Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays.

Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.

Microfluidics contrasted to thrombelastography: perplexities in defining hypercoagulability.

BACKGROUND: Elevated clot strength (maximum amplitude [MA]) measured by thrombelastography (TEG) is associated with thrombotic complications. However, it remains unclear how MA translates to thrombotic risks, as this measurement is independent of time, blood flow, and clot degradation. We hypothesize that under flow conditions, increased clot strength correlates to time-dependent measurements of coagulation and resistance to fibrinolysis. MATERIALS AND METHODS: Surgical patients at high risk of thrombotic complications were analyzed with TEG and total thrombus-formation analysis system (T-TAS). TEG hypercoagulability was defined as an r <10.2 min, angle >59, MA >66 or LY30 <0.2% (based off of healthy control data, n = 141). The T-TAS AR and PL chips were used to measure clotting at arterial shear rates. T-TAS measurements include occlusion start time, occlusion time (OT), occlusion speed (OSp), and total clot generation (area under the curve). These measurements were correlated to TEG indices (R time, angle, MA, and LY30). Both T-TAS and TEG assays were challenged with tissue plasminogen activator (t-PA) to assess clot resistance to fibrinolysis. RESULTS: Thirty subjects were analyzed, including five controls. TEG-defined hypercoagulability by MA was detected in 52% of the inflammatory bowel disease/cancer patients; 0% was detected in the controls. There were no TEG measurements that significantly correlated with T-TAS AR and PL chip. However, in the presence of t-PA, T-TAS AR determined OSp to have an inverse relationship with TEG angle (-0.477, P = 0.012) and LY30 (-0.449, P = 0.019), and a positive correlation with R time (0.441 P = 0.021). In hypercoagulability determined by TEG MA, T-TAS PL had a significantly reduced OT (4:07 versus 6:27 min, P = 0.043). In hypercoagulability defined by TEG LY30, T-TAS PL had discordant findings, with a significantly prolonged OT (6:36 versus 4:30 min, P = 0.044) and a slower OSp (10.5 versus 19.0 kPa/min, P = 0.030). CONCLUSIONS: Microfluidic coagulation assessment with T-TAS has an overall poor correlation with most TEG measurements in a predominantly hypercoagulable patient population, except in the presence of t-PA. The one anticipated finding was an elevated MA having a shorter time to platelet-mediated microfluidic occlusion, supporting the role of platelets and hypercoagulability. However, hypercoagulability defined by LY30 had opposing results in which a low LY30 was associated with a longer PL time to occlusion and slower OSp. These discordant findings warrant ongoing investigation into the relationship between clot strength and fibrinolysis under different flow conditions.

3D printed microfluidics for biological applications.

The term "Lab-on-a-Chip," is synonymous with describing microfluidic devices with biomedical applications. Even though microfluidics have been developing rapidly over the past decade, the uptake rate in biological research has been slow. This could be due to the tedious process of fabricating a chip and the absence of a "killer application" that would outperform existing traditional methods. In recent years, three dimensional (3D) printing has been drawing much interest from the research community. It has the ability to make complex structures with high resolution. Moreover, the fast building time and ease of learning has simplified the fabrication process of microfluidic devices to a single step. This could possibly aid the field of microfluidics in finding its "killer application" that will lead to its acceptance by researchers, especially in the biomedical field. In this paper, a review is carried out of how 3D printing helps to improve the fabrication of microfluidic devices, the 3D printing technologies currently used for fabrication and the future of 3D printing in the field of microfluidics.

Magnetic bead droplet immunoassay of oligomer amyloid ß for the diagnosis of Alzheimer's disease using micro-pillars to enhance the stability of the oil-water interface.

Despite scientific progress in the study of Alzheimer's disease (AD), it is still challenging to develop a robust and sensitive methodology for the early diagnosis of AD due to the lack of a decisive biomarker in blood. Recent reports on the oligomer amyloid ß (Aß) as a biomarker demonstrated its possibility for identifying early onset of AD in patients, but its low concentration in blood requires highly reliable detection techniques. To overcome the low reliability and labor-intensive procedures of conventional enzyme-linked immunosorbent assay (ELISA), we present a magnetic bead-droplet immunoassay platform for simple and highly sensitive detection of oligomer Aß for the diagnosis of AD. This microchip consists of chambers that contain water-based reagents or oil for consecutive assay procedures, and there are arrays of micro-pillars fabricated between the two adjacent chambers to form robust water-oil interfaces. With the aid of these micro-pillars, magnetic beads can stably pass through each chamber by linearly actuating a magnet along the microchip. The robust water-oil interface and simple procedures of the assay make it possible to obtain reliable results from this microchip. The intensity of the fluorescence at the read-out chamber increased quantitatively and linearly, depending on the amount of serially-diluted standard Aß solution. The results of the assay indicated that the limit of detection was about 10 pg/mL even though it was done with manual manipulation of the magnet. This platform simplified the complicated ELISA procedure and achieved high sensitivity that was no lower than that of the conventional magnetic bead immunoassay. The magnetic bead-droplet platform reduced the assay time to 45 min, and it also reduced the amount of antibody usage in a single diagnosis significantly (10-30 ng of antibody per single assay). Consequently, this microfluidic chip has strong potential as a feasible system for use in the diagnosis of AD with a fast and easy immunoassay process, since the suggested platform can be automated with ease for point-of-care testing as well as high-throughput diagnostic equipment.

Approaching near real-time biosensing: microfluidic microsphere based biosensor for real-time analyte detection.

In this study we describe a simple lab-on-a-chip (LOC) biosensor approach utilizing well mixed microfluidic device and a microsphere-based assay capable of performing near real-time diagnostics of clinically relevant analytes such cytokines and antibodies. We were able to overcome the adsorption kinetics reaction rate-limiting mechanism, which is diffusion-controlled in standard immunoassays, by introducing the microsphere-based assay into well-mixed yet simple microfluidic device with turbulent flow profiles in the reaction regions. The integrated microsphere-based LOC device performs dynamic detection of the analyte in minimal amount of biological specimen by continuously sampling micro-liter volumes of sample per minute to detect dynamic changes in target analyte concentration. Furthermore we developed a mathematical model for the well-mixed reaction to describe the near real time detection mechanism observed in the developed LOC method. To demonstrate the specificity and sensitivity of the developed real time monitoring LOC approach, we applied the device for clinically relevant analytes: Tumor Necrosis Factor (TNF)-α cytokine and its clinically used inhibitor, anti-TNF-α antibody. Based on the reported results herein, the developed LOC device provides continuous sensitive and specific near real-time monitoring method for analytes such as cytokines and antibodies, reduces reagent volumes by nearly three orders of magnitude as well as eliminates the washing steps required by standard immunoassays.

Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system.

A highly sensitive immunoassay utilizing surface-enhanced Raman scattering (SERS) has been developed with a new Raman reporter and a unique SERS-active substrate incorporated into a microfluidic device. An appropriately designed Raman reporter, basic fuchsin (FC), gives strong SERS enhancement and has the ability to bind both the antibody and gold nanostructures. The fuchsin-labeled immuno-Au nanoflowers can form a sandwich structure with the antigen and the antibody immobilized on the SERS-active substrate based on Au-Ag coated GaN. Our experimental results indicate that this SERS-active substrate with its strong surface-enhancement factor, high stability and reproducibility plays a crucial role in improving the efficiency of SERS immunoassay. This SERS assay was applied to the detection of Hepatitis B virus antigen (HBsAg) in human blood plasma. A calibration curve was obtained by plotting the intensity of SERS signal of FC band at 1178cm(-1) versus the concentration of antigen. The low detection limit for Hepatitis B virus antigen was estimated to be 0.01IU/mL. The average relative standard deviation (RSD) of this method is less than 10%. This SERS immunoassay gives exact results over a broad linear range, reflecting clinically relevant HBsAg concentrations. It also exhibits high biological specificity for the detection of Hepatitis B virus antigen.

Engineering particle trajectories in microfluidic flows using particle shape.

Recent advances in microfluidic technologies have created a demand for techniques to control the motion of flowing microparticles. Here we consider how the shape and geometric confinement of a rigid microparticle can be tailored for 'self-steering' under external flow. We find that an asymmetric particle, weakly confined in one direction and strongly confined in another, will align with the flow and focus to the channel centreline. Experimentally and theoretically, we isolate three viscous hydrodynamic mechanisms that contribute to particle dynamics. Through their combined effects, a particle is stably attracted to the channel centreline, effectively behaving as a damped oscillator. We demonstrate the use of self-steering particles for microfluidic device applications, eliminating the need for external forces or sheath flows.

Picoinjection enables digital detection of RNA with droplet rt-PCR.

The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization.

Improvement of the positional dispensing in the washing machine MW 2001.

This article is about how to improve the positional dispensing accuracy of low-volume (microliters) drops and to understand the factors affecting them. These same parameters can be investigated to reduce deleterious effects on dispensing performance. Many applications, such as those in immunoassay diagnostics, require accurate volumetric dispensing and are also less tolerant of other phenomena that cause the reagent to be located outside the target area of interest. In this article, we work with liquid inertia. This phenomenon is present in positional dispensing and affects its accuracy. The negative effect of liquid inertia is reduced by changing parameters such as pressure and the diameter of the involved conducts.

Evaluation of the fully automated hematological analyzer Sysmex XE-5000 for flow cytometric analysis of peritoneal fluid.

Although microscopy still represents the gold standard for cytometric analysis of peritoneal fluids, automated flow cytometry may improve throughput and accuracy. We evaluated the performance of total nucleated cell (TNC), white blood cell (WBC), polymorphonuclear cell (PMN), and mononuclear cell (MONO) counts of Sysmex XE-5000 on peritoneal fluids. The imprecision was excellent, being always lower than 11%, whereas linearity studies yielded correlation coefficients of 1.00 for all parameters. The carryover was always lower than 0.2%. The comparison between XE-5000 and microscopic analysis of 117 ascitic fluids yielded correlation coefficients always greater than 0.96, with mean biases <11/µL. The diagnostic accuracy versus manual microscopy was greater than that of XE-2100, especially at thresholds for septic ascites (100 versus 98% for ≥500 WBC/µL; 98 versus 93% for ≥250 PMN/µL). The correlation with manual microscopy for macrophages and mesothelial cell count was also higher for XE-5000 than for XE-2100 (0.63 versus 0.55). The results of this evaluation show optimal performance of XE-5000 for routine analysis of ascitic fluids, which are combined with the advantages of automated analysis such as high throughout, shortened turnaround time, no need of sample preparation and trained staff, reduced sample volume, and less likelihood of transcriptional errors.

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices.

In this work, we first employ a drying method combining with the bienzyme colorimetric detection of glucose and uric acid on microfluidic paper-based analysis devices (µPADs). The channels of 3D µPADs are also designed by us to get better results. The color results are recorded by both Gel Documentation systems and a common camera. By using Gel Documentation systems, the limits of detection (LOD) of glucose and uric acid are 3.81 × 10(-5)M and 4.31 × 10(-5)M, respectively one order of magnitude lower than that of the reported methods on µPADs. By using a common camera, the limits of detection (LOD) of glucose and uric acid are 2.13 × 10(-4)M and 2.87 × 10(-4)M, respectively. Furthermore, the effects of detection conditions have been investigated and discussed comprehensively. Human serum samples are detected with satisfactory results, which are comparable with the clinical testing results. A low-cost, simple and rapid colorimetric method for the simultaneous detection of glucose and uric acid on the µPADs has been developed with enhanced sensitivity.

Ultimate resolution for refractometric sensing with whispering gallery mode microcavities.

Many proposed microfluidic biosensor designs are based on the measurement of the resonances of an optical microcavity. Fluorescence-based resonators tend to be simpler and more robust than setups that use evanescent coupling from tuneable laser to probe the cavity. In all sensor designs the detection limits depend on the wavelength resolution of the detection system, which is a limitation of fluorescence-based devices. In this work, we explore the ultimate resolution and detection limits of refractometric microcavity sensor structures. Because many periodic modes are collected simultaneously from fluorescent resonators, standard Fourier methods can be best suited for rapid and precise analysis of the resonance shifts. Simple numerical expressions to calculate the ultimate sensor resolution and detection limits were found, and the results compared to experiments in which the resonances of fluorescent-core microcapillaries responded to various sucrose concentrations in water.

Geometrical optimisation of a biochip microchannel fluidic separator.

This article reports on the geometric optimisation of a T-shaped biochip microchannel fluidic separator aiming to maximise the separation efficiency of plasma from blood through the improvement of the unbalanced separation performance among different channel bifurcations. For this purpose, an algebraic analysis is firstly implemented to identify the key parameters affecting fluid separation. A numerical optimisation is then carried out to search the key parameters for improved separation performance of the biochip. Three parameters, the interval length between bifurcations, the main channel length from the outlet to the bifurcation region and the side channel geometry, are identified as the key characteristic sizes and defined as optimisation variables. A balanced flow rate ratio between the main and side channels, which is an indication of separation effectiveness, is defined as the objective. It is found that the degradation of the separation performance is caused by the unbalanced channel resistance ratio between the main and side channel routes from bifurcations to outlets. The effects of the three key parameters can be summarised as follows: (a) shortening the interval length between bifurcations moderately reduces the differences in the flow rate ratios; (b) extending the length of the main channel from the main outlet is effective for achieving a uniformity of flow rate ratio but ineffective in changing the velocity difference of the side channels and (c) decreasing the lengths of side channels from upstream to downstream is effective for both obtaining a uniform flow rate ratio and reducing the differences in the flow velocities between the side branch channels. An optimisation process combining the three parameters is suggested as this integration approach leads to fast convergent process and also offers flexible design options for satisfying different requirements.

Multichannel bipotentiostat integrated with a microfluidic platform for electrochemical real-time monitoring of cell cultures.

An electrochemical detection system specifically designed for multi-parameter real-time monitoring of stem cell culturing/differentiation in a microfluidic system is presented. It is composed of a very compact 24-channel electronic board, compatible with arrays of microelectrodes and coupled to a microfluidic cell culture system. A versatile data acquisition software enables performing amperometry, cyclic voltammetry and impedance spectroscopy in each of the 12 independent chambers over a 100 kHz bandwidth with current resolution down to 5 pA for 100 ms measuring time. The design of the platform, its realization and experimental characterization are reported, with emphasis on the analysis of impact of input capacitance (i.e., microelectrode size) and microfluidic pump operation on current noise. Programmable sequences of successive injections of analytes (ferricyanide and dopamine) and rinsing buffer solution as well as the impedimetric continuous tracking for seven days of the proliferation of a colony of PC12 cells are successfully demonstrated.

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels.

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/µL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence.

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum.

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 µm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL⁻¹ levels. MPs were conjugated with ∼90,000 antibodies and ∼200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL⁻¹ for PSA and 0.30 pg mL⁻¹ for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.

High-sensitivity electrochemical enzyme-linked assay on a microfluidic interdigitated microelectrode.

A novel enzyme-linked DNA hybridization assay on an interdigitated array (IDA) microelectrode integrated into a microfluidic channel is demonstrated with sub-nM detection limit. To improve the detection limit as compared to conventional electrochemical biosensors, a recyclable redox product, 4-aminophenol (PAP) is used with an IDA microelectrode. The IDA has a modest and easily fabricated inter-digit spacing of 10 µm, yet we were able to demonstrate 97% recycling efficiency of PAP due to the integration in a microfluidic channel. With a 70 nL sample volume, the characterized detection limit for PAP of 1.0 × 10⁻¹° M is achieved, with a linear dynamic range that extends from 1.0 × 10⁻9 to 1.0 × 10⁻5 M. This detection limit, which is the lowest reported detection limit for PAP, is due to the increased sensitivity provided by the sample confinement in the microfluidic channel, as well as the increased repeatability due to perfectly static flow in the microchannel and an additional anti-fouling step in the protocol. DNA sequence detection is achieved through a hybridization sandwich of an immobilized complementary probe, the target DNA sequence, and a second complementary probe labeled with ß-galactosidase (ß-GAL); the ß-GAL converts its substrate, 4-aminophenyl-d-galactopyranoside (PAPG), into PAP. In this report we present the lowest reported observed detection limit (1.0 × 10⁻¹° M) for an enzyme-linked DNA hybridization assay using an IDA microelectrode and a redox signaling paradigm. Thus, we have demonstrated highly sensitive detection of a targeted DNA sequence using a low-cost easily fabricated electrochemical biosensor integrated into a microfluidic channel.

Compact optical microfluidic uric acid analysis system.

We designed, fabricated and tested a novel compact fluorescence analysis system for quantification of uric acid (UA) in clinical samples at the point-of-care. To perform an analysis, diluted saliva, urine or blood samples are simply placed in a disposable thin-film sample holder using a dropper. A new enzyme immobilization technique was developed to retain within the sample holder two enzymes and a molecule, which transforms into a fluorescer in amounts depending on the UA concentration. The small instrument (7.5 cm × 5 cm × 5 cm) into which the sample holder is placed for analysis contains an LED, a narrow-band filter and an amplified photodiode. The analysis time is 30s, and the dynamic range of the system is 4-400 µM of UA. The calibration curve for transparent saliva and urine was made using solutions of UA. The calibration curve for opaque blood was obtained with spiked samples of blood. The three different types of clinical samples were collected from three subjects and simply diluted before their measurements. Analysis with our instrument yielded UA concentrations within the expected concentration ranges. Development of instruments based on the current laboratory prototype is expected to result in products for clinical trials and point-of-care.