The use of cell culture techniques to model human disease is an indispensable tool that has helped improve the health and well-being of the world. Monolayer cultures have most often been used for biomedical research, although not accurately recapitulating an in vivo human tumor. Tumor spheroids are a form of three-dimensional cell culture that better mimics an avascularized human tumor through their cell-cell contacts in all directions, development of various chemical gradients, and distinct populations of cells found within the spheroid. In this review, we highlight how mass spectrometry has propelled the utility of the spheroid model to understand cancer biology. We discuss how mass spectrometry imaging can be utilized to determine the penetration efficiency of various chemotherapeutics, how proteomics can be used to understand the biology in the various layers of a spheroid, and how metabolomics and lipidomics are used to elucidate how various spheroids behave toward chemotherapeutics.

Protein glycosylation, the covalent attachment of carbohydrate, or glycan, structures onto the protein backbone, is one of the most complex and heterogeneous post-translational modifications (PTMs). Extracellular protein glycosylation, in particular N- and mucin-type O-glycosylation, plays pivotal roles in a number of biophysical and biochemical processes, such as protein folding and stability, cell adhesion, signaling, and protection. As such, aberrant glycosylation is implicated in a variety of diseases, including cancer. However, the nontemplated nature and structural heterogeneity of protein glycosylation hinder glycoprotein characterization with traditional methods. Recent advances in analytical techniques have improved capabilities for decoding glycan complexity, a promising step toward understanding the role of glycosylation in human health and disease. In this review, we highlight key and emerging techniques to study protein glycosylation, and we emphasize how these techniques have improved our understanding of glycosylation in a biologically relevant context.

Mass spectrometry-based proteomics and metaproteomics have long been used in the study of human microbiomes, with the potential of metaproteomics only recently being fully harnessed. This progress is due to the advancements of high-performance mass spectrometers, innovative proteomics strategies, and the development of dedicated bioinformatics tools. In this review, we critically examine the recent technological developments that enhance the application of metaproteomics in clinical microbiome analysis. We also summarize significant advancements in the application of metaproteomics to study human microbiomes across various body sites under disease conditions. Despite these, the potential of metaproteomics remains underutilized due to typically small sample sizes and insufficient data mining. We thereby highlight some key aspects that could facilitate the broader and more effective application of mass spectrometry-based metaproteomics in clinical microbiome analysis, including the development of microbiome assays for translational research and application.

Mass spectrometry (MS)-based top-down proteomics (TDP) characterizes proteoforms in cells, tissues, and biological fluids (e.g., human plasma) to better our understanding of protein function and to discover new protein biomarkers for disease diagnosis and therapeutic development. Separations of proteoforms with high peak capacity are needed due to the high complexity of biological samples. Capillary electrophoresis (CE)-MS has been recognized as a powerful analytical tool for protein analysis since the 1980s owing to its high separation efficiency and sensitivity of CE-MS for proteoforms. Here, we review benefits of CE-MS for advancing TDP, challenges and solutions of the method, and the main research areas in which CE-MS-based TDP can make significant contributions. We provide a brief perspective of CE-MS-based TDP moving forward.

Amyloid-related diseases, such as Alzheimer's and Parkinson's disease, are devastating conditions caused by the accumulation of abnormal protein aggregates known as amyloid fibrils. While assays involving animal models are essential for understanding the pathogenesis and developing therapies, a wide array of standard analytical techniques exists to enhance our understanding of these disorders. These techniques provide valuable information on the formation and propagation of amyloid fibrils, as well as the pharmacokinetics and pharmacodynamics of candidate drugs. Despite ethical concerns surrounding animal use, animal models remain vital tools in the search for treatments. Regardless of the specific animal model chosen, the analytical methods used are usually standardized. Therefore, the main objective of this review is to categorize and outline the primary analytical methods used in in vivo assays for amyloid-related diseases, highlighting their critical role in furthering our understanding of these disorders and developing effective therapies.

The electrochemical interface formed between an electrode and an electrolyte significantly affects the rate and mechanism of the electrode reaction through its structure and properties, which vary across the interface. The scope of the interface has been expanded, along with the development of energy electrochemistry, where a solid-electrolyte interphase may form on the electrode and the active materials change properties near the surface region. Developing a comprehensive understanding of electrochemical interfaces and interphases necessitates three-dimensional spatial resolution characterization. Atomic force microscopy (AFM) offers advantages of imaging and long-range force measurements. Here we assess the capabilities of AFM by comparing the force curves of different regimes and various imaging modes for in situ characterizing of electrochemical interfaces and interphases. Selected examples of progress on work related to the structures and processes of electrode surfaces, electrical double layers, and lithium battery systems are subsequently illustrated. Finally, this review provides perspectives on the future development of electrochemical AFM.

Single-particle (or digital) measurements enhance sensitivity (10- to 100-fold improvement) and uncover heterogeneity within a population (one event in 100 to 10,000). Many biological systems are significantly influenced by rare or infrequent events, and determining what species is present, in what quantity, and the role of that species is critically important to unraveling many questions. To develop these measurement systems, resistive-pulse sensing is used as a label-free, single-particle detection technique and can be combined with a range of functional elements, e.g., mixers, reactors, filters, separators, and pores. Virtually, any two-dimensional layout of the micro- and nanofluidic conduits can be envisioned, designed, and fabricated in the plane of the device. Multiple nanopores in series lead to higher-precision measurements of particle size, shape, and charge, and reactions coupled directly with the particle-size measurements improve temporal response. Moreover, other detection techniques, e.g., fluorescence, are highly compatible with the in-plane format. These integrated in-plane nanofluidic devices expand the toolbox of what is possible with single-particle measurements.

The ability to measure dynamic changes in neurochemicals with high spatiotemporal resolution is essential for understanding the diverse range of functions mediated by the brain. We review recent advances in genetically encoded sensors for detecting neurochemicals and discuss their in vivo applications. For example, notable progress has been made with respect to sensors for second messengers such as cyclic adenosine monophosphate, enabling in vivo real-time monitoring of these messengers at single-cell and even subcellular resolution. Moreover, the emergence of highly sensitive sensors for neurotransmitters and neuromodulators has greatly accelerated the study of these signaling molecules in a wide variety of behavioral models using an array of powerful imaging techniques. Finally, we discuss the future direction of neurochemical sensors, including their ability to measure neurochemical concentrations and the potential for multiplex imaging.

White light endoscopic imaging allows for the examination of internal human organs and is essential in the detection and treatment of early-stage cancers. To facilitate diagnosis of precancerous changes and early-stage cancers, label-free optical technologies that provide enhanced malignancy-specific contrast and depth information have been extensively researched. The rapid development of technology in the past two decades has enabled integration of these optical technologies into clinical endoscopy. In recent years, the significant advantages of using these adjunct optical devices have been shown, suggesting readiness for clinical translation. In this review, we provide an overview of the working principles and miniaturization considerations and summarize the clinical and preclinical demonstrations of several such techniques for early-stage cancer detection. We also offer an outlook for the integration of multiple technologies and the use of computer-aided diagnosis in clinical endoscopy.

Ribonucleic acids (RNAs) are key biomolecules responsible for the transmission of genetic information, the synthesis of proteins, and modulation of many biochemical processes. They are also often the key components of viruses. Synthetic RNAs or oligoribonucleotides are becoming more widely used as therapeutics. In many cases, RNAs will be chemically modified, either naturally via enzymatic systems within a cell or intentionally during their synthesis. Analytical methods to detect, sequence, identify, and quantify RNA and its modifications have demands that far exceed requirements found in the DNA realm. Two complementary platforms have demonstrated their value and utility for the characterization of RNA and its modifications: mass spectrometry and next-generation sequencing. This review highlights recent advances in both platforms, examines their relative strengths and weaknesses, and explores some alternative approaches that lie at the horizon.

A frontier of analytical sciences is centered on the continuous measurement of molecules in or near cells, tissues, or organs, within the biological context in situ, where the molecular-level information is indicative of health status, therapeutic efficacy, and fundamental biochemical function of the host. Following the completion of the Human Genome Project, current research aims to link genes to functions of an organism and investigate how the environment modulates functional properties of organisms. New analytical methods have been developed to detect chemical changes with high spatial and temporal resolution, including minimally invasive surface-enhanced Raman scattering (SERS) nanofibers using the principles of endoscopy (SERS nanoendoscopy) or optical physiology (SERS optophysiology). Given the large spectral data sets generated from these experiments, SERS nanoendoscopy and optophysiology benefit from advances in data science and machine learning to extract chemical information from complex vibrational spectra measured by SERS. This review highlights new opportunities for intracellular, extracellular, and in vivo chemical measurements arising from the combination of SERS nanosensing and machine learning.

Bacteriophages, which are viral predators of bacteria, have evolved to efficiently recognize, bind, infect, and lyse their host, resulting in the release of tens to hundreds of propagated viruses. These abilities have attracted biosensor developers who have developed new methods to detect bacteria. Recently, several comprehensive reviews have covered many of the advances made regarding the performance of phage-based biosensors. Therefore, in this review, we first describe the landscape of phage-based biosensors and then cover advances in other aspects of phage biology and engineering that can be used to make high-impact contributions to biosensor development. Many of these advances are in fields adjacent to analytical chemistry such as synthetic biology, machine learning, and genetic engineering and will allow those looking to develop phage-based biosensors to start taking alternative approaches, such as a bottom-up design and synthesis of custom phages with the singular task of detecting their host.

Point-of-care (POC) devices have become rising stars in the biosensing field, aiming at prognosis and diagnosis of diseases with a positive impact on the patient but also on healthcare and social care systems. Putting the patient at the center of interest requires the implementation of noninvasive technologies for collecting biofluids and the development of wearable platforms with integrated artificial intelligence-based tools for improved analytical accuracy and wireless readout technologies. Many electrical and electrochemical transducer technologies have been proposed for POC-based sensing, but several necessitate further development before being widely deployable. This review focuses on recent innovations in electrochemical and electrical biosensors and their growth opportunities for nanotechnology-driven multidisciplinary approaches. With a focus on analytical aspects to pave the way for future electrical/electrochemical diagnostics tests, current limitations and drawbacks as well as directions for future developments are highlighted.

Nature has inspired the development of biomimetic membrane sensors in which the functionalities of biological molecules, such as proteins and lipids, are harnessed for sensing applications. This review provides an overview of the recent developments for biomembrane sensors compatible with either bulk or planar sensing applications, namely using lipid vesicles or supported lipid bilayers, respectively. We first describe the individual components required for these sensing platforms and the design principles that are considered when constructing them, and we segue into recent applications being implemented across multiple fields. Our goal for this review is to illustrate the versatility of nature's biomembrane toolbox and simultaneously highlight how biosensor platforms can be enhanced by harnessing it.

Analytical techniques operating at the nanoscale introduce confinement as a tool at our disposal. This review delves into the phenomenon of accelerated reactivity within micro- and nanodroplets. A decade of accelerated reactivity observations was succeeded by several years of fundamental studies aimed at mechanistic enlightenment. Herein, we provide a brief historical context for rate enhancement in and around micro- and nanodroplets and summarize the mechanisms that have been proposed to contribute to such extraordinary reactivity. We highlight recent electrochemical reports that make use of restricted mass transfer to enhance electrochemical reactions and/or quantitatively measure reaction rates within droplet-confined electrochemical cells. A comprehensive approach to nanodroplet reactivity is paramount to understanding how nature takes advantage of these systems to provide life on Earth and, in turn, how to harness the full potential of such systems.

Imaging mass spectrometry (IMS) enables highly multiplexed, untargeted tissue mapping for a broad range of molecular classes, facilitating in situ biological discovery. Yet, challenges persist in molecular specificity, which is the ability to discern one molecule from another, and spatial specificity, which is the ability to link untargeted imaging data to specific tissue features. Instrumental developments have dramatically improved IMS spatial resolution, allowing molecular observations to be more readily associated with distinct tissue features across spatial scales, ranging from larger anatomical regions to single cells. High-performance mass analyzers and systems integrating ion mobility technologies are also becoming more prevalent, further improving molecular coverage and the ability to discern chemical identity. This review provides an overview of recent advancements in high-specificity IMS that are providing critical biological context to untargeted molecular imaging, enabling integrated analyses, and addressing advanced biomedical research applications.

The last decade has been incredibly fruitful in proving the multifunctionality of paper for delivering innovative electrochemical (bio)sensors. The paper material exhibits unprecedented versatility to deal with complex liquid matrices and facilitate analytical detection in aerosol and solid phases. Such remarkable capabilities are feasible by exploiting the intrinsic features of paper, including porosity, capillary forces, and its easy modification, which allow for the fine designing of a paper device. In this review, we shed light on the most relevant paper-based electrochemical (bio)sensors published in the literature so far to identify the smart functional roles that paper can play to bridge the gap between academic research and real-world applications in the biomedical, environmental, agrifood, and security fields. Our analysis aims to highlight how paper's multifarious properties can be artfully harnessed for breaking the boundaries of the most classical applications of electrochemical (bio)sensors.

Spatial comprehensive three-dimensional chromatography (3D-LC) offers an innovative approach to achieve unprecedented resolving power in terms of peak capacity and sample throughput. This advanced technique separates components within a 3D separation space, where orthogonal retention mechanisms are incorporated. The parallel development of the second- and third-dimension stages effectively overcomes the inherent limitation of conventional multidimensional approaches, where sampled fractions are analyzed sequentially. This review focuses on the design aspects of the microchip for spatial 3D-LC and the selection of orthogonal separation modes to enable the analysis of intact proteins. The design considerations for the flow distributor and channel layout are discussed, along with various approaches to confine the flow during the subsequent development stages. Additionally, the integration of stationary phases into the microchip is addressed, and interfacing to mass spectrometry detection is discussed. According to Pareto optimality, the integration of isoelectric focusing, size-exclusion chromatography, and reversed-phase chromatography in a spatial 3D-LC approach is predicted to achieve an exceptional peak capacity of over 30,000 within a 1-h analysis, setting a new benchmark in chromatographic performance.

Bioluminescence imaging (BLI) is a powerful method for visualizing biological processes and tracking cells. Engineered bioluminescent bacteria that utilize luciferase-catalyzed biochemical reactions to generate luminescence have become useful analytical tools for in vitro and in vivo bacterial imaging. Accordingly, this review initially introduces the development of engineered bioluminescent bacteria that use different luciferase-luciferin pairs as analytical tools and their applications for in vivo BLI, including real-time bacterial tracking of infection, probiotic investigation, tumor-targeted therapy, and drug screening. Applications of engineered bioluminescent bacteria as whole-cell biosensors for sensing biological changes in vitro and in vivo are then discussed. Finally, we review the optimizations and future directions of bioluminescent bacteria for imaging. This review aims to provide fundamental insights into bacterial BLI and highlight the potential development of this technique in the future.

In this review, we discuss the cutting-edge developments in mass spectrometry proteomics and metabolomics that have brought improvements for the identification of new disease-based biomarkers. A special focus is placed on psychiatric disorders, for example, schizophrenia, because they are considered to be not a single disease entity but rather a spectrum of disorders with many overlapping symptoms. This review includes descriptions of various types of commonly used mass spectrometry platforms for biomarker research, as well as complementary techniques to maximize data coverage, reduce sample heterogeneity, and work around potentially confounding factors. Finally, we summarize the different statistical methods that can be used for improving data quality to aid in reliability and interpretation of proteomics findings, as well as to enhance their translatability into clinical use and generalizability to new data sets.

We critically evaluate the current status of portable mass spectrometry (pMS), particularly where this aligns with ambient ionization. Assessing the field of pMS can be quite subjective, especially in relation to the portable aspects of design, deployment, and operation. In this review, we discuss what it means to be portable and introduce a set of criteria by which pMS and ambient ionization sources can be assessed. Moreover, we consider the recent literature in terms of the most popular and significant advances in portable instrumentation for ambient ionization and miniature mass spectrometers. Finally, emerging trends and exciting future prospects are discussed and some recommendations are offered.