboratories for a range of research problems beyond photocatalysis. A mobile robot autonomously operates analytical instruments in a wet chemistry laboratory, performing a photocatalyst optimization task much faster than a human would be able to.
Despite the precise controllability of droplet samples in digital microfluidic (DMF) systems, their capability in isolating single cells for long-time culture is still limited: typically, only a few cells can be captured on an electrode. Although fabricating small-sized hydrophilic micropatches on an electrode aids single-cell capture, the actuation voltage for droplet transportation has to be significantly raised, resulting in a shorter lifetime for the DMF chip and a larger risk of damaging the cells. In this work, a DMF system with 3D microstructures engineered on-chip is proposed to form semi-closed micro-wells for efficient single-cell isolation and long-time culture. Our optimum results showed that approximately 20% of the micro-wells over a 30 × 30 array were occupied by isolated single cells. In addition, low-evaporation-temperature oil and surfactant aided the system in achieving a low droplet actuation voltage of 36V, which was 4 times lower than the typical 150 V, minimizing the potential damage to the cells in the droplets and to the DMF chip. To exemplify the technological advances, drug sensitivity tests were run in our DMF system to investigate the cell response of breast cancer cells (MDA-MB-231) and breast normal cells (MCF-10A) to a widely used chemotherapeutic drug, Cisplatin (Cis). The results on-chip were consistent with those screened in conventional 96-well plates. This novel, simple and robust single-cell trapping method has great potential in biological research at the single cell level.
Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s⁻¹ is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.
Microcoil nuclear magnetic resonance (NMR) has been interfaced with digital microfluidics (DMF) and is applied to monitor organic reactions in organic solvents as a proof of concept. DMF permits droplets to be moved and mixed inside the NMR spectrometer to initiate reactions while using sub‐microliter volumes of reagent, opening up the potential to follow the reactions of scarce or expensive reagents. By setting up the spectrometer shims on a reagent droplet, data acquisition can be started immediately upon droplet mixing and is only limited by the rate at which NMR data can be collected, allowing the monitoring of fast reactions. Here we report a cyclohexene carbonate hydrolysis in dimethylformamide and a Knoevenagel condensation in methanol/water. This is to our knowledge the first time rapid organic reactions in organic solvents have been monitored by high field DMF‐NMR. The study represents a key first step towards larger DMF‐NMR arrays that could in future serve as discovery platforms, where computer controlled DMF automates mixing/titration of chemical libraries and NMR is used to study the structures formed and kinetics in real time.
There is a growing drive in the chemistry community to exploit rapidly growing robotic technologies along with artificial intelligence-based approaches. Applying this to chemistry requires a holistic approach to chemical synthesis design and execution. Here, we outline a universal approach to this problem beginning with an abstract representation of the practice of chemical synthesis that then informs the programming and automation required for its practical realization. Using this foundation to construct closed-loop robotic chemical search engines, we can generate new discoveries that may be verified, optimized, and repeated entirely automatically. These robots can perform chemical reactions and analyses much faster than can be done manually. As such, this leads to a road map whereby molecules can be discovered, optimized, and made on demand from a digital code.
This work describes the interfacing of electrowetting-on-dielectric based digital microfluidic (DMF) sample preparation devices with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). The DMF droplet manipulation technique was adopted to facilitate drug distribution and metabolism assays in droplet scale, while ambient mass spectrometry (MS) was exploited for the analysis of dried samples directly on the surface of the DMF device. Although ambient MS is well-established for bio- and forensic analyses directly on surfaces, its interfacing with DMF is scarce and requires careful optimization of the surface-sensitive processes, such as sample precipitation and the subsequent desorption/ionization. These technical challenges were addressed and resolved in this study by making use of the high mechanical, thermal, and chemical stability of SU-8. In our assay design, SU-8 served as the dielectric layer for DMF as well as the substrate material for DAPPI-MS. The feasibility of SU-8 based DMF devices for DAPPI-MS was demonstrated in the analysis of selected pharmaceuticals following on-chip liquid-liquid extraction or an enzymatic dealkylation reaction. The lower limits of detection were in the range of 1–10 pmol per droplet (0.25–1.0 µg/mL) for all pharmaceuticals tested.
In this paper, we present a digital microfluidic droplet sorting platform to achieve automated droplet sorting based on fluorescent detection. We design and fabricate a kind of digital microfluidic chip for manipulating nano-liter-sized liquid droplets, and the chip is integrated with a fluorescence-initiated feedback system for real-time sorting control. The driving and sorting characteristics of fluorescent droplets encapsulating fluorescent-labeled particles are studied on this platform. The droplets dispensed from on-chip reservoir electrode are transported to a fluorescence detection site and sorted according to their fluorescence signals. The fluorescent droplets and non-fluorescent droplets are successfully separated and the number of fluorescent particles inside each droplet is quantified by its fluorescent intensity. We realize droplet sorting at 20 Hz and obtain a linear relationship between the fluorescent particle concentrations and the fluorescence signals. This work is easily adapted for sorting out fluorescent-labeled microparticles, cells and bacteria and thus has the potential of quantifying catalytic or regulatory bio-activities.
We characterize shapes and volumes of droplets generated in PDMS T-junctions and assess the use of this type of microfluidic device to generate droplets suitable for the study of nucleation. Water droplets were formed in oil in a PDMS T-junction and subsequently stored. Droplet volume reproducibility and stability were investigated from acquired micrographs. By theoretically analyzing the influence of the mean volume of a population of droplets on the estimation of nucleation rates, we have shown that deviations in mean volumes can seriously affect the estimates, unless such deviation is smaller than 10%. This condition is fulfilled if experiments are repeated using the same microdevice. Measured droplet polydispersity remained low enough to treat the droplets as monodisperse. Immersing the microdevice in a water bath mitigates solvent evaporation, and allows for very accurate temperature control. Finally, a screening procedure was used to select the ideal operating conditions to obtain droplets with the desired sizes. Applying this method in devices with increasing T-junction cross sectional area, we have demonstrated a scaling-up of droplet volumes close to an order of magnitude while tuning the droplet shape, i.e., the average length to width ratio, at values between 1 and 1.2.
The discovery of chemical reactions is an inherently unpredictable and time-consuming process1. An attractive alternative is to predict reactivity, although relevant approaches, such as computer-aided reaction design, are still in their infancy2. Reaction prediction based on high-level quantum chemical methods is complex3, even for simple molecules. Although machine learning is powerful for data analysis4,5, its applications in chemistry are still being developed6. Inspired by strategies based on chemists' intuition7, we propose that a reaction system controlled by a machine learning algorithm may be able to explore the space of chemical reactions quickly, especially if trained by an expert8. Here we present an organic synthesis robot that can perform chemical reactions and analysis faster than they can be performed manually, as well as predict the reactivity of possible reagent combinations after conducting a small number of experiments, thus effectively navigating chemical reaction space. By using machine learning for decision making, enabled by binary encoding of the chemical inputs, the reactions can be assessed in real time using nuclear magnetic resonance and infrared spectroscopy. The machine learning system was able to predict the reactivity of about 1,000 reaction combinations with accuracy greater than 80 per cent after considering the outcomes of slightly over 10 per cent of the dataset. This approach was also used to calculate the reactivity of published datasets. Further, by using real-time data from our robot, these predictions were followed up manually by a chemist, leading to the discovery of four reactions.
By modification of glasses with ultrafast laser radiation and subsequent wet-chemical etching (here named SLE = selective laser-induced etching), precise 3D structures have been produced, especially in quartz glass (fused silica), for more than a decade. By the combination of a three-axis system to move the glass sample and a fast 3D system to move the laser focus, the SLE process is now suitable to produce more complex structures in a shorter time. Here we present investigations which enabled the new possibilities. We started with investigations of the optimum laser parameters to enable high selective laser-induced etching: surprisingly, not the shortest pulse duration is best suited for the SLE process. Secondly we investigated the scaling of the writing velocity: a faster writing speed results in higher selectivity and thus higher precision of the resulting structures, so the SLE process is now even suitable for the mass production of 3D structures. Finally we programmed a printer driver for commercial CAD software enabling the automated production of complex 3D glass parts as new examples for lab-on-a-chip applications such as nested nozzles, connectors and a cell-sorting structure.
We introduce digital microfluidic interface between solid-phase microextraction (SPME) and liquid chromatography. In the new system, a SPME fiber is used to extract analytes from a complex sample, and is then inserted into a DMF device. Solvent droplets are then used to extract analytes from the fiber with continuous actuation, followed by analysis with liquid chromatography (LC) and mass spectrometry (MS). Compared to conventional methods, the new technique allows for fast, efficient desorption of analytes from the SPME fiber using a small volume of solvent. The miniaturized system allows for preconcentration of analytes and was applied the analysis of steroid hormones in human urine.
The emerging technology of digital microfluidics is opening up the possibility of performing radiochemistry at the microliter scale to produce tracers for positron emission tomography (PET) labeled with fluorine-18 or other isotopes. Working at this volume scale not only reduces reagent costs but also improves specific activity (SA) by reducing contamination by the stable isotope. This technology could provide a practical means to routinely prepare high-SA tracers for applications such as neuroimaging and could make it possible to routinely achieve high SA using synthesis strategies such as isotopic exchange. Reagent droplets are controlled electronically, providing high reliability, a compact control system, and flexibility for diverse syntheses with a single-chip design. The compact size may enable the development of a self-shielded synthesizer that does not require a hot cell. This article reviews the progress of this technology and its application to the synthesis of PET tracers.
We introduce an automated method to facilitate in-line coupling of digital microfluidics (DMF) with HPLC-MS, using a custom, 3D-printed manifold and a custom plugin to the popular open-source control system, DropBot. The method was designed to interface directly with commercial autosamplers (with no prior modification), suggesting that it will be widely accessible for end-users. The system was demonstrated to be compatible with samples dissolved in aqueous buffers and neat methanol and was validated by application to a common steroid-labeling derivatization reaction. We propose that the methods described here will be useful for a wide range of applications, combining the automated sample processing power of DMF with the resolving and analytical capacity of HPLC-MS.
Nanostructured microelectrodes (NMEs) are three-dimensional electrodes that have superb sensitivity for electroanalysis. Here we report the integration of NMEs with the versatile fluid-handling system digital microfluidics (DMF), for eventual application to distributed diagnostics outside of the laboratory. In the new methods reported here, indium tin oxide DMF top plates were modified to include Au NMEs as well as counter and pseudoreference electrodes. The new system was observed to outperform planar sensing electrodes of the type that are typically integrated with DMF. A rubella virus (RV) IgG immunoassay was developed to evaluate the diagnostic potential for the new system, relying on magnetic microparticles coated with RV particles and analysis by differential pulse voltammetry. The limit of detection of the assay (0.07 IU mL(-1)) was >100× below the World Health Organization defined cut-off for rubella immunity. The sensitivity of the integrated device and its small size suggest future utility for distributed diagnostics.
We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3[thinsp]s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations[mdash]for example, a 10[thinsp]s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population.
We present a theoretical model to calculate the volume of non-wetting bubbles and droplets in segmented microflows from given dimensions of the microchannel and measured lengths of bubbles and droplets. Despite the importance of these volumes in interpreting experiments of reaction kinetics and transport phenomena, an accurate model like the one we present here did not yet exist. The model has its theoretical basis in the principle of interfacial energy minimization and is set up such that volume calculations are possible for a wide variety of channel geometries. We succesfully validated our model with the 3D numerical energy minimization code Surface Evolver for the three most commonly used channel geometries in the field of microfluidics and provide accurate user-friendly equations for these geometries.
Microfluidic devices offer great advantages in integrating sample processes, minimizing sample and reagent volumes, and increasing analysis speed, while mass spectrometry detection provides high information content, is sensitive, and can be used in quantitative analyses. The coupling of microfluidic devices to mass spectrometers is becoming more common with the strengths of both systems being combined to analyze precious and complex samples. This review summarizes select achievements published between 2010 and July 2014 in novel coupling between microfluidic devices and mass spectrometers. The review is subdivided by the types of ionization sources employed, and the different microfluidic systems used.
Copyright © 2014 Elsevier B.V. All rights reserved.
The past two decades have seen far-reaching progress in the development of microfluidic systems for use in the chemical and biological sciences. Here we assess the utility of microfluidic reactor technology as a tool in chemical synthesis in both academic research and industrial applications. We highlight the successes and failures of past research in the field and provide a catalogue of chemistries performed in a microfluidic reactor. We then assess the current roadblocks hindering the widespread use of microfluidic reactors from the perspectives of both synthetic chemistry and industrial application. Finally, we set out seven challenges that we hope will inspire future research in this field.
Instrument miniaturization is one of the critical issues to improve sensitivity, speed, throughput, and to reduce the cost of analysis. Microfluidics possesses the ability to handle small sample amounts, with minimal concerns related to sample loss and cross-contamination, problems typical for standard fluidic manipulations. Moreover, the native properties of microfluidics provide the potential for high-density, parallel sample processing, and high-throughput analysis. Recently, the coupling of microfluidic devices to mass spectrometry, especially electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), has attracted an increasing interest and produced tremendous achievements. The interfaces between microfluidics and mass spectrometry are one of the primary focused problems. In this review, we summarize the latest achievements since 2008 in the field of the technologies and applications in the combining of microfluidics with ESI-MS and MALDI-MS. The integration of several analytical functions on a microfluidic device such as sample pretreatment and separations before sample introduction into the mass spectrometer is also discussed.
In this work, we introduce an approach to merge droplet microfluidics with an HPLC/MS functionality on a single chip to analyze the contents of individual droplets. This is achieved by a mechanical rotor-stator interface that precisely positions a microstructured PEEK rotor on a microfluidic chip in a pressure-tight manner. The developed full-body fused silica chip, manufactured by selective laser-induced etching, contained a segmented microflow compartment followed by a packed HPLC channel, which were interconnected by the microfluidic PEEK rotor on the fused silica lid with hair-thin through-holes. This enabled the targeted and leakage-free transfer of 10 nL fractions of droplets as small as 25 nL from the segmented microflow channel into the HPLC compartment that operated at pressures of up to 60 bar. In a proof of concept study, this approach was successfully applied to monitor reactions at the nanoliter scale and to distinguish the formed enantiomers.
The molecules of today — the medicines that cure diseases, the agrochemicals that protect our crops, the materials that make life convenient — are becoming increasingly sophisticated thanks to advancements in chemical synthesis. As tools for synthesis improve, molecular architects can be bold and creative in the way they design and produce molecules. Several emerging tools at the interface of chemical synthesis and data science have come to the forefront in recent years, including algorithms for retrosynthesis and reaction prediction, and robotics for autonomous or high-throughput synthesis. This Primer covers recent additions to the toolbox of the data-savvy organic chemist. There is a new movement in retrosynthetic logic, predictive models of reactivity and chemistry automata, with considerable recent engagement from contributors in diverse fields. The promise of chemical synthesis in the information age is to improve the quality of the molecules of tomorrow through data-harnessing and automation. This Primer is written for organic chemists and data scientists looking to understand the software, hardware, data sets and tactics that are commonly used as well as the capabilities and limitations of the field. The Primer is split into three main components covering retrosynthetic logic, reaction prediction and automated synthesis. The former of these topics is about distilling the strategy of multistep synthesis to a logic that can be taught to a computer. The section on reaction prediction details modern tools and models for developing reaction conditions, catalysts and even new transformations based on information-rich data sets and statistical tools such as machine learning. Finally, we cover recent advances in the use of liquid handling robotics and autonomous systems that can physically perform experiments in the chemistry laboratory.
ConspectusThe digitization of chemistry is not simply about using machine learning or artificial intelligence systems to process chemical data, or about the development of ever more capable automation hardware; instead, it is the creation of a hard link between an abstracted process ontology of chemistry and bespoke hardware for performing reactions or exploring reactivity. Chemical digitization is therefore about the unambiguous development of an architecture, a chemical state machine, that uses this ontology to connect precise instruction sets to hardware that performs chemical transformations. This approach enables a universal standard for describing chemistry procedures via a chemical programming language and facilitates unambiguous dissemination of these procedures. We predict that this standard will revolutionize the ability of chemists to collaborate, increase reproducibility and safety, as we all as optimize for cost and efficiency. Most importantly, the digitization of chemistry will dramatically reduce the labor needed to make new compounds and broaden accessible chemical space. In recent years, the developments of automation in chemistry have gone beyond flow chemistry alone, with many bespoke workflows being developed not only for automating chemical synthesis but also for materials, nanomaterials, and formulation production. Indeed, the leap from fixed-configuration synthesis machines like peptide, nucleic acid, or dedicated cross-coupling engines is important for developing a truly universal approach to "dial-a-molecule". In this case, a key conceptual leap is the use of a batch system that can encode the chemical reagents, solvent, and products as packets which can be moved around the system, and a graph-based approach for the description of hardware modules that allows the compilation of chemical code that runs on, in principle, any hardware. Further, the integration of sensor systems for monitoring and controlling the state of the chemical synthesis machine, as well as high resolution spectroscopic tools, is vital if these systems are to facilitate closed-loop autonomous experiments. Systems that not only make molecules and materials, but also optimize their function, and use algorithms to assist with the development of new synthetic pathways and process optimization are also possible. Here, we discuss how the digitization of chemistry is happening, building on the plethora of technological developments in hardware and software. Importantly, digital-chemical robot systems need to integrate feedback from simple sensors, e.g., conductivity or temperature, as well as online analytics in order to navigate process space autonomously. This will open the door to accessing known molecules (synthesis), exploring whether known compounds/reactions are possible under new conditions (optimization), and searching chemical space for unknown and unexpected new molecules, reactions, and modes of reactivity (discovery). We will also discuss the role of chemical knowledge and how this can be used to challenge bias, as well as define and expand synthetically accessible chemical space using programmable robotic chemical state machines.
Digital microfluidics has the potential to minimize and automate reactions in biochemical labs. However, the complexity of drop manipulation and sample preparation on-chip has limited its incorporation into daily workflow. In this paper, we report a novel method for flexible sample delivery on digital microfluidics in a wide volume range spanning four orders of magnitude from picoliters to nanoliters. The method is based on the phenomenon of satellite droplet ejection, triggered by a sudden change in the strength of the electric field across a drop on a hydrophobic dielectric surface. By precisely modulating the actuation signal with convenient external electric controls, satellite droplet ejection can be turned on to dispense samples or turned off to transport picking-up drops. A pico-dosing design is presented and validated in this work to demonstrate the direct and flexible on-chip sample delivery. This approach could pave the way for the acceptance of microfluidics as a common platform for daily reactions to realize lab-on-a-chip.
Microdrop generation with excellent controllability and volume precision is of paramount significance for a large variety of microfluidic applications. In this work, we propose a new configuration comprising only stripped electrodes of rectangular shape for the closed electrowetting-on-dielectric digital microfluidic (EWOD DMF) system and investigate its parallel microdrop generation outcomes via a numerical approach. The microfluidic droplet motion is solved by a finite-volume scheme on a fixed computational domain. The numerical model is verified by an experimental study of microdrop production from an EWOD DMF device with three different electrode designs. After model verification, we examine the influences of the equilibrium contact angle and the spacing of the microchannel on stripped electrode based microdrop generation outcomes, and discover five different regimes including the phenomena of satellite droplet formation and separation cessation. Despite the various generation outcomes, the daughter droplet size is found to vary linearly with a dimensionless EWOD parameter κ*. More importantly, for all successful generations, the deviation of the daughter droplet size from that of the stripped electrode is smaller than 3.5%, which even reaches zero in proper conditions. This new configuration can be utilized as a convenient alternative for electrowetting-induced parallel microdrop production with excellent precision and controllability.
Machine learning (ML) has emerged as a general, problem-solving paradigm with many applications in computer vision, natural language processing, digital safety, or medicine. By recognizing complex patterns in data, ML bears the potential to modernise the way how many chemical challenges are approached. In this review, an introduction to ML is given from the perspective of synthetic chemistry: starting from the fundamentals regarding algorithms and best-practice workflows, the review covers different applications of machine learning in synthesis planning, property prediction, molecular design, and reactivity prediction. In particular, different approaches of representing and utilizing organic molecules will be discussed – providing synthetic chemists both with the understanding and the tools required to apply machine learning in the context of their research, and pointers for further studying.
Digital microfluidics (DMF) is a liquid handling technique that has been demonstrated to automate biological experimentation in a low-cost, rapid, and programmable manner. This review discusses the role of DMF as a “digital bioconverter”—a tool to connect the digital aspects of the design–build–learn cycle with the physical execution of experiments. Several applications are reviewed to demonstrate the utility of DMF as a digital bioconverter, namely, genetic engineering, sample preparation for sequencing and mass spectrometry, and enzyme-, immuno-, and cell-based screening assays. These applications show that DMF has great potential in the role of a centralized execution platform in a fully integrated pipeline for the production of novel organisms and biomolecules. In this paper, we discuss how the function of a DMF device within such a pipeline is highly dependent on integration with different sensing techniques and methodologies from machine learning and big data. In addition to that, we examine how the capacity of DMF can in some cases be limited by known technical and operational challenges and how consolidated efforts in overcoming these challenges will be key to the development of DMF as a major enabling technology in the computer-aided biology framework.
With illustrative examples from our laboratories, we make the case for how our digital and machine-based world is impacting on the assembly of our functional molecules in a more sustainable fashion.
Our equipment today is better able to deliver efficiencies through labour-saving methods, optimisation and scale-up, helping to minimise solvent usage, improve reaction telescoping and enhance other principles for process intensification. The machinery gives us confidence in safely handling reactive gases, exotherms and the other potentially hazardous events through remote control methods. Continuous flow chemistry processing plays a central role in delivering on this new holistic systems approach to synthesis.
While LC-MS-based proteomics with high nanograms to micrograms of total protein has become routine, the analysis of samples derived from low cell numbers is challenged by factors such as sample losses, or difficulties encountered with the manual manipulation of small liquid volumes. Digital microfluidics (DMF) is an emerging technique for miniaturized and automated droplet manipulation, which has been proposed as a promising tool for proteomic sample preparation. However, proteome analysis of samples prepared on-chip by DMF has previously been unfeasible, due to incompatibility with down-stream LC-MS instrumentation. To overcome these limitations, we here developed protocols for bottom-up LC-MS based proteomics sample preparation of as little as 100 mammalian cells on a commercially available digital microfluidics device. To this end, we developed effective cell lysis conditions optimized for DMF, as well as detergent-buffer systems compatible with downstream proteolytic digestion on DMF chips and subsequent LC-MS analysis. A major step was the introduction of the single-pot, solid-phase-enhanced sample preparation (SP3) approach on-chip, which allowed to remove salts and anti-fouling polymeric detergents, thus rendering sample preparation by DMF compatible with LC-MS-based proteome analysis. Application of DMF-SP3 to the proteome analysis of Jurkat T cells led to the identification of up to 2,500 proteins from approximately 500 cells, and up to 1,200 proteins from approximately 100 cells on an Orbitrap mass spectrometer, emphasizing the high compatibility of DMF-SP3 with low protein input and minute volumes handled by DMF. Taken together, we demonstrate the first sample preparation workflow for proteomics on a DMF chip device reported so far, allowing the sensitive analysis of limited biological material.
Pairing prediction and robotic synthesis
Progress in automated synthesis of organic compounds has been proceeding along parallel tracks. One goal is algorithmic prediction of viable routes to a desired compound; the other is implementation of a known reaction sequence on a platform that needs little to no human intervention. Coley et al. now report preliminary integration of these two protocols. They paired a retrosynthesis prediction algorithm with a robotically reconfigurable flow apparatus. Human intervention was still required to supplement the predictor with practical considerations such as solvent choice and precise stoichiometry, although predictions should improve as accessible data accumulate for training.
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In recent years microcoils and related structures have been developed to increase the mass sensitivity of nuclear magnetic resonance spectroscopy, allowing this extremely powerful analytical technique to be extended to small sample volumes (<5 μl). In general, microchannels have been used to deliver the samples of interest to these microcoils; however, these systems tend to have large dead volumes and require more complex fluidic connections. Here, we introduce a two-plate digital microfluidic (DMF) strategy to interface small-volume samples with NMR microcoils. In this system, a planar microcoil is surrounded by a copper plane that serves as the counter-electrode for the digital microfluidic device, allowing for precise control of droplet position and shape. This feature allows for the user-determination of the orientation of droplets relative to the main axes of the shim stack, permitting improved shimming and a more homogeneous magnetic field inside the droplet below the microcoil, which leads to improved spectral lineshape. This, along with high-fidelity droplet actuation, allows for rapid shimming strategies (developed over decades for vertically oriented NMR tubes) to be employed, permitting the determination of reaction-product diffusion coefficients as well as quantitative monitoring of reactive intermediates. We propose that this system paves the way for new and exciting applications for in situ analysis of small samples by NMR spectroscopy.
A cryogenic ion trap vibrational spectrometer is combined with a microfluidic chip reactor in a proof-of-principle experiment on the Hantzsch cyclization reaction forming 2-amino-4-phenyl thiazole from phenacyl bromide and thiourea. First, the composition of the reaction solution is characterized using electrospray-ionization mass spectrometry combined with two-color infrared photodissociation (IRPD) spectroscopy. The latter yields isomer-specific vibrational spectra of the reaction intermediates and products. A comparison to results from electronic structure calculations then allows for an unambiguous structural assignment and molecular-level insights into the reaction mechanism. Subsequently, we demonstrate that isomeric and isobaric ions can be selectively monitored online with low process time, i.e. using a single IRPD wavelength per isomer, as the chip reaction parameters are varied.
In vitro cofactor supply and regeneration has been a major obstacle for biocatalytic processes, in particular on a large scale. Peroxidases often suffer from inactivation by their oxidative co‐factor. Combining photocatalysis and biocatalysis offers an innovative solution to this problem, but lacks atom economy due to the sacrificial electron donors needed. Herein, we show that redox‐active buffers or even water alone can serve as efficient, biocompatible electron sources, when combined with photocatalysis. Mechanistic investigations revealed first insights into the possibilities and limitations of this approach and allowed for adjusting the reaction conditions to the specific needs of biocatalytic transformations. Proof‐of‐concept for the applicability of this photobiocatalytic reaction setup was given by enzymatic halogenations.
A polyimide microfluidic chip with a microhole emitter (Ø 10–12 μm) created on top of a microchannel by scanning laser ablation has been designed for nanoelectrospray ionization (spyhole-nanoESI) to couple microfluidics with mass spectrometry. The spyhole-nanoESI showed higher sensitivity compared to standard ESI and microESI from the end of the microchannel. The limits of detection (LOD) for peptide with the spyhole-nanoESI MS reached 50 pM, which was 600 times lower than that with standard ESI. The present microchip emitter allows the analysis of small volumes of samples. As an example, a small cell lung cancer biomarker, neuron-specific enolase (NSE), was detected by monitoring the transition of its unique peptide with the spyhole-nanoESI MS/MS. NSE at 0.2 nM could be well identified with a signal to noise ratio (S/N) of 50, and thereby its LOD was estimated to be 12 pM. The potential application of the spyhole-nanoESI MS/MS in cancer diagnosis was further demonstrated with the successful detection of 2 nM NSE from 1 μL of human serum. Before the detection, the serum sample spiked with NSE was first depleted with immune spin column, then desalted by centrifugal filter device, and finally digested by trypsin, without any other complicated preparation steps. The concentration matched the real condition of clinical samples. In addition, the microchips can be disposable to avoid any cross contamination. The present technique provides a highly efficient way to couple microfluidics with MS, which brings additional values to various microfluidics and MS-based analysis.
A range of academic and industrial fields exploit interfacial polymerization in producing fibers, capsules and films. Although widely used, measurements of reaction kinetics remain challenging and rarely reported, due to film thinness and reaction rapidity. Here, polyamide film formation is studied using microfluidic interferometry, measuring monomer concentration profiles near the interface during the reaction. Our results reveal the reaction is initially controlled by a reaction-diffusion boundary layer within the organic phase, which allows the first measurements of the rate constant for this system.
We introduce an approach for the integration of high performance liquid chromatography and droplet microfluidics on a single high pressure resistant microfluidic glass chip. By coupling these two functionalities, separated analyte bands eluting from the HPLC column are fractionated into numerous droplets in a continuous flowing oil phase. The compartmentalisa-tion of the HPLC-eluate in a segmented flow was performed with droplet sizes of approximately 1 nL and with droplet fre-quencies reaching up to 45 Hz. This approach prevents peak dispersion and facilitates post column processing of chromato-graphic fractions on chip. A reliable generation of droplets is also possible in reversed phase gradient elution mode as demonstrated by applying a solvent gradient from 20% to 100% acetonitrile. A chip design with an incorporated dosing unit enabled the directed post-column addition of reagents to individual droplet fractions. The capability of this dosing function was successfully evidenced by post column addition of a reagent which quenches the fluorescence signal of the analytes. The chip-integration of gradient HPLC, fractionation, detection and post column addition of reagents opens up new ave-nues to perform multistep chemical processes on a single lab-on-a-chip device.
A droplet-based microfluidic device with seamless hyphenation to electrospray mass spectrometry was developed to rapidly investigate organic reactions in segmented flow providing a versatile tool for drug development. A chip-MS interface with an integrated counterelectrode allowed for a flexible poitionioning of the chip-emitter in front of the MS orifice as well as an independent adjustment of the electrospray potentials. This was necessary to avoid contamination a the mass spectrometer as well as sample overloading due to the high analyte concentrations. The device was exemplarily applied to study the scope of an amino-catalyzed domino reaction with low picomole amount of catalyst in individual nanoliter sized droplets.
Nuclear magnetic resonance (NMR) spectroscopy is extremely powerful for chemical analysis but it suffers from lower mass sensitivity compared to many other analytical detection methods. NMR microcoils have been developed in response to this limitation, but interfacing these coils with small sample volumes is a challenge. We introduce here the first digital microfluidic system capable of interfacing droplets of analyte with microcoils in a high-field NMR spectrometer. A finite element simulation was performed to assist in determining appropriate system parameters. After optimization, droplets inside the spectrometer could be controlled remotely, permitting the observation of processes such as xylose-borate complexation and glucose oxidase catalysis. We propose that the combination of DMF and NMR will be a useful new tool for a wide range of applications in chemical analysis.
Vanadium-dependent haloperoxidases (VHPOs) are an exquisite class of halogenating enzymes found in fungi, lichen, algae and bacteria. We report the cloning, purification and characterization of a functional VHPO from the cyanobacterium Acaryochloris marina (AmVHPO), including its structure determination by X-ray crystallography. The AmVHPO features a unique set of disulfide bonds, which stabilize the dodecameric assembly of the protein. Easy access by high-yield recombinant expression as well as resistance towards organic solvents and temperature, together with a distinct halogenation reactivity, make this enzyme a promising starting point for the development of biocatalytic transformations.
Following the development of microfluidic systems, there has been a high tendency towards developing lab-on-a-chip devices for biochemical applications. A great deal of effort has been devoted to improve and advance these devices with the goal of performing complete sets of biochemical assays on the device and possibly developing portable platforms for point of care applications. Among the different microfluidic systems used for such a purpose, digital microfluidics (DMF) shows high flexibility and capability of performing multiplex and parallel biochemical operations, and hence, has been considered as a suitable candidate for lab-on-a-chip applications. In this review, we discuss the most recent advances in the DMF platforms, and evaluate the feasibility of developing multifunctional packages for performing complete sets of processes of biochemical assays, particularly for point-of-care applications. The progress in the development of DMF systems is reviewed from eight different aspects, including device fabrication, basic fluidic operations, automation, manipulation of biological samples, advanced operations, detection, biological applications, and finally, packaging and portability of the DMF devices. Success in developing the lab-on-a-chip DMF devices will be concluded based on the advances achieved in each of these aspects.
The employment of continuous-flow platforms for synthetic chemistry is becoming increasingly popular in research and industrial environments. Integrating analytics in-line enables obtaining a large amount of information in real-time about the reaction progress, catalytic activity and stability, etc. Furthermore, it is possible to influence the reaction progress and selectivity via manual or automated feedback optimisation, thus constituting a dial-a-molecule approach employing digital synthesis. This contribution gives an overview of the most significant contributions in the field to date.
Digital synthesis has been applied to the formation of peptide-based macrocycles and their analogues with side chains appended during late-stage aziridine ring-opening. Discrete nanoliter- to microliter-sized droplets of samples and reagents are controlled in parallel by applying a series of electrical potentials to an array of electrodes coated with a hydrophobic insulator.
Organic synthesis is changing; in a world where budgets are constrained and the environmental impacts of practice are scrutinized, it is increasingly recognized that the efficient use of human resource is just as important as material use. New technologies and machines have found use as methods for transforming the way we work, addressing these issues encountered in research laboratories by enabling chemists to adopt a more holistic systems approach in their work. Modern developments in this area promote a multi-disciplinary approach and work is more efficient as a result. This Review focuses on the concepts, procedures and methods that have far-reaching implications in the chemistry world. Technologies have been grouped as topics of opportunity and their recent applications in innovative research laboratories are described.
For efficient coupling of droplet-based microfluidics with mass spectrometry (MS), a spyhole drilled on the top of a microchip is used to sample the passing droplets by electrostatic-spray ionization (ESTASI) MS. The technique involves placing an electrode below the chip under the spyhole and applying high-voltage pulses. Electrospray occurs directly from the spyhole, and the droplet content is analyzed by MS without a dilution or oil removal step. To demonstrate the versatility of this technique, we have successfully monitored a droplet-based tryptic digestion, as well as a biphasic reaction between β-lactoglobulin in water and α-tocopheryl acetate in 1,2-dichloroethane, where the protein extracts the antioxidant from the oil phase and becomes reduced.
In this work, for the first time, we demonstrate nanoscale droplet generation from a continuous electrowetting microchannel using a simple and precise image-based droplet volume metering technique. One of the most popular ways of droplet generation in electrowetting devices is to split a droplet from a preloaded volume as a fluid reservoir. This method is effective, but lowers volume consistency after multiple droplets are generated. Impedance- and capacitance-based methods of volume metering have been successfully used in digital microfluidics, but require complex circuitry and feedback signal processing. In this work, we demonstrate nanoliter droplet generation from a continuous electrowetting channel used as a replenishable fluid reservoir which compensates for the loss of reservoir volume as droplets are sequentially split. This improves volume consistency especially for applications requiring multi-droplet generation. Based on the area of the electrode, the volume of each droplet split from the electrowetting channel can be obtained by a simple and precise image processing technique with no need for additional hardware and measurement errors of ±0.05 %. This simple technique can be used in a wide range of applications that require precise volume metering, such as immunoassay.
A microfluidic technique for combinatorial chemical synthesis of peptidomimetics has been developed. The new method is fast, automated and includes an integrated magnetic separation of inorganic catalysts from reaction products. This proof-of-concept study should lead to methods for generating libraries of compounds suitable for screening for bioactivity.