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The early-stage diagnosis of cancer is a crucial clinical need. The inadequacies of surgery tissue biopsy have prompted a transition to a less invasive profiling of molecular biomarkers from biofluids, known as liquid biopsy. Exosomes are phospholipid bilayer vesicles present in many biofluids with a biologically active cargo, being responsible for cell-to-cell communication in biological systems. An increase in their excretion and changes in their cargo are potential diagnostic biomarkers for an array of diseases, including cancer, and they constitute a promising analyte for liquid biopsy. The number of exosomes released, the morphological properties, the membrane composition, and their content are highly related to the physiological and pathological states. The main analytical challenge to establishing liquid biopsy in clinical practice is the development of biosensors able to detect intact exosomes concentration and simultaneously analyze specific membrane biomarkers and those contained in their cargo. Before analysis, exosomes also need to be isolated from biological fluids. Microfluidic systems can address several issues present in conventional methods (i.e., ultracentrifugation, size-exclusion chromatography, ultrafiltration, and immunoaffinity capture), which are time-consuming and require a relatively high amount of sample; in addition, they can be easily integrated with biosensing systems. A critical review of emerging microfluidic-based devices for integrated biosensing approaches and following the major analytical need for accurate diagnostics is presented here. The design of a new miniaturized biosensing system is also reported. A device based on hollow-fiber flow field-flow fractionation followed by luminescence-based immunoassay is applied to isolate intact exosomes and characterize their cargo as a proof of concept for colon cancer diagnosis.

Introduction: Gold nanoparticles (AuNPs) and their conjugates are used for many applications in the field of sensors. Literature lacks procedures able to separate, purify and characterize these species in native conditions without altering them while assuring a high throughput. This technological gap can be reduced by exploiting Asymmetrical Flow Field Flow Fractionation multidetection platforms (AF4 multidetection).Method: This work describes a complete set of strategies based on the AF4 system, from nanoparticle synthesis to separative method optimization to conjugates screening and characterization, achieving quantitative control and purification of ready-to-use conjugated Gold nanoparticles and ensuring effectiveness in biosensing.Results and Discussion: AF4-multidetection was used to study AuNPs with different types of surface coating [Poly ethylene glycol, (PEG) and Citrate], their binding behaviour with protein (Bovine serum albumin, BSA) and their stability after conjugation to BSA. A robust but flexible method was developed, able to be applied to different AuNPs and conjugating molecules. The morphology and conjugation mechanism of AuNPs-BSA conjugates were evaluated by combining online Multiangle light scattering (MALS) and offline Dynamic Light Scattering (DLS) measures, which provided an important feature for the quality control required to optimize bio-probe synthesis and subsequent bioassays.

Field-flow fractionation (FFF) is a family of single-phase separative techniques exploited to gently separate and characterize nano- and microsystems in suspension. These techniques cover an extremely wide dynamic range and are able to separate analytes in an interval between a few nm to 100 µm size-wise (over 15 orders of magnitude mass-wise). They are flexible in terms of mobile phase and can separate the analytes in native conditions, preserving their original structures/properties as much as possible. Molecular biology is the branch of biology that studies the molecular basis of biological activity, while biotechnology deals with the technological applications of biology. The areas where biotechnologies are required include industrial, agri-food, environmental, and pharmaceutical. Many species of biological interest belong to the operational range of FFF techniques, and their application to the analysis of such samples has steadily grown in the last 30 years. This work aims to summarize the main features, milestones, and results provided by the application of FFF in the field of molecular biology and biotechnology, with a focus on the years from 2000 to 2022. After a theoretical background overview of FFF and its methodologies, the results are reported based on the nature of the samples analyzed.

Summary: Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.

Instrument automation, technological advancements and improved computational power made separation science an extremely data-rich approach, requiring the use of statistical and data analysis tools that are able to optimize processes and combine multiple outputs. The use of chemometrics is growing, greatly improving the ability to extract meaningful information. Separation–multidetection generates multidimensional data, whose elaboration should not be left to the discretion of the operator. However, some applications or techniques still suffer from the lack of method optimization through DoE and downstream multivariate analysis, limiting their potential. This review aims at summarizing how chemometrics can assist analytical chemists in terms of data elaboration and method design, focusing on what can be achieved by applying chemometric approaches to separation science. Recent applications of chemometrics in separation analyses, in particular in gas, liquid and size-exclusion chromatography, together with field flow fractionation, will be detailed to visualize the state of the art of separation chemometrics, encompassing volatile, soluble and solid (colloidal) analytes. The samples considered will range from food chemistry and environmental chemistry to bio/pharmaceutical science.

Dissolved oxygen (DO) is deeply involved in preserving the life of cellular tissues and human beings due to its key role in cellular metabolism: its alterations may reflect important pathophysiological conditions. DO levels are measured to identify pathological conditions, explain pathophysiological mechanisms, and monitor the efficacy of therapeutic approaches. This is particularly relevant when the measurements are performed in vivo but also in contexts where a variety of biological and synthetic media are used, such as ex vivo organ perfusion. A reliable measurement of medium oxygenation ensures a high-quality process. It is crucial to provide a high-accuracy, real-time method for DO quantification, which could be robust towards different medium compositions and temperatures. In fact, biological fluids and synthetic clinical fluids represent a challenging environment where DO interacts with various compounds and can change continuously and dynamically, and further precaution is needed to obtain reliable results. This study aims to present and discuss the main oxygen detection and quantification methods, focusing on the technical needs for their translation to clinical practice. Firstly, we resumed all the main methodologies and advancements concerning dissolved oxygen determination. After identifying the main groups of all the available techniques for DO sensing based on their mechanisms and applicability, we focused on transferring the most promising approaches to a clinical in vivo/ex vivo setting.

Magnetic nanoparticles (MNPs) present outstanding properties making them suitable as therapeutic agents for hyperthermia treatments. Since the main safety concerns of MNPs are represented by their inherent instability in a biological medium, strategies to both achieve long-term stability and monitor hazardous MNP degradation are needed. We combined a dynamic approach relying on flow field flow fractionation (FFF)-multidetection with conventional techniques to explore frame-by-frame changes of MNPs injected in simulated biological medium, hypothesize the interaction mechanism they are subject to when surrounded by a saline, protein-rich environment, and understand their behaviour at the most critical point of intravenous administration. In the first moments of MNPs administration in the patient, MNPs change their surrounding from a favorable to an unfavorable medium, i.e., a complex biological fluid such as blood; the particles evolve from a synthetic identity to a biological identity, a transition that needs to be carefully monitored. The dynamic approach presented herein represents an optimal alternative to conventional batch techniques that can monitor only size, shape, surface charge, and aggregation phenomena as an averaged information, given that they cannot resolve different populations present in the sample and cannot give accurate information about the evolution or temporary instability of MNPs. The designed FFF method equipped with a multidetection system enabled the separation of the particle populations providing selective information on their morphological evolution and on nanoparticle–proteins interaction in the very first steps of infusion. Results showed that in a dynamic biological setting and following interaction with serum albumin, PP-MNPs retain their colloidal properties, supporting their safety profile for intravenous administration.

Among the in vitro and ex vivo models used to study human cancer biology, cancer cell lines are widely utilized. The standardization of a correct tumor model including the stage of in vitro testing would allow for the development of new high-efficiency drug systems. The poor correlation between preclinical in vitro and in vivo data and clinical trials is still an open issue, hence the need for new systems for the quality control (QC) of these cell products. In this work, we present a new technology, Celector®, capable of the label-free analysis and separation of cells based on their physical characteristics with full preservation of their native properties. Two types of cancer cell lines were used: HL60 as cells growing in suspension and SW620 as adherent cells. Cell lines in general show a growth variability depending on the passage and method of culture. Celector® highlights physical differences that can be correlated to cell viability. This work demonstrates the use of Celector® as an analytical platform for the QC of cells used for drug screening, with fundamental improvement of preclinical tests. Cells with a stable doubling time under analysis can be collected and used as standardized systems for high-quality drug monitoring.

Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, particularly when the API is marketed as a ready-to-use solution. Consequently, appropriate non-destructive techniques able to replicate the peptide environment must be employed. In our work, we exploited Asymmetrical Flow Field-Flow Fractionation (AF4), connected to UV, dRI, fluorescence, and MALS detectors, to fully characterize the aggregation state of Liraglutide, a peptide API used for the treatment of diabetes type 2 and chronic obesity. In previous studies, Liraglutide was hypothesized to assemble into hexa-octamers in phosphate buffer, but no information on its behavior in the formulation medium was provided up to now. The method used allowed researchers to work using formulation as the mobile phase with excellent recoveries and LoQ/LoD, discerning between stable and degraded samples, and detecting, when present, aggregates up to 108 Da. The native state of Liraglutide was assessed and found to be an association into pentamers, with a non-spherical conformation. Combined to benchmark analyses, the sameness study was complete and descriptive, also giving insight on the aggregation process and covalent/non-covalent aggregate types.

Tomato sauce is a world famous food product. Despite standards regulating the production of tomato derivatives, the market suffers frpm fraud such as product adulteration, origin mislabelling and counterfeiting. Methods suitable to discriminate the geographical origin of food samples and identify counterfeits are required. Chemometric approaches offer valuable information: data on tomato sauce is usually obtained through chromatography (HPLC and GC) coupled to mass spectrometry, which requires chemical pretreatment and the use of organic solvents. In this paper, a faster, cheaper, and greener analytical procedure has been developed for the analysis of volatile organic compounds (VOCs) and the colloidal fraction via multivariate statistical analysis. Tomato sauce VOCs were analysed by GC coupled to flame ionisation (GC-FID) and to ion mobility spectrometry (GC-IMS). Instead of using HPLC, the colloidal fraction was analysed by asymmetric flow field-fractionation (AF4), which was applied to this kind of sample for the first time. The GC and AF4 data showed promising perspectives in food-quality control: the AF4 method yielded comparable or better results than GC-IMS and offered complementary information. The ability to work in saline conditions with easy pretreatment and no chemical waste is a significant advantage compared to environmentally heavy techniques. The method presented here should therefore be taken into consideration when designing chemometric approaches which encompass a large number of samples.

The interaction of heme with blood serum proteins plays an important role in many physiological and pathological processes involving enzyme activity, gene expression and cell proliferation. The mechanisms underlying these interactions are; however, not yet fully understood. New analytical methods able to investigate protein-heme binding in native, biologically representative conditions are thus required. In this work, we present a method based on miniaturized, hollow-fiber flow field-flow fractionation with multiple spectrophotometric and light-scattering detection for size separation of high-abundance serum proteins and selective detection of heme-bound subpopulations. Heme is found to mainly interact with serum albumin, whereas a low amount also binds to other proteins such as IgM. The ability to bind heme in physiological conditions is also investigated for individual serum proteins. IgG is found unable to bind heme at clinically relevant concentrations. The proposed method allows separation, quantitation, and mass/size characterization of serum high-abundance proteins, providing information of heme-protein complex stability and preferred heme-clearing pathways. The same approach could be in perspective extended to the investigation of specific heme-antibody binding, and to further studies involving other molecules of pharmaceutical/clinical interest.

(1) Background: Hydrogen sulfide (H2S) is a widely recognized gasotransmitter, with key roles in physiological and pathological processes. The accurate quantification of H2S and reactive sulfur species (RSS) may hold important implications for the diagnosis and prognosis of diseases. However, H2S species quantification in biological matrices is still a challenge. Among the sulfide detection methods, monobromobimane (MBB) derivatization coupled with reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most reported. However, it is characterized by a complex preparation and time-consuming process, which may alter the actual H2S level; moreover, a quantitative validation has still not been described. (2) Methods: We developed and validated an improved analytical protocol for the MBB RP-HPLC method. MBB concentration, temperature and sample handling were optimized, and the calibration method was validated using leave-one-out cross-validation and tested in a clinical setting. (3) Results: The method shows high sensitivity and allows the quantification of H2S species, with a limit of detection of 0.5 µM. Finally, it can be successfully applied in measurements of H2S levels in the serum of patients subjected to inhalation with vapors rich in H2S. (4) Conclusions: These data demonstrate that the proposed method is precise and reliable for measuring H2S species in biological matrices and can be used to provide key insights into the etiopathogenesis of several diseases and sulfur-based treatments.

Adipose tissue is an attractive source of stem cells due to its wide availability. They contribute to the stromal vascular fraction (SVF), which is composed of pre-adipocytes, tissue-progenitors, and pericytes, among others. Because its direct use in medical applications is increasing worldwide, new quality control systems are required. We investigated the ability of the Non-Equilibrium Earth Gravity Assisted Dynamic Fractionation (NEEGA-DF) method to analyze and separate cells based solely on their physical characteristics, resulting in a fingerprint of the biological sample. Adipose tissue was enzymatically digested, and the SVF was analyzed by NEEGA-DF. Based on the fractogram (the UV signal of eluting cells versus time of analysis) the collection time was set to sort alive cells. The collected cells (F-SVF) were analyzed for their phenotype, immunomodulation ability, and differentiation potential. The SVF profile showed reproducibility, and the alive cells were collected. The F-SVF showed intact adhesion phenotype, proliferation, and differentiation potential. The methodology allowed enrichment of the mesenchymal component with a higher expression of mesenchymal markers and depletion of debris, RBCs, and an extracellular matrix still present in the digestive product. Moreover, cells eluting in the last minutes showed higher circularity and lower area, proving the principles of enrichment of a more homogenous cell population with better characteristics. We proved the NEEGA-DF method is a “gentle” cell sorter that purifies primary cells obtained by enzymatic digestion and does not alter any stem cell function.

Advances in nanotechnology have opened up new horizons in nanomedicine through the synthesis of new composite nanomaterials able to tackle the growing drug resistance in bacterial strains. Among these, nanosilver antimicrobials sow promise for use in the treatment of bacterial infections. The use of polydopamine (PDA) as a biocompatible carrier for nanosilver is appealing; however, the synthesis and functionalization steps used to obtain Ag-PDA nanoparticles (NPs) are complex and require time-consuming cleanup processes. Post-synthesis treatment can also hinder the stability and applicability of the material, and dry, offline characterization is time-consuming and unrepresentative of real conditions. The optimization of Ag-PDA preparation and purification together with well-defined characterization are fundamental goals for the safe development of these new nanomaterials. In this paper, we show the use of field-flow fractionation with multi-angle light scattering and spectrophotometric detection to improve the synthesis and quality control of the production of Ag-PDA NPs. An ad hoc method was able to monitor particle growth in a TLC-like fashion; characterize the species obtained; and provide purified, isolated Ag-PDA nanoparticles, which proved to be biologically active as antibacterial agents, while achieving a short analysis time and being based on the use of green, cost-effective carriers such as water.

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM). Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM; it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications.

The use of stem cells for regenerative applications and immunomodulatory effect is increasing. Amniotic epithelial cells (AECs) possess embryonic-like proliferation ability and multipotent differentiation potential. Despite the simple isolation procedure, inter-individual variability and different isolation steps can cause differences in isolation yield and cell proliferation ability, compromising reproducibility observations among centers and further applications. We investigated the use of a new technology as a diagnostic tool for quality control on stem cell isolation. The instrument label-free separates cells based on their physical characteristics and, thanks to a micro-camera, generates a live fractogram, the fingerprint of the sample. Eight amniotic membranes were processed by trypsin enzymatic treatment and immediately analysed. Two types of profile were generated: a monomodal and a bimodal curve. The first one represented the unsuccessful isolation with all recovered cell not attaching to the plate; while for the second type, the isolation process was successful, but we discovered that only cells in the second peak were alive and resulted adherent. We optimized a Quality Control (QC) method to define the success of AEC isolation using the fractogram generated. This predictive outcome is an interesting tool for laboratories and cell banks that isolate and cryopreserve fetal annex stem cells for research and future clinical applications.

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without detrimental toxic side effects. However, there is not a clear and complete protocol to define and relate the properties of the particles (size, shape, surface charge, ionic content) with their specific activity. In this paper, we propose an effective multi-step approach for the identification of a ‘purpose-specific active applicability window’ to maximize the antimicrobial activity of medical devices containing silver nanoparticles (Ag NPs) (such as surface coaters), minimizing any consequent risk for human health (safety by design strategy). The antimicrobial activity and the cellular toxicity of four types of Ag NPs, differing in their coating composition and concentration have been quantified. Through the implementation of flow-field flow fractionation, Ag NPs have been characterized in terms of metal release, size and shape. The particles are fractionated in the process while being left unmodified, allowing for the identification of biological particle-specific contribution. Toxicity and inflammatory response in vitro have been assessed on human skin models, while antimicrobial activity has been monitored with both non-pathogenic and pathogenic Escherichia coli. The main benefit associated with such approach is the comprehensive assessment of the maximal effectiveness of candidate nanomaterials, while simultaneously indexing their properties against their safety.