Application of microfluidic technologies in forensic analysis.

The application of microfluidic technology in forensic medicine has steadily expanded over the last two decades due to the favorable features of low cost, rapidity, high throughput, user-friendliness, contamination-free, and minimum sample and reagent consumption. In this context, bibliometric methods were adopted to visualize the literature information contained in the Science Citation Index Expanded from 1989 to 2022, focusing on the co-occurrence analysis of forensic and microfluidic topics. A deep interpretation of the literature was conducted based on co-occurrence results, in which microfluidic technologies and their applications in forensic medicine, particularly forensic genetics, were elaborated. The purpose of this review is to provide an impartial evaluation of the utilization of microfluidic technology in forensic medicine. Additionally, the challenges and future trends of implementing microfluidic technology in forensic genetics are also addressed.

Innovative Quantification of Critical Quality Attributes.

Potency testing is an important part of the evaluation of cellular therapy products. In vitro quantification of identified quality-related biomarkers is a technique often used at the laboratory. Nonetheless, the limited stability of most cellular therapy products, the lot variability and the limited time within which to perform testing are currently hindering their widespread use. Fortunately, within the last two decades, the evolution of material technology and miniaturisation processes has enabled the research community to shift the spotlight of attention towards the Lab-on-Chip concept for diagnostic applications. Such devices enable portable, rapid, sensitive, automated and affordable biochemical analyses aiming to advance the healthcare services across a broad application spectrum. However, it could be argued that the aspirations on their affordability are far from being exceeded, mainly due to the lack of a practical manufacturing technology. The Lab-on-Printed Circuit Board (Lab-on-PCB) approach has demonstrated enormous potential for developing economical diagnostic platforms leveraging the advantage provided by economy of scale manufacturing of the long-standing PCB industry. The integration capabilities that the PCB platform introduces to the Lab-on-Chip concept concerning the electronics and microfluidics seem to be unique. In this chapter, we will be reviewing the progress of Lab-on-PCB prototypes quantifying within miniaturised microchips a range of critical quality attributes with potential in potency testing. We will focus on their technology and applications whilst addressing the potential of this approach in practical use and commercialisation.

Microfluidic Approaches for Affinity-Based Exosome Separation.

As a subspecies of extracellular vesicles (EVs), exosomes have provided promising results in diagnostic and theranostic applications in recent years. The nanometer-sized exosomes can be extracted by liquid biopsy from almost all body fluids, making them especially suitable for mainly non-invasive point-of-care (POC) applications. To achieve this, exosomes must first be separated from the respective biofluid. Impurities with similar properties, heterogeneity of exosome characteristics, and time-related biofouling complicate the separation. This practical review presents the state-of-the-art methods available for the separation of exosomes. Furthermore, it is shown how new separation methods can be developed. A particular focus lies on the fabrication and design of microfluidic devices using highly selective affinity separation. Due to their compactness, quick analysis time and portable form factor, these microfluidic devices are particularly suitable to deliver fast and reliable results for POC applications. For these devices, new manufacturing methods (e.g., laminating, replica molding and 3D printing) that use low-cost materials and do not require clean rooms are presented. Additionally, special flow routes and patterns that increase contact surfaces, as well as residence time, and thus improve affinity purification are displayed. Finally, various analyses are shown that can be used to evaluate the separation results of a newly developed device. Overall, this review paper provides a toolbox for developing new microfluidic affinity devices for exosome separation.

A review on microscale polymerase chain reaction based methods in molecular diagnosis, and future prospects for the fabrication of fully integrated portable biomedical devices.

Since the advent of microfabrication technology and soft lithography, the lab-on-a-chip concept has emerged as a state-of-the-art miniaturized tool for conducting the multiple functions associated with micro total analyses of nucleic acids, in series, in a seamless manner with a miniscule volume of sample. The enhanced surface-to-volume ratio inside a microchannel enables fast reactions owing to increased heat dissipation, allowing rapid amplification. For this reason, PCR has been one of the first applications to be miniaturized in a portable format. However, the nature of the basic working principle for microscale PCR, such as the complicated temperature controls and use of a thermal cycler, has hindered its total integration with other components into a micro total analyses systems (µTAS). This review (with 179 references) surveys the diverse forms of PCR microdevices constructed on the basis of different working principles and evaluates their performances. The first two main sections cover the state-of-the-art in chamber-type PCR microdevices and in continuous-flow PCR microdevices. Methods are then discussed that lead to microdevices with upstream sample purification and downstream detection schemes, with a particular focus on rapid on-site detection of foodborne pathogens. Next, the potential for miniaturizing and automating heaters and pumps is examined. The review concludes with sections on aspects of complete functional integration in conjunction with nanomaterial based sensing, a discussion on future prospects, and with conclusions. Graphical abstract In recent years, thermocycler-based PCR systems have been miniaturized to palm-sized, disposable polymer platforms. In addition, operational accessories such as heaters and mechanical pumps have been simplified to realize semi-automatted stand-alone portable biomedical diagnostic microdevices that are directly applicable in the field. This review summarizes the progress made and the current state of this field.

SAW-Based Phononic Crystal Microfluidic Sensor-Microscale Realization of Velocimetry Approaches for Integrated Analytical Platform Applications.

The current work demonstrates a novel surface acoustic wave (SAW) based phononic crystal sensor approach that allows the integration of a velocimetry-based sensor concept into single chip integrated solutions, such as Lab-on-a-Chip devices. The introduced sensor platform merges advantages of ultrasonic velocimetry analytic systems and a microacoustic sensor approach. It is based on the analysis of structural resonances in a periodic composite arrangement of microfluidic channels confined within a liquid analyte. Completed theoretical and experimental investigations show the ability to utilize periodic structure localized modes for the detection of volumetric properties of liquids and prove the efficacy of the proposed sensor concept.

Development of on-chip fully automated immunoassay system "µTASWako i30" to measure the changes in glycosylation profiles of alpha-fetoprotein in patients with hepatocellular carcinoma.

Glycosylation profiles significantly change during oncogenesis. Aberrant glycosylation can be used as a cancer biomarker in clinical settings. Different glycoforms can be separately detected using lectin affinity electrophoresis and lectin array-based methods. However, most methodologies and procedures need experienced technique to perform the assays and expertise to interpret the results. To apply glycomarkers for clinical practice, a robust assay system with an easy-to-use workflow is required. Wako's µTASWako i30, a fully automated immunoanalyzer, was developed for in vitro diagnostics based on microfluidic technology. It utilizes the principles of liquid-phase binding assay, where immunoreactions are performed in a liquid phase, and electrokinetic analyte transport assay. Capillary electrophoresis on microfluidic chip has enabled the detection of different glycoform types of alpha-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma. AFP with altered glycosylation can be separated based on the reactivity to Lens culinaris agglutinin on electrophoresis. The glycoform AFP-L3 was reportedly more specific in hepatocellular carcinoma. This assay system can provide a high sensitivity and rapid results in 9 min. The test results for ratio of AFP-L3 to total AFP using µTASWako i30 are correlated with those of conventional methodology. The µTASWako assay system and the technology can be utilized for glycosylation analysis in the postgenomic era.

Microfluidics Integrated Biosensors: A Leading Technology towards Lab-on-a-Chip and Sensing Applications.

A biosensor can be defined as a compact analytical device or unit incorporating a biological or biologically derived sensitive recognition element immobilized on a physicochemical transducer to measure one or more analytes. Microfluidic systems, on the other hand, provide throughput processing, enhance transport for controlling the flow conditions, increase the mixing rate of different reagents, reduce sample and reagents volume (down to nanoliter), increase sensitivity of detection, and utilize the same platform for both sample preparation and detection. In view of these advantages, the integration of microfluidic and biosensor technologies provides the ability to merge chemical and biological components into a single platform and offers new opportunities for future biosensing applications including portability, disposability, real-time detection, unprecedented accuracies, and simultaneous analysis of different analytes in a single device. This review aims at representing advances and achievements in the field of microfluidic-based biosensing. The review also presents examples extracted from the literature to demonstrate the advantages of merging microfluidic and biosensing technologies and illustrate the versatility that such integration promises in the future biosensing for emerging areas of biological engineering, biomedical studies, point-of-care diagnostics, environmental monitoring, and precision agriculture.

Coupling of fluorescence correlation spectroscopy with capillary and microchannel analytical systems and its applications.

In the past decade, fluorescence correlation spectroscopy (FCS) has become an ultrasensitive and noninvasive single-molecule detection technique, which is widely applied in the physical, chemical, and life-science research. The coupling of FCS with narrow channel flow systems including the ones based on capillary provide the important, convenient, and sensitive assay platforms for probing and understanding the behavior of single molecules or nanoparticles with improved temporal and spatial resolution and need for less sample volume among other advantages. This review focus on different approaches for FCS with capillary and microchannel analytical systems and its applications in confined diffusion study, flow profiles, and imaging of narrow channel, multicomponent analyses such as protein, DNA analysis, and characterization on nanoparticles.

Microfluidic isolation of nucleic acids.

The detection of nucleic acids (NAs) within micro total analysis systems (µTASs) for point-of-care use is a rapidly developing research area. The efficient isolation of NAs from a raw sample is crucial for these systems to be maximally effective. The use of microfluidics assists in reducing sample sizes and reagent consumption, increases speed, avoids contamination, and enables automation. Through miniaturization into microchips, new techniques have been realized that would be unfavorable and inconvenient to use on a macroscopic scale, but provide an excellent platform for the purification of NAs on a microscopic scale. This Review considers the complexities of NA isolation with miniaturized and microfluidic devices, as well as the considerations when choosing a technique for microfluidic NA isolation, along with their advantages, disadvantages, and potential applications. The techniques presented include using silica-based surfaces, functionalized paramagnetic beads, oligonucleotide-modified polymer surfaces, pH-dependent charged surfaces, Al2O3 membranes, and liquid-phase isolation. This Review provides a basis to develop the chemistry to improve NA isolation and move it toward achieving 100% efficiencies.

Microfluidic Paper-Based Analytical Devices (µPADs) and Micro Total Analysis Systems (µTAS): Development, Applications and Future Trends.

Microfluidic paper-based analytical devices and micro total analysis systems are relatively new group of analytical tools, capable of analyzing complex biochemical samples containing macromolecules, proteins, nucleic acids, toxins, cells or pathogens. Within one analytical run, fluidic manipulations like transportation, sorting, mixing or separation are available. Recently, microfluidic devices are a subject of extensive research, mostly for fast and non-expensive biochemical analysis but also for screening of medical samples and forensic diagnostics. They are used for neurotransmitter detection, cancer diagnosis and treatment, cell and tissue culture growth and amplification, drug discovery and determination, detection and identification of microorganisms. This review summarizes development history, basic fabrication methods, applications and also future development trends for production of such devices.

The System's Point of View Applied to Dielectrophoresis in Plate Capacitor and Pointed-versus-Pointed Electrode Chambers.

The DEP force is usually calculated from the object's point of view using the interaction of the object's induced dipole moment with the inducing field. Recently, we described the DEP behavior of high- and low-conductive 200-µm 2D spheres in a square 1 × 1-mm chamber with a plane-versus-pointed electrode configuration from the system's point of view. Here we extend our previous considerations to the plane-versus-plane and pointed-versus-pointed electrode configurations. The trajectories of the sphere center and the corresponding DEP forces were calculated from the gradient of the system's overall energy dissipation for given starting points. The dissipation's dependence on the sphere's position in the chamber is described by the numerical "conductance field", which is the DC equivalent of the capacitive charge-work field. While the plane-versus-plane electrode configuration is field-gradient free without an object, the presence of the highly or low-conductive spheres generates structures in the conductance fields, which result in very similar DEP trajectories. For both electrode configurations, the model describes trajectories with multiple endpoints, watersheds, and saddle points, very high attractive and repulsive forces in front of pointed electrodes, and the effect of mirror charges. Because the model accounts for inhomogeneous objectpolarization by inhomogeneous external fields, the approach allows the modeling of the complicated interplay of attractive and repulsive forces near electrode surfaces and chamber edges. Non-reversible DEP forces or asymmetric magnitudes for the highly and low-conductive spheres in large areas of the chamber indicate the presence of higher-order moments, mirror charges, etc.

Trajectories and Forces in Four-Electrode Chambers Operated in Object-Shift, Dielectrophoresis and Field-Cage Modes-Considerations from the System's Point of View.

In two previous papers, we calculated the dielectrophoresis (DEP) force and corresponding trajectories of high- and low-conductance 200-µm 2D spheres in a square 1 × 1-mm chamber with plane-versus-pointed, plane-versus-plane and pointed-versus-pointed electrode configurations by applying the law of maximum entropy production (LMEP) to the system. Here, we complete these considerations for configurations with four-pointed electrodes centered on the chamber edges. The four electrodes were operated in either object-shift mode (two adjacent electrodes opposite the other two adjacent electrodes), DEP mode (one electrode versus the other three electrodes), or field-cage mode (two electrodes on opposite edges versus the two electrodes on the other two opposite edges). As in previous work, we have assumed DC properties for the object and the external media for simplicity. Nevertheless, every possible polarization ratio of the two media can be modeled this way. The trajectories of the spherical centers and the corresponding DEP forces were calculated from the gradients of the system's total energy dissipation, described by numerically-derived conductance fields. In each of the three drive modes, very high attractive and repulsive forces were found in front of pointed electrodes for the high and low-conductance spheres, respectively. The conductance fields predict bifurcation points, watersheds, and trajectories with multiple endpoints. The high and low-conductance spheres usually follow similar trajectories, albeit with reversed orientations. In DEP drive mode, the four-point electrode chamber provides a similar area for DEP measurements as the classical plane-versus-pointed electrode chamber.

Dielectrophoresis from the System's Point of View: A Tale of Inhomogeneous Object Polarization, Mirror Charges, High Repelling and Snap-to-Surface Forces and Complex Trajectories Featuring Bifurcation Points and Watersheds.

Microscopic objects change the apparent permittivity and conductivity of aqueous systems and thus their overall polarizability. In inhomogeneous fields, dielectrophoresis (DEP) increases the overall polarizability of the system by moving more highly polarizable objects or media to locations with a higher field. The DEP force is usually calculated from the object's point of view using the interaction of the object's induced dipole or multipole moments with the inducing field. Recently, we were able to derive the DEP force from the work required to charge suspension volumes with a single object moving in an inhomogeneous field. The capacitance of the volumes was described using Maxwell−Wagner's mixing equation. Here, we generalize this system's-point-of-view approach describing the overall polarizability of the whole DEP system as a function of the position of the object with a numerical "conductance field". As an example, we consider high- and low conductive 200 µm 2D spheres in a square 1 × 1 mm chamber with plain-versus-pointed electrode configuration. For given starting points, the trajectories of the sphere and the corresponding DEP forces were calculated from the conductance gradients. The model describes watersheds; saddle points; attractive and repulsive forces in front of the pointed electrode, increased by factors >600 compared to forces in the chamber volume where the classical dipole approach remains applicable; and DEP motions with and against the field gradient under "positive DEP" conditions. We believe that our approach can explain experimental findings such as the accumulation of viruses and proteins, where the dipole approach cannot account for sufficiently high holding forces to defeat Brownian motion.

Microfluidic systems for biosensing.

In the past two decades, Micro Fluidic Systems (MFS) have emerged as a powerful tool for biosensing, particularly in enriching and purifying molecules and cells in biological samples. Compared with conventional sensing techniques, distinctive advantages of using MFS for biomedicine include ultra-high sensitivity, higher throughput, in-situ monitoring and lower cost. This review aims to summarize the recent advancements in two major types of micro fluidic systems, continuous and discrete MFS, as well as their biomedical applications. The state-of-the-art of active and passive mechanisms of fluid manipulation for mixing, separation, purification and concentration will also be elaborated. Future trends of using MFS in detection at molecular or cellular level, especially in stem cell therapy, tissue engineering and regenerative medicine, are also prospected.

High-Aspect-Ratio SU-8-Based Optofluidic Device for Ammonia Detection in Cell Culture Media.

Miniaturization of sensing technology has led to the development of multifunctional micro total analysis systems (µTAS) that benefit from microfluidic technology. Optical sensing is one of the most commonly used sensing approaches integrated into µTAS devices and features high sensitivity and low detection limits. Different materials have been used for the fabrication of µTAS devices, each having their advantages and disadvantages. Herein, a high-aspect-ratio optofluidic waveguide fabricated from SU-8 is presented for the first time. The suitable optical properties and chemical inertness of SU-8 provide a durable device made by a flexible and cost-efficient fabrication process. The optofluidic device was used for colorimetric ammonia (NH3) sensing with a dynamic range of 3-70 µM, a detection limit of 2.5 µM, a response time of 8 min, and close to 10 times better analytical performance compared to using a standard microplate reader. The µTAS device was capable of monitoring NH3 accumulating in the cell culture media of prostatic epithelial cell (BPH-1) culture.

µPump: An open-source pressure pump for precision fluid handling in microfluidics.

An open-source precision pressure pump system and control software is presented, primarily designed for the experimental microfluidics community, although others may find additional uses for this precision pressure source. This mechatronic system is coined 'µPump,' and its performance rivals that of commercially available systems, at a fraction of the cost. The pressure accuracy, stability, and resolution are 0.09%, 0.02%, and 0.02% of the full span, respectively. The settling time to reach 2 bar from zero and stabilize is less than 2 s. Material for building a four-channel µPump (approx. $3000 USD) or an eight-channel µPump (approx. $5000 USD) is approximately a quarter, or a third of the cost of buying a high-end commercial system, respectively. The design rationale is presented, together with documented design details and software, so that the system may be replicated or customized to particular applications. µPump can be used for two-phase droplet microfluidics, single-phase microfluidics, gaseous flow microfluidics and any other applications requiring precise fluid handling. µPump provides researchers, students, and startups with a cost-effective solution for precise fluid control.

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems.

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform.

An Open-Source, Programmable Pneumatic Setup for Operation and Automated Control of Single- and Multi-Layer Microfluidic Devices.

Microfluidic technologies have been used across diverse disciplines (e.g. high-throughput biological measurement, fluid physics, laboratory fluid manipulation) but widespread adoption has been limited in part due to the lack of openly disseminated resources that enable non-specialist labs to make and operate their own devices. Here, we report the open-source build of a pneumatic setup capable of operating both single and multilayer (Quake-style) microfluidic devices with programmable scripting automation. This setup can operate both simple and complex devices with 48 device valve control inputs and 18 sample inputs, with modular design for easy expansion, at a fraction of the cost of similar commercial solutions. We present a detailed step-by-step guide to building the pneumatic instrumentation, as well as instructions for custom device operation using our software, Geppetto, through an easy-to-use GUI for live on-chip valve actuation and a scripting system for experiment automation. We show robust valve actuation with near real-time software feedback and demonstrate use of the setup for high-throughput biochemical measurements on-chip. This open-source setup will enable specialists and novices alike to run microfluidic devices easily in their own laboratories.

A Closed System for Pico-Liter Order Substance Transport from a Giant Liposome to a Cell.

In single cell analysis, transport of foreign substances into a cell is an important technique. In particular, for accurate analysis, a method to transport a small amount (pico-liter order) of substance into the cell without leakage while retaining the cell shape is essential. Because the fusion of the cell and the giant liposome is a closed system to the outside, it may be possible to transport a precise, small amount of substances into the cell. Additionally, there is no possibility that a leaked substance would affect other systems. To develop the liposome-cell transportation system, knowledge about the behavior of substances in the liposome and the cell is important. However, only a few studies have observed the substance transport between a liposome and a cell. Here, we report observation of small amount of substance transport into a single C2C12 cell by using a giant liposome. Substance transport occurred by electrofusion between the cell and the giant liposome containing the substance, which is a closed system. First, to observe the electrofusion and substance transport from the moment of voltage application, we fabricated a microfluidic device equipped with electrodes. We introduced suspensions of cells and liposomes into the microfluidic device and applied alternating current (AC) and direct current (DC) voltages for electrofusion. We observed a small amount (22.4 ± 0.1%, 10.3 ± 0.4% and 9.1 ± 0.1%) of fluorescent substance (Calcein) contained in the liposomes was transported into the cell without leakage outside the cell, and we obtained the diffusion coefficient of Calcein in the cell as 137 ± 18 µm²/s. We anticipate that this system and the knowledge acquired will contribute to future realization of more accurate single cell analysis in a wide range of fields.

Direct coupling of supported liquid membranes to capillary electrophoresis for analysis of complex samples: a tutorial.

This tutorial provides an overview of direct coupling of extraction techniques based on supported liquid membranes (SLMs) to capillary electrophoresis (CE) for treatment and subsequent analysis of complex samples. Pros and cons of using each of the described instrumental arrangement are addressed and where relevant, comments with personal experience of the authors are presented. Solid porous membrane based extraction techniques coupled directly to CE are also presented in this tutorial and a comprehensive discussion is included on their instrumental set-ups and their possible adaptation for use with SLMs.