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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

Convergence of Surface-Enhanced Raman Scattering with Molecular Diagnostics: A Perspective on Future Directions.

Molecular diagnostics (MD) is widely employed in multiple scientific disciplines, such as oncology, pathogen detection, forensic investigations, and the pharmaceutical industry. Techniques such as polymerase chain reaction (PCR) revolutionized the rapid and accurate identification of nucleic acids (DNA, RNA). More recently, CRISPR and its CRISPR-associated protein (Cas) have been a ground-breaking discovery that is the latest revolution in molecular biology, including MD. Surface-enhanced Raman scattering (SERS) is a very attractive alternative to fluorescence as the currently most widely used optical readout in MD. In this Perspective, milestones in the development of MD, SERS-PCR, and next-generation approaches to MD, such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), are briefly summarized. Our perspective on the future convergence of SERS with MD is focused on SERS-based CRISPR/Cas (SERS-CRISPR) since we anticipate many promising applications in this rapidly emerging field. We predict that major future developments will exploit the advantages of real-time monitoring with the superior brightness, photostability, and spectral multiplexing potential of SERS nanotags in an automated workflow for rapid assays under isothermal, amplification-free conditions.

Numerical and experimental investigation on the performance of rapid ultrasonic-assisted nucleic acid extraction based on dispersive two-phase flow.

BACKGROUND: Nucleic acid extraction (NAE) is an essential step in the whole process of nucleic acid detection (NAT). Traditional manual extraction methods are time-consuming and laborious, unfavorable to the point-of-care testing of nucleic acids. Ultrasound has been emphasized due to its noncontact and easy-to-manipulate characteristics, and integration with microfluidic chip can realize rapid NAE through acoustic streaming effect. The uniformity of magnetic bead mixing in this process is a critical factor affecting the extraction effect. In this study, we developed an ultrasound-assisted NAE technique based on the magnetic bead method and optimized the chip structure to achieve rapid NAE. RESULT: We use ultrasonic-assisted coupled with magnetic bead method for ultra-fast NAE. The mixing process of magnetic beads driven by acoustic streaming is simulated by a dispersive two-phase flow model, and the ultrasonic incidence angle (θin), cone structure aspect ratio (Dc/Hc) and sheet structure thickness (Hp) are optimized to enhance the mixing performance. Furthermore, the effectiveness of NAE is validated by utilizing quantitative real-time PCR (qPCR) detection. The findings reveal that a θin value of 10° yields superior mixing performance compared to other incidence angles, resulting in a maximum increase of 84 % in mixing intensity. When Dc/Hc = 0.5 and Hp = 0.5 mm, the maximum mixing index in the localized region of the chamber after 1 s of ultrasound action can reach 83.6 % and 92.5 %, respectively. Compared to the original chamber, the CT values extracted after 5 s of ultrasound action shifted forward by up to 1.9 ct and 4.1 ct, respectively. SIGNIFICANCE: The dispersed two-phase flow model can effectively simulate the mixing process of magnetic beads, which plays an important role in assisting the structural design of chip extraction chambers. The single-step mixing of ultrasound-assisted NAE takes only 15s to achieve an extraction performance comparable to manual extraction. The extraction process can be completed within 7 min after integrating this technology with microfluidic chips and automated equipment, providing a solution for automated and efficient NAE.

Predicting Anaerobic Membrane Bioreactor Performance Using Flow-Cytometry-Derived High and Low Nucleic Acid Content Cells.

Having a tool to monitor the microbial abundances rapidly and to utilize the data to predict the reactor performance would facilitate the operation of an anaerobic membrane bioreactor (AnMBR). This study aims to achieve the aforementioned scenario by developing a linear regression model that incorporates a time-lagging mode. The model uses low nucleic acid (LNA) cell numbers and the ratio of high nucleic acid (HNA) to LNA cells as an input data set. First, the model was trained using data sets obtained from a 35 L pilot-scale AnMBR. The model was able to predict the chemical oxygen demand (COD) removal efficiency and methane production 3.5 days in advance. Subsequent validation of the model using flow cytometry (FCM)-derived data (at time t - 3.5 days) obtained from another biologically independent reactor did not exhibit any substantial difference between predicted and actual measurements of reactor performance at time t. Further cell sorting, 16S rRNA gene sequencing, and correlation analysis partly attributed this accurate prediction to HNA genera (e.g., Anaerovibrio and unclassified Bacteroidales) and LNA genera (e.g., Achromobacter, Ochrobactrum, and unclassified Anaerolineae). In summary, our findings suggest that HNA and LNA cell routine enumeration, along with the trained model, can derive a fast approach to predict the AnMBR performance.

Quality and Quantity of Nucleic Acids Extracted from Formalin-Fixed Paraffin-Embedded Lymphoma Biopsies from Nigerian Archived Biopsy.

BACKGROUND: Integrity of nucleic acids derived from archived formalin-fixed paraffin-embedded (FFPE) cancer specimens affects diagnosis, prognosis, and therapy. Several factors affect the quality and quantity of extracted nucleic acids and one of such factors is storage period. AIM: We investigated the impact of storage duration on the quality and quantity of nucleic acids extracted from archived FFPE lymphoma biopsies in Nigeria. MATERIALS AND METHODS: A total of 53 FFPE biopsies diagnosed as lymphoma stored over several years (2008-2019) were analyzed. They were 22 chronic lymphocytic leukemia (CLL) cases, 17 Hodgkin lymphoma (HL) cases, and 14 diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS). DNA was extracted from all the lymphoma samples which were analyzed for integrity and amplifiability using the four pairs of control genes polymerase chain reaction (PCR) primers of BIOMED-2 protocol, whereas RNA extraction was from 6 CLL cases used for qPCR analysis of RNU43. RESULTS: For CLL, the mean DNA yield was 193.6 ng/µl (range: 3.0-533.0 ng/µl), whereas the mean A260/A280 ratio was 1.7 (1.2-1.9). For DLBCL, NOS, and HL, 255.5 ng/µl (range: 32.9-605.4 ng/µl), 1.8 (1.5-2.0) and 242.7 ng/µl (range: 1.3-886.0 ng/µl), and 1.7 (0.9-1.8), respectively. The extracted DNA gave amplifiable products of at least 200bp, whereas the RNA analysis showed CT values of <38 in all the samples. The mean RNA yield was 462.2 ng/µl (range: 74.7-1082.1), whereas the mean A260/A280 was 1.7 (1.5-1.8). CONCLUSION: Quantity and quality of nucleic acids from FFPE tissues stored for different time periods showed no significant difference in yield and quality.

Solid-State Nanopore Sensors with Enhanced Sensitivity through Nucleic Acid Amplification.

Solid-state nanopores have wide applications in DNA sequencing, energy conversion and storage, seawater desalination, sensors, and reactors due to their high stability, controllable geometry, and a variety of pore-forming materials. Solid-state nanopore sensors can be used for qualitative and quantitative analyses of ions, small molecules, proteins, and nucleic acids. The combination of nucleic acid amplification and solid-state nanopores to achieve trace detection of analytes is gradually attracting attention. This review outlines nucleic acid amplification strategies for enhancing the sensitivity of solid-state nanopore sensors by summarizing the articles published in the past 10 years. The future development prospects and challenges of nucleic acid amplification in solid-state nanopore sensors are discussed. This review helps readers better understand the field of solid-state nanopore sensors. We believe that solid-state nanopore sensors will break through the bottleneck of traditional detection and become a powerful single-molecule detection platform.

Simple and Multiplexed Detection of Nucleic Acid Targets Based on Fluorescent Ring Patterns and Deep Learning Analysis.

Simple diagnostic tests for nucleic acid targets can provide great advantages for applications such as rapid pathogen detection. Here, we developed a membrane assay for multiplexed detection of nucleic acid targets based on the visualization of two-dimensional fluorescent ring patterns. A droplet of the assay solution is applied to a cellulose nitrate membrane, and upon radial chromatographic flow and evaporation of the solvent, fluorescent patterns appear under UV irradiation. The target nucleic acid is isothermally amplified and is immediately hybridized with fluorescent oligonucleotide probes in a one-pot reaction. We established the fluorescent ring assay integrated with isothermal amplification (iFluor-RFA = isothermal fluorescent ring-based radial flow assay), and feasibility was tested using nucleic acid targets of the receptor binding domain (RBD) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2. We demonstrate that the iFluor-RFA method is capable of specific and sensitive detection in the subpicomole range, as well as multiplexed detection even in complex solutions. Furthermore, we applied deep learning analysis of the fluorescence images, showing that patterns could be classified as positive or negative and that quantitative amounts of the target could be predicted. The current technique, which is a membrane pattern-based nucleic acid assay combined with deep learning analysis, provides a novel approach in diagnostic platform development that can be versatilely applied for the rapid detection of infectious pathogens.