Self-Powered Microfluidics for Point-of-Care Solutions: From Sampling to Detection of Proteins and Nucleic Acids.

Self-powered microfluidics presents a revolutionary approach to address the challenges of healthcare in decentralized and point-of-care settings where limited access to resources and infrastructure prevails or rapid clinical decision-making is critical. These microfluidic systems exploit physical and chemical phenomena, such as capillary forces and surface tension, to manipulate tiny volumes of fluids without the need for external power sources, making them cost-effective and highly portable. Recent technological advancements have demonstrated the ability to preprogram complex multistep liquid operations within the microfluidic circuit of these standalone systems, which enabled the integration of sensitive detection and readout principles. This chapter first addresses how the accessibility to in vitro diagnostics can be improved by shifting toward decentralized approaches like remote microsampling and point-of-care testing. Next, the crucial role of self-powered microfluidic technologies to enable this patient-centric healthcare transition is emphasized using various state-of-the-art examples, with a primary focus on applications related to biofluid collection and the detection of either proteins or nucleic acids. This chapter concludes with a summary of the main findings and our vision of the future perspectives in the field of self-powered microfluidic technologies and their use for in vitro diagnostics applications.

Catalytic Hairpin Assembly-Based Self-Ratiometric Gel Electrophoresis Detection Platform for Reliable Nucleic Acid Analysis.

The development of gel electrophoresis-based biodetection assays for point-of-care analysis are highly demanding. In this work, we proposed a ratiometric gel electrophoresis-based biosensing platform by employing catalytic hairpin assembly (CHA) process functions as both the signal output and the signal amplification module. Two types of nucleic acids, DNA and miRNA, are chosen for demonstration. The proposed strategy indeed provides a new paradigm for the design of a portable detection platform and may hold great potential for sensitive diagnoses.

Sensitive, precise fingerprint profiling for monosaccharide analysis of Bacillus Calmette-Guérin polysaccharide and nucleic acid isolates.

A sensitive and precise HPLC-DAD method with pre-column PMP derivatization was established and validated, for analyzing the polysaccharides in Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN) isolates, after acid hydrolysis. And the HPLC fingerprint profiling was used to analyze its monosaccharide composition. The monosaccharide concentration-peak area calibration curve was of good linearity (R2 > 0.99), over the range of 0.016-0.08 mg/mL for mannose or 0.24-1.20 mg/mL for glucose, with high recovery of 93-105 % for quality control samples. The intra-day RSD values of mannose and glucose concentration were less than 2.5 % and 2.1 %, respectively, and their inter-day RSD values were less than 4.3 % and 2.2 %, respectively, and remained stable for up to 14 days. This method also remained durable against changes in chromatographic parameters, but it's susceptible to the flow rate of mobile phase. Additionally, the method was applied to analyze the content of mannose and glucose in 22 batches BCG-PSN powder and 17 batches BCG-PSN injection. The results showed that the HPLC-DAD fingerprint spectra of all the BCG-PSN powder and BCG-PSN injection samples had a high degree of similarity, with the similar indexes up to 0.999 and 0.998, respectively. The HPLC-DAD method with pre-column PMP derivatization is highly rapid, effective, visual, and accurate for determination of monosaccharide contents. The validated method was successfully applied to the analysis of polysaccharide in both BCG-PSN powder and injection.

Sampling efficiency and nucleic acid stability during long-term sampling with different bioaerosol samplers.

Aerosol microbiome studies have received increased attention as technological advancements have made it possible to dive deeper into the microbial diversity. To enhance biomass collection for metagenomic sequencing, long-term sampling is a common strategy. While the impact of prolonged sampling times on microorganisms' culturability and viability is well-established, its effect on nucleic acid stability remains less understood but is essential to ensure representative sample collection. This study evaluated four air samplers (SKC BioSampler, SASS3100, Coriolis µ, BioSpot-VIVAS 300-P) against a reference sampler (isopore membrane filters) to identify nucleic acid stability during long-term sampling. Physical sampling efficiencies determined with a fluorescent tracer for three particle sizes (0.8, 1, and 3 µm), revealed high efficiencies (> 80% relative to reference) for BioSampler, SASS3100, and BioSpot-VIVAS for all particle sizes, and for Coriolis with 3 µm particles. Coriolis exhibited lower efficiency for 0.8 µm (7%) and 1 µm (50%) particles. During 2-h sampling with MS2 and Pantoea agglomerans, liquid-based collection with Coriolis and BioSampler showed a decrease in nucleic acid yields for all test conditions. BioSpot-VIVAS displayed reduced sampling efficiency for P. agglomerans compared to MS2 and the other air samplers, while filter-based collection with SASS3100 and isopore membrane filters, showed indications of DNA degradation for 1 µm particles of P. agglomerans after long-term sampling. These findings show that long-term air sampling affects nucleic acid stability in both liquid- and filter-based collection methods. These results highlight bias produced by bioaerosol collection and should be considered when selecting an air sampler and interpreting aerosol microbiome data.

Nucleic acid-based electrochemical biosensors.

Colorectal cancer, breast cancer, oxidative DNA damage, and viral infections are all significant and major health threats to human health, presenting substantial challenges in early diagnosis. In this regard, a wide range of nucleic acid-based electrochemical platforms have been widely employed as point-of-care diagnostics in health care and biosensing technologies. This review focuses on biosensor design strategies, underlying principles involved in the development of advanced electrochemical genosensing devices, approaches for immobilizing DNA on electrode surfaces, as well as their utility in early disease diagnosis, with a particular emphasis on cancer, leukaemia, oxidative DNA damage, and viral pathogen detection. Notably, the role of biorecognition elements and nanointerfaces employed in the design and development of advanced electrochemical genosensors for recognizing biomarkers related to colorectal cancer, breast cancer, leukaemia, oxidative DNA damage, and viral pathogens has been extensively reviewed. Finally, challenges associated with the fabrication of nucleic acid-based biosensors to achieve high sensitivity, selectivity, a wide detection range, and a low detection limit have been addressed. We believe that this review will provide valuable information for scientists and bioengineers interested in gaining a deeper understanding of the fabrication and functionality of nucleic acid-based electrochemical biosensors for biomedical diagnostic applications.

Size exclusion chromatography of biopharmaceutical products: From current practices for proteins to emerging trends for viral vectors, nucleic acids and lipid nanoparticles.

The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.

Deciphering biomolecular complexities: the indispensable role of surface-enhanced Raman spectroscopy in modern bioanalytical research.

Surface-enhanced Raman spectroscopy (SERS) has emerged as an indispensable analytical tool in biomolecular research, providing unmatched sensitivity critical for the elucidation of biomolecular structures. This review presents a thorough examination of SERS, outlining its fundamental principles, cataloging its varied applications within the biomolecular sphere, and contemplating its future developmental trajectories. We begin with a detailed analysis of SERS's mechanistic principles, emphasizing both the phenomena of surface enhancement and the complexities inherent in Raman scattering spectroscopy. Subsequently, we delve into the pivotal role of SERS in the structural analysis of diverse biomolecules, including proteins, nucleic acids, lipids, carbohydrates, and biochromes. The remarkable capabilities of SERS extend beyond mere detection, offering profound insights into biomolecular configurations and interactions, thereby enriching our comprehension of intricate biological processes. This review also sheds light on the application of SERS in real-time monitoring of various bio-relevant compounds, from enzymes and coenzymes to metal ion-chelate complexes and cellular organelles, thereby providing a holistic view and empowering researchers to unravel the complexities of biological systems. We also address the current challenges faced by SERS, such as enhancing sensitivity and resolution, developing stable and reproducible substrates, and conducting thorough analyses in complex biological matrices. Nonetheless, the continual advancements in nanotechnology and spectroscopy solidify the standing of SERS as a formidable force in biomolecular research. In conclusion, the versatility and robustness of SERS not only deepen our understanding of biomolecular intricacies but also pave the way for significant developments in medical research, therapeutic innovation, and diagnostic approaches.

Paper-based sensors: affordable, versatile, and emerging analyte detection platforms.

Paper-based sensors, often referred to as paper-based analytical devices (PADs), stand as a transformative technology in the field of analytical chemistry. They offer an affordable, versatile, and accessible solution for diverse analyte detection. These sensors harness the unique properties of paper substrates to provide a cost-effective and adaptable platform for rapid analyte detection, spanning chemical species, biomolecules, and pathogens. This review highlights the key attributes that make paper-based sensors an attractive choice for analyte detection. PADs demonstrate their versatility by accommodating a wide range of analytes, from ions and gases to proteins, nucleic acids, and more, with customizable designs for specific applications. Their user-friendly operation and minimal infrastructure requirements suit point-of-care diagnostics, environmental monitoring, food safety, and more. This review also explores various fabrication methods such as inkjet printing, wax printing, screen printing, dip coating, and photolithography. Incorporating nanomaterials and biorecognition elements promises even more sophisticated and sensitive applications.

Fluorogenic cell surface glycan labelling with fluorescence molecular rotor dyes and nucleic acid stains.

We show that covalent labelling of sialic acids on live cell surfaces or mucin increases the fluorescence of the fluorescence molecular rotors (FMRs) CCVJ, Cy3 and thioazole orange, enabling wash-free imaging of cell surfaces. Dual labelling with an FMR and an environmentally insensitive dye allows detection of changes that occur, for example, when cross-linking is altered.

Establishment of a graphene oxide-assisted nucleic acid chromatography strip detection technology for Prorocentrum minimum.

In recent decades, the harmful algal blooms (HABs) caused by Prorocentrum minimum have caused serious environmental damage and economic losses. The detection of P. minimum plays an important role in warning the outbreak of P. minimum-forming HABs. By utilizing the powerful absorption of graphene oxide (GO) on short-stranded DNA, a GO-assisted nucleic acid chromatography strip (GO-NACS) was proposed here to achieve a highly sensitive, specific, intuitive, and convenient detection of P. minimum. In particular, this study used our previously reported conventional-NACS (C-NACS) as a control to evaluate the improvement of detection performance with the use of GO. The performance of GO-NACS was evaluated from the perspectives of specificity, sensitivity, stability, and practicality. The specificity test demonstrated that it had a high degree of specificity and did not display cross-reacting with non-target algal species. The sensitivity test with the genomic DNA indicated that it had a detection limit of 1.30 × 10-3 ng µL-1, representing a 10-fold higher sensitivity than C-NACS and a 100-fold higher sensitivity than agarose gel electrophoresis (AGE). The interference test with non-target algal species demonstrated that it had a good detection stability, and the interfering algal species had no obvious effect on the detection of P. minimum. The practicality test with simulated natural water samples showed that the cellular detection limit of GO-NACS was 6.8 cells mL-1, which was 10-fold and 100-fold lower than that of C-NACS and AGE, respectively. In conclusion, the established GO-NACS may offer a novel alternative technique for the detection of P. minimum while guaranteeing specificity and enhancing sensitivity without requiring extensive apparatus.

Recent advances in nucleic acid signal amplification-based aptasensors for sensing mycotoxins.

Mycotoxin contamination in food products may cause serious health hazards and economic losses. The effective control and accurate detection of mycotoxins have become a global concern. Even though a variety of methods have been developed for mycotoxin detection, most conventional methods suffer from complicated operation procedures, low sensitivity, high cost, and long assay time. Therefore, the development of simple and sensitive methods for mycotoxin assay is highly needed. The introduction of nucleic acid signal amplification technology (NASAT) into aptasensors significantly improves the sensitivity and facilitates the detection of mycotoxins. Herein, we give a comprehensive review of the recent advances in NASAT-based aptasensors for assaying mycotoxins and summarize the principles, features, and applications of NASAT-based aptasensors. Moreover, we highlight the challenges and prospects in the field, including the simultaneous detection of multiple mycotoxins and the development of portable devices for field detection.

A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets.

Digital PCR (dPCR) has become indispensable in nucleic acid (NA) detection across various fields, including viral diagnostics and mutant detection. However, misclassification of partitions in dPCR can significantly impact accuracy. Despite existing methods to minimize misclassification bias, accurate classification remains elusive, especially for nonamplified target partitions. To address these challenges, this study introduces an innovative microdroplet-based competitive PCR platform for nucleic acid quantification in microfluidic devices independent of Poisson statistics. In this approach, the target concentration (T) is determined from the concentration of competitor DNA (C) at the equivalence point (E.P.), where C/T is 1. Competitive PCR ensures that the ratio of target to competitor DNA remains constant during amplification, reflected in the resultant fluorescence intensity, allowing the quantification of target DNA concentration at the equivalence point. The unique amplification technique eliminates Poisson distribution, addressing misclassification challenges. Additionally, our approach reduces the need for post-PCR procedures and shortens analytical time. We envision this platform as versatile, reproducible, and easily adaptable for driving significant progress in molecular biology and diagnostics.

Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets.

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

Beyond the Gold-Thiol Paradigm: Exploring Alternative Interfaces for Electrochemical Nucleic Acid-Based Sensing.

Nucleic acid-based electrochemical sensors (NBEs) use oligonucleotides as affinity reagents for the detection of a variety of targets, ranging from small-molecule therapeutics to whole viruses. Because of their versatility in molecular sensing, NBEs are being developed broadly for diagnostic and biomedical research applications. Benchmark NBEs are fabricated via self-assembly of thiol-based monolayers on gold. Although robust for rapid prototyping, thiol monolayers suffer from limitations in terms of stability under voltage modulation and in the face of competitive ligands such as thiolated molecules naturally occurring in biofluids. Additionally, gold cannot be deployed as an NBE substrate for all biomedical applications, such as in cases where molecular measurements coupled to real-time, under-the-sensor tissue imaging is needed. Seeking to overcome these limitations, the field of NBEs is pursuing alternative ligands and electrode surfaces. In this perspective, I discuss new interface fabrication strategies that have successfully achieved NBE sensing, or that have the potential to allow NBE sensing on conductive surfaces other than gold. I hope this perspective will provide the reader with a fresh view of how future NBE interfaces could be constructed and will serve as inspiration for the pursuit of collaborative developments in the field of NBEs.

A comprehensive guide to extract information from extracellular vesicles: a tutorial review towards novel analytical developments.

In the medical field, extracellular vesicles (EVs) are gaining importance as they act as cells mediators. These are phospholipid bilayer vesicles and contain crucial biochemical information about their mother cells being carrier of different biomolecules such as small molecules, proteins, lipids, and nucleic acids. After release into the extracellular matrix, they enter the systemic circulation and can be found in all human biofluids. Since EVs reflect the state of the cell of origin, there is exponential attention as potential source of new circulating biomarkers for liquid biopsy. The use of EVs in clinical practice faces several challenges that need to be addressed: these include the standardization of lysis protocols, the availability of low-cost reagents and the development of analytical tools capable of detecting biomarkers. The process of lysis is a crucial step that can impact all subsequent analyses, towards the development of novel analytical strategies. To aid researchers to support the evolution of measurement science technology, this tutorial review evaluates and discuss the most commonly protocols used to characterize the contents of EVs, including their advantages and disadvantages in terms of experimental procedures, time and equipment. The purpose of this tutorial review is to offer practical guide to researchers which are intended to develop novel analytical approaches. Some of the most significant applications are considered, highlighting their main characteristics divided per mechanism of action. Finally, comprehensive tables which provide an overview at a glance are provided to readers.

Tunable nanofluidic device for digital nucleic acid analysis.

Nano/microfluidic-based nucleic acid tests have been proposed as a rapid and reliable diagnostic technology. Two key steps for many of these tests are target nucleic acid (NA) immobilization followed by an enzymatic reaction on the captured NAs to detect the presence of a disease-associated sequence. NA capture within a geometrically confined volume is an attractive alternative to NA surface immobilization that eliminates the need for sample pre-treatment (e.g. label-based methods such as lateral flow assays) or use of external actuators (e.g. dielectrophoresis) that are required for most nano/microfluidic-based NA tests. However, geometrically confined spaces hinder sample loading while making it challenging to capture, subsequently, retain and simultaneously expose target NAs to required enzymes. Here, using a nanofluidic device that features real-time confinement control via pneumatic actuation of a thin membrane lid, we demonstrate the loading of digital nanocavities by target NAs and exposure of target NAs to required enzymes/co-factors while the NAs are retained. In particular, as proof of principle, we amplified single-stranded DNAs (M13mp18 plasmid vector) in an array of nanocavities via two isothermal amplification approaches (loop-mediated isothermal amplification and rolling circle amplification).

Fully integrated and automated centrifugal microfluidic chip for point-of-care multiplexed molecular diagnostics.

Public health events caused by pathogens have imposed significant economic and societal burdens. However, conventional methods still face challenges including complex operations, the need for trained operators, and sophisticated instruments. Here, we proposed a fully integrated and automated centrifugal microfluidic chip, also termed IACMC, for point-of-care multiplexed molecular diagnostics by harnessing the advantages of active and passive valves. The IACMC incorporates multiple essential components including a pneumatic balance module for sequential release of multiple reagents, a pneumatic centrifugation-assisted module for on-demand solution release, an on-chip silicon membrane module for nucleic acid extraction, a Coriolis force-mediated fluid switching module, and an amplification module. Numerical simulation and visual validation were employed to iterate and optimize the chip's structure. Upon sample loading, the chip automatically executes the entire process of bacterial sample lysis, nucleic acid capture, elution quantification, and isothermal LAMP amplification. By optimizing crucial parameters including centrifugation speed, direction of rotation, and silicone membrane thickness, the chip achieves exceptional sensitivity (twenty-five Salmonella or forty Escherichia coli) and specificity in detecting Escherichia coli and Salmonella within 40 min. The development of IACMC will drive advancements in centrifugal microfluidics for point-of-care testing and holds potential for broader applications in precision medicine including high-throughput biochemical analysis immune diagnostics, and drug susceptibility testing.

In Situ Detection of Nucleic Acids in Extracellular Vesicles via Membrane Fusion.

Extracellular vesicles (EVs) carry diverse biomolecules (e. g., nucleic acids, proteins) for intercellular communication, serving as important markers for diseases. Analyzing nucleic acids derived from EVs enables non-invasive disease diagnosis and prognosis evaluation. Membrane fusion, a fundamental cellular process wherein two lipid membranes merge, facilitates cell communication and cargo transport. Building on this natural phenomenon, recent years have witnessed the emergence of membrane fusion-based strategies for the detection of nucleic acids within EVs. These strategies entail the encapsulation of detection probes within either artificial or natural vesicles, followed by the induction of membrane fusion with EVs to deliver probes. This innovative approach not only enables in situ detection of nucleic acids within EVs but also ensures the maintenance of structural integrity of EVs, thus preventing nucleic acid degradation and minimizing the interference from free nucleic acids. This concept categorizes approaches into universal and targeted membrane fusion strategies, and discusses their application potential, and challenges and future prospects.

Isothermal amplification-based microfluidic devices for detecting foodborne pathogens: a review.

The gold standard for nucleic acid amplification-based diagnosis is the polymerase chain reaction (PCR). The PCR recognizes the targets such as foodborne pathogens by amplifying their specific genes. The integration of nucleic acid amplification-based assays on microfluidic platforms represents a highly promising solution for convenient, cheap, and effective control of foodborne pathogens. However, the application of the PCR is limited to on-site detection because the method requires sophisticated equipment for temperature control, which makes it complicated for microfluidic integration. Alternatively, isothermal amplification methods are promising tools for integrating microfluidic platforms for on-site detection of foodborne pathogens. This review summarized advances in isothermal amplification-based microfluidic devices for detecting foodborne pathogens. Different nucleic acid extraction approaches and the integration of these approaches in microfluidic platforms were first reviewed. Microfluidic platforms integrated with three common isothermal amplification methods including loop-mediated isothermal amplification, recombinase polymerase amplification, and recombinase-aided amplification were then described and discussed.