Microfluidic systems for diagnostic applications: a review

Because of intensive developments in recent years, the microfluidic system has become a powerful tool for biological analysis. Entire analytic protocols including sample pretreatment, sample/reagent manipulation, separation, reaction, and detection can be integrated into a single chip platform. A lot of demonstrations on the diagnostic applications related to genes, proteins, and cells have been reported because of their advantages associated with miniaturization, automation, sensitivity, and specificity. The aim of this article is to review recent developments in microfluidic systems for diagnostic applications. Based on the categories of various fluid-manipulating mechanisms and biological detection approaches, in-depth discussion of the microfluidic-based diagnostic systems is provided. Moreover, a brief discussion on materials and manufacturing techniques will be included. The current excellent integration of microfluidic systems and diagnostic applications suggests a solid foundation for the development of practical point-of-care devices.     

Sample Acquisition and Control On-Chip Microfluidic Sample Preparation

Microfluidic flow conditions allow the design of highly effective, yet simple devices for on-chip sample preparation and cleanup. Over the past few years, Micronics has developed a number of novel microfluidic structures that are compatible with complex samples such as whole blood or contaminated environmental fluids. The H-Filter is a technology based on the parallel laminar flow of two or more miscible streams in contact with each other. Such streams do not mix, but chemicals contained in these streams can diffuse from one stream into the other, with smaller molecules diffusing faster than larger ones. This principle can be used, for example, to remove salt from a solution containing DNA, or to extract smaller molecules from whole blood. The T-Sensor is based on the same laminar flow diffusion principle, but combines sample preparation with self-calibration and detection. These devices can be used not only in stand-alone research and point-of-use testing applications, but they can also be integrated, as sample preparation modules, into existing laboratory systems.     

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.     

Sample preconcentration in microfluidic devices

Microfluidic systems have attracted considerable attention and have experienced rapid growth in the past two decades due to advantages associated with miniaturization, integration, and automation. Poor detection sensitivities mainly attributed to the small dimensions of these lab-on-a-chip (LOC) devices; however, sometimes can greatly hinder their practical applications in detecting low-abundance analytes, particularly those in bio-samples. Although off-chip sample pretreatment strategies can be used to address this problem prior to analysis, they may introduce contaminants or lead to an undesirable loss of some original sample volume. Moreover, they are often time-consuming and labor-intensive. Toward the goals of automation, improvement in analytical efficiency, and reductions in sample loss and contamination, many on-chip sample preconcentration techniques based on different working principles for improving the detection sensitivity have been developed and implemented in microchips. The aim of this article is to review recent works in microchip-based sample preconcentration techniques and give detailed discussions about these techniques. We start with a brief introduction regarding the importance of preconcentration techniques in microfluidics and the classification of these techniques based on their concentration mechanisms, followed by in-depth discussions of about these techniques. Finally, personal perspectives on microfluidic-based sample preconcentration will be provided. These advancements in microfluidic sample preconcentration techniques may provide promising strategies for improving the detection sensitivities of LOC devices in many practical applications.     

High throughput blood plasma separation using a passive PMMA microfluidic device

Since plasma is rich in many biomarkers used in clinical diagnostic experiments, microscale blood plasma separation is a primitive step in most of microfluidic analytical chips. In this paper, a passive microfluidic device for on-chip blood plasma separation based on Zweifach–Fung effect and plasma skimming was designed and fabricated by hot embossing of microchannels on a PMMA substrate and thermal bonding process. Human blood was diluted in various times and injected into the device. The main novelty of the proposed microfluidic device is the design of diffuser-shaped daughter channels. Our results demonstrated that this design exerted a considerable positive influence on the separation efficiency of the passive separator device, and the separation efficiency of 66.6 % was achieved. The optimum purity efficiency of 70 % was achieved for 1:100 dilution times.     

微流控芯片技术在心肌标志物检测中的应用综述

心肌标志物的检测异常,是急性心肌梗死的重要诊断指标之一。对心肌标志物的监测可直接影响心血管疾病患者的临床诊断、危险分层、治疗方案选择和预后判断。微流控芯片集进样、预处理、分离和检测于一体,具有样品需求量小、便携、分析快速等特点,是理想的心肌标志物检测平台。文章根据检测方法的不同,综述了近年来利用微流控芯片平台对心肌标志物的检测。已有检测方法中主要是光学和电学方法。随着传感器技术的发展,更多检测方法被采用。通过及时的综述概括,既可对已有技术和方法起到归纳作用,又可促进微流控芯片在心肌标志物即时诊断领域的发展。     

Particle/cell separation on microfluidic platforms based on centrifugation effect: a review

Particle/cell separation in heterogeneous mixtures including biological samples is a standard sample preparation step for various biomedical assays. A wide range of microfluidic-based methods have been proposed for particle/cell sorting and isolation. Two promising microfluidic platforms for this task are microfluidic chips and centrifugal microfluidic disks. In this review, we focus on particle/cell isolation methods that are based on liquid centrifugation phenomena. Under this category, we reviewed particle/cell sorting methods which have been performed on centrifugal microfluidic platforms, and inertial microfluidic platforms that contain spiral channels and multi-orifice channels. All of these platforms implement a form of centrifuge-based particle/cell separation: either physical platform centrifugation in the case of centrifugal microfluidic platforms or liquid centrifugation due to Dean drag force in the case of inertial microfluidics. Centrifugal microfluidic platforms are suitable for cases where the preparation step of a raw sample is required to be integrated on the same platform. However, the limited available space on the platform is the main disadvantage, especially when high sample volume is required. On the other hand, inertial microfluidics (spiral and multi-orifice) showed various advantages such as simple design and fabrication, the ability to process large sample volume, high throughput, high recovery rate, and the ability for multiplexing for improved performance. However, the utilization of syringe pump can reduce the portability options of the platform. In conclusion, the requirement of each application should be carefully considered prior to platform selection.     

Effect of microstructure on blood cell clogging in blood separators based on capillary action

In this study, the blood cell clogging phenomenon occurring in blood separators based on capillary action is carefully investigated and how to minimize the reduction in plasma separation speed caused by clogging is discussed. Four different blood separators are fabricated on optically transparent glass substrates to clearly observe the blood plasma separation and the blood cell clogging in the microfluidic devices. Each separation experiment is captured by a high-speed video camera. The captured images are analyzed using a theoretical model proposed in this study to quantify the effect of the microstructure on the degree of blood cell clogging. Finally, design guidelines for the microstructure of the micro blood separator are discussed based on the analysis. D. Kim and J. Y. Yun contributed equally to this work.     

Centrifugal automation of a triglyceride bioassay on a low-cost hybrid paper-polymer device

We present a novel paper-polymer hybrid construct for the simple automation of fundamental microfluidic operations in a lab-on-a-disc platform. The novel design, we term a paper siphon, consists of chromatographic paper strips embedded along a siphon microchannel. The paper siphon relies on two main interplaying forces to create unique valving and liquid-sampling methods in centrifugal microfluidics. At sufficiently low speeds, the inherent wicking of the paper overcomes the rotationally induced centrifugal force to drive liquids towards inwards positions of the disc. At elevated speeds, the dominant centrifugal force will extract liquid from the siphon paper strip towards the edge of the disc. Distinct modes of flow control have been developed to account for water (reagent) and more viscous plasma samples. The system functionality is demonstrated by the automation of sequential sample preparation steps in a colorimetric triglyceride assay: plasma is metered from a whole blood sample and incubated with a specific enzymatic mixture, followed by detection of triglyceride levels through (off-disc) absorbance measurements. The successful quantification of triglycerides and the simple fabrication offer attractive directions for such hybrid devices in low-cost bioanalysis.     

On-chip whole blood plasma separator based on microfiltration,sedimentation and wetting contrast

Miniaturized on-chip blood separators have a great value for point-of-care diagnosis. In our work, a combined design strategy—microfiltration, sedimentation in a retarded flow, and wetting contrast—was taken to overcome the known limitations of on-chip blood separators. Our microfluidic chip consists of a polydimethylsiloxane micropillar array and an etched glass with microchannel branches. The red blood cells are significantly slowed and gradually settled down due to micropillars and enlarged dimension of a chamber. An etched glass microchannel allows the extraction of blood plasma exclusively due to the capillary effect. The fabricated microfluidic device can separate blood plasma from a whole blood sample without any external driving force or dilution. The measured plasma separation efficiency was close to 100 % from human whole blood. Autonomous on-chip separation and collection of blood plasma was demonstrated.     

Micro-injection moulding of polymer microfluidic devices

Microfluidic devices have several applications in different fields, such as chemistry, medicine and biotechnology. Many research activities are currently investigating the manufacturing of integrated microfluidic devices on a mass-production scale with relatively low costs. This is especially important for applications where disposable devices are used for medical analysis. Micromoulding of thermoplastic polymers is a developing process with great potential for producing low-cost microfluidic devices. Among different micromoulding techniques, micro-injection moulding is one of the most promising processes suitable for manufacturing polymeric disposable microfluidic devices. This review paper aims at presenting the main significant developments that have been achieved in different aspects of micro-injection moulding of microfluidic devices. Aspects covered include device design, machine capabilities, mould manufacturing, material selection and process parameters. Problems, challenges and potential areas for research are highlighted.     

Deterministic lateral displacement (DLD) in the high Reynolds number regime: high-throughput and dynamic separation characteristics

Recent progress in the development of biosensors has created a demand for high-throughput sample preparation techniques that can be easily integrated into microfluidic or lab-on-a-chip platforms. One mechanism that may satisfy this demand is deterministic lateral displacement (DLD), which uses hydrodynamic forces to separate particles based on size. Numerous medically relevant cellular organisms, such as circulating tumor cells (10–15 µm) and red blood cells (6–8 µm), can be manipulated using microscale DLD devices. In general, these often-viscous samples require some form of dilution or other treatment prior to microfluidic transport, further increasing the need for high-throughput operation to compensate for the increased sample volume. However, high-throughput DLD devices will require a high flow rate, leading to an increase in Reynolds numbers (Re) much higher than those covered by existing studies for microscale (≤?100 µm) DLD devices. This study characterizes the separation performance for microscale DLD devices in the high-Re regime (10?<?Re?<?60) through numerical simulation and experimental validation. As Re increases, streamlines evolve and microvortices emerge in the wake of the pillars, resulting in a particle trajectory shift within the DLD array. This differs from previous DLD works, in that traditional models only account for streamlines that are characteristic of low-Re flow, with no consideration for the transformation of these streamlines with increasing Re. We have established a trend through numerical modeling, which agrees with our experimental findings, to serve as a guideline for microscale DLD performance in the high-Re regime. Finally, this new phenomenon could be exploited to design passive DLD devices with a dynamic separation range, controlled simply by adjusting the device flow rate.     

Thread-based microfluidic system for detection of rapid blood urea nitrogen in whole blood

This study develops a thread-based microfluidic device with variable volume injection capability and 3-dimensional (3D) detection electrodes for capillary electrophoresis electrochemical (CE–EC) detection of blood urea nitrogen (BUN) in whole blood. A poly methyl methacrylate (PMMA) substrate with concave 3D electrodes produced by the hot embossing method is used to enhance the sensing performance of the CE–EC system. Results show that the chip with 3D sensing electrodes exhibits a measured current response nine times higher and signal-to-noise ratio five times higher when compared to the peak responses obtained using a chip with conventional 2D sensing electrodes. In addition, the developed thread-based microfluidic system is capable of injecting variable sample volumes into the separation thread simply by wrapping the injection thread different numbers of times around the separation thread. The peak S/N ratio can be further enhanced with this simple approach. Results also indicate that the CE–EC system exhibits good linear dynamic range for detecting a urea sample in concentrations from 0.1 to 10.0 mM (R ² = 0.9848), which is suitable for adoption in detecting the BUN concentration in human blood (1.78–7.12 mM). Separation and detection of the ammonia ions converted from BUN in whole blood is successfully demonstrated in the present study, with the developed thread-based microfluidic system providing a low-cost, high-performance method for detecting BUN in human blood.     

Passive microfluidic devices for plasma extraction from whole human blood

Our goal is to analyze and compare different continuous microfluidic principles dedicated to plasma extraction from hardly diluted human blood for lab-on-chip applications. First, the strengths and weaknesses of various emerging passive microfluidic methods (microfiltration- and centrifugation-based methods) were analyzed. Various devices were designed, microfabricated and tested with beads or blood. Filtration may be efficient, but with a high sample dilution, low flow rate and optimized geometry. Due to fast cell clogging, this remains a short-term solution. Separation effects resulting from centrifugal acceleration in curved channel flows are hindered by Dean vortices and anyhow are not pronounced with blood. An innovative device is then proposed and investigated experimentally. This is based on the lateral migration of red cells and the resulting cell-free layer, which is used to supply geometric singularities (an ear-cavity or a corner-edge) and locally enhance the clear plasma region. A maximum extraction of 10.7% is obtained for 1/20 diluted blood, injected at 100 μL/min in the corner-edge design.     

Electrochemical detection techniques in micro- and nanofluidic devices

Electrochemical techniques are widely used in microfluidic and nanofluidic devices because they are suitable for miniaturization, have better sensitivity compared to optical detection techniques, and their components can be reliably microfabricated. In addition to the detection and quantification of analytes, electrochemical techniques can be used to monitor processes such as biological cell death and protein/DNA separations/purifications. Such techniques are combined with micro- and nanofluidic devices with point-of-care (POC) applications in mind, where cost, footprint, ease of use, and independence from peripheral equipment are critical for a viable design. A large variety of electrode materials and device configurations have been employed to meet these requirements. This review introduces the reader to the major electrochemical techniques, materials, and fabrication methods for working and reference electrodes, and to surface modifications of electrodes to facilitate electrochemical measurements, in the context of micro- and nanofluidic devices. The continuing development of these techniques holds promise for the next-generation lab-on-a-chip devices, which can realize the goals of this technology such as POC clinical analysis.     

Electrophoresis separation and electrochemical detection on a novel thread-based microfluidic device

This paper describes a thread-based microfluidic system for rapid and low-cost electrophoresis separation and electrochemical (EC) detection of ion samples. Instead of using liquid channel for sample separation, thin polyester threads of various diameters are used as the routes for separating the samples with electrophoresis. Hot-pressed PMMA chip with protruding sleeper structures are adopted to set up the polyester threads and for electrochemical detection of the ion samples on the thread. Plasma treatment greatly improves the wetability of thin threads and surface quality of the threads. The measured electrical currents on plasma treated threads are 10 times greater than the threads without treatment. Results indicate that nice redox signals can be obtained by measuring ferric cyanide salt on the polyester thread. The estimated detection limit for EC sensing of potassium ferricyanide (K₃Fe(CN)₆) is around 6.25 μM using the developed thread-based microfluidic device. Mixed ion samples (Cl^?, Br^? and I^?) and bio-sample are successfully separated and detected using the developed thread-based microfluidic device.     

Microfluidic applications of functionalized magnetic particles for environmental analysis: focus on waterborne pathogen detection

The continuous surveillance of drinking water is extremely important to provide early warning of contamination and to ensure continuous supplies of healthy drinking water. Isolation and detection of a particular type of pathogen present at low concentration in a large volume of water, concentrating the analyte in a small detection volume, and removing detection inhibiting factors from the concentrated sample, present the three most important challenges for water quality monitoring laboratories. Combining advanced biological detection methods (e.g., nucleic acid-based or immunology-based protocols) with microfluidics and immunomagnetic separation techniques that exploit functionalized magnetic particles has tremendous potential for realization of an integrated system for pathogen detection, in particular, of waterborne pathogens. Taking advantage of the unique properties of magnetic particles, faster, more sensitive, and more economical diagnostic assays can be developed that can assist in the battle against microbial pathogenesis. In this review, we highlight current technologies and methods used for realization of magnetic particle-based microfluidic integrated waterborne pathogen isolation and detection systems, which have the potential to comply in future with regulatory water quality monitoring requirements.     

No more bonding,no more clamping,magnetically assisted membrane integration in microfluidic devices

The integration of porous membranes with microfluidic devices allows a simple but high-throughput mass transport control for numerous microfluidic applications, such as single-cell separation, sample analysis, and purification. In this study, we demonstrate a novel integration process of porous membranes into microfluidic devices by applying a magnetic field and hydrodynamically stabilizing them. This new approach simplifies the integration process by removing physicochemical bonding between membranes and microfluidic devices, but overcomes many practical issues observed in current methods, such as device leakage, membrane replacement, and membrane material selection. More importantly, our approach allows us to install membranes with diverse physicochemical features and spatial configurations into a single microfluidic device. This additional ability can significantly improve its performance and capability in applications. Finally, we successfully demonstrate the utilization of our membrane device for simple particle separation.     

Magnetic particle dosing and size separation in a microfluidic channel

Separation of functional magnetic particles or magnetically labeled entities is a key feature for bioanalytical or biomedical applications and therefore also an important component of lab-on-a-chip devices for biological applications. We present a novel integrated microfluidic magnetic bead manipulation device, comprising dosing of magnetic particles, controlled release and subsequent magnetophoretic size separation with high resolution. The system is designed to meet the requirements of specific bioassays, in particular of on-chip agglutination assays for the detection of rare analytes by particle coupling as doublets. Integrated soft-magnetic microtips with different shapes provide the magnetic driving force of the bead manipulation protocol. The magnetic tips that serve as field concentrators of an external electromagnetic field, are positioned in close contact to a microfluidic channel in order to generate high magnetic actuation forces. Mixtures of 1.0 μm and 2.8 μm superparamagnetic beads have been used to characterize the system. Magnetophoretic size separation with high resolution was performed in static conditions and in continuous flow mode. In particular, we could demonstrate the separation of 1.0 μm single beads and doublets in a sample flow.     

Towards numerical prototyping of labs-on-chip: modeling for integrated microfluidic devices

This review article presents an overview of some of the tools, techniques and applications of numerical simulation for integrated microfluidic devices. Provided is a broad overview of the different areas to which numerical techniques have been applied in the development of these devices from detailed studies of fundamental microfluidic problems (e.g., species mixing and sample dispersion) to unique approaches that take a more global overview of the entire system. While the majority of the work to date has been in these areas, also reviewed is some recent progress into other equally important areas of microscale transport such as thermal analysis and chemical reactivity and specificity. An overview of the advantages and disadvantages of common numerical techniques is also presented along with a brief discussion of some of the existing numerical tools, focusing on those best suited for microscale transport analysis. As microfluidic devices become increasingly complex, optimal fluidic and transport designs become more and more difficult to do experimentally. Thus, it is believed that future demand in the field will be for highly integrated simulation tools that allow users without a significant computational fluids background to numerical prototype highly integrated devices.