Search

Many advanced microfluidic Lab-on-disc (LOD) devices require an on-board power supply for powering active components. LODs with an on-board electrical power supply are called electrified-LODs (eLODs) and are the subject of the present review. This survey comprises two main parts. First, we discuss the different means of delivering electrical energy to a spinning disc including slip-ring, wireless power transmission, and on-board power supply. In the second part, we focus on utilizing electrical power on eLODs for three electrokinetic microfluidic processes: electrophoresis, electroosmotic flow, and dielectrophoresis. Electrokinetic phenomena enable propulsion, separation, and manipulation of different fluids and various types of microparticles/cells. We summarize the theoretical and experimental results for all three electrokinetic phenomena enacted on centrifugal platforms. While extensive numerical modeling and experimental research are available for electrokinetics on stationary platforms, there is a noticeable lack of development in this area when executed on rotating platforms. The review concludes by comparing the strengths and weaknesses of different electrokinetic techniques implemented on centrifugal platforms, and additionally, the most promising applications of electrokinetic-assisted eLOD devices are singled out., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Upregulated expression of P-selectin on activated endothelium and platelets significantly contributes to the initiation and progression of vaso-occlusive crises (VOC), a major cause of morbidity in sickle cell disease (SCD). Crizanlizumab (ADAKVEO®), a humanized monoclonal antibody against P-selectin, primarily inhibits the interaction between leukocytes and P-selectin, and has been shown to decrease the frequency of VOCs in clinical trials. However, the lack of reliable in vitro assays that objectively measure leukocyte adhesion to P-selectin remains a critical barrier to evaluating and improving the therapeutic treatment in SCD. Here, we present a standardized microfluidic BioChip whole blood adhesion assay to assess leukocyte adhesion to P-selectin under physiologic flow conditions. Our results demonstrated heterogeneous adhesion by leukocytes to immobilized P-selectin, and dose-dependent inhibition of this adhesion following pre-exposure to Crizanlizumab. Importantly, treatment with Crizanlizumab following adhesion to P-selectin promoted detachment of rolling, but not of firmly adherent leukocytes. Taken together, our results suggest that the microfluidic BioChip system is a promising in vitro assay with which to screen patients, monitor treatment response, and guide current and emerging anti-adhesive therapies in SCD., Competing Interests: Declaration of competing interest YM, UG, EK, JAL, and UAG are inventors of intellectual property (PCT/US2018/022888 filed on March 16, 2018 and provisional patent application 62/928,109 filed on November 1, 2019) on the microfluidic BioChip technology presented in this manuscript. Competing interests of Case Western Reserve University employees are overseen and managed by the Conflict of Interests Committee according to a Conflict of Interest Management Plan., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices., Study Design and Methods: The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C., Results: The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average., Conclusions: The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method., (© 2020 AABB.)

Background: Immunosuppressant therapeutic drug monitoring (TDM) usually requires outpatient travel to hospitals or phlebotomy sites for venous blood collection; however Mitra® Microsampling Device (MSD) sampling could allow self-collection and shipping of samples to a laboratory for analysis. This study examined the feasibility of using volumetric microsampling by MSD for TDM of tacrolimus (TaC) and cyclosporin A (CsA) in transplant patients, along with their feedback on the process., Methods: MSD was used to collect TaC and CsA from venous (VB) or capillary (CB) blood. The MSDs were rehydrated, extracted, and analyzed using on-line solid phase extraction coupled to tandem mass spectrometry (SPE-MS/MS). We report an abbreviated method validation of the MSD including: accuracy, precision, linearity, carry-over, and stability using residual venous whole blood (VB) samples. Subsequent clinical validation compared serially collected MSD + CB against VB (200 µL) from transplant patients., Results: Accuracy comparing VB vs. MSD+VB showed high clinical concordance (TaC = 89% and CsA = 98%). Inter- and intra-precision was ≤11.5 %CV for TaC and CsA. Samples were stable for up to 7 days at room temperature with an average difference of <10%. Clinical validation with MSD+CB correlated well with VB for CsA (slope = 0.95, r2 = 0.88, n = 47) and TaC (slope = 0.98, r2 = 0.82, n = 49). CB vs. VB gave concordance of 94% for CsA and 79% for TaC. A satisfaction survey showed 82% of patients preferred having the capillary collection option., Conclusion: Transplant patients favored having the ability to collect capillary samples at home for TaC/CsA monitoring. Our results demonstrate good concordance between MSD+CB and VB for TaC and CsA TDM, but additional studies are warranted., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s-4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 10 5 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R 2 ≥ 0.994; ddLAMP: R 2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance.

Understanding the contribution of relevant factors to the analytical variability of the micro-flow imaging (MFI) technique is of prime importance because of the significance of the subvisible particulate data in biopharmaceutical product development. The current study was performed to determine the contribution of several key variables to the variability of the subvisible particle counts (e.g., day-to-day, vial-to-vial, sample-to-sample, and measurement-to-measurement variabilities) using a nested statistical analysis. The variability was measured in the <10 μm, ≥10 μm, ≥25 μm, and ≥50 μm size ranges along with the total particle count and the maximum and the mean particle size. The contribution of the vial to the variability of the subvisible particle counts was found to be greater than those of the other factors evaluated in the current study. The analytical method variability in terms of percent relative standard deviation with respect to the particle count in the <10 μm, ≥10 μm, and ≥25 μm size ranges was found to be 16%, 40%, and 44%, respectively. A thorough understanding of the contribution of key factors to the analytical variability revealed how the corresponding contribution can be minimized, that is, by increasing the number of vials, samples, and measurements. The results of the current study may be leveraged for the optimization of the analytical method or for minimization of the analytical variability with the MFI technique., (© PDA, Inc. 2020.)

The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. Polydimethylsiloxane (PDMS)-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.

VereBeef™ Detection Kit, incorporating both multiplex PCR and microarray technologies on a lab-on-chip platform, is intended for qualitative detection and differentiation of Escherichia coli O157:H7, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145, Shiga toxin-producing E. coli (STEC) virulence factors ( stx1A, stx2A, eae ), and Salmonella species in one test using raw beef trim samples. This product underwent extensive evaluations, including inclusivity-exclusivity, method comparison, robustness, lot-to-lot variability, and stability studies. The inclusivity/exclusivity study demonstrated that VereBeef Detection Kit specifically detects and identifies target analytes without occurrence of false-positive and false-negative detection. In the method comparison study, the performance of the VereBeef Detection Kit was compared with U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook's methods for target organism detection in raw beef trim using E. coli O157:H7 single inoculation and Salmonella and non-O157 STEC dual inoculation. Data demonstrated equivalence in both methods. The robustness study showed that changes in the test parameters do not impact assay performance. Collectively, VereBeef Detection Kit is able to detect target pathogens in raw beef trim with a minimum enrichment time of 8 h for E. coli O157:H7 detection and 10 h for Salmonella and non-O157 STEC detection.

The low specificity of the prostate-specific antigen (PSA) for early detection of prostate cancer (PCa) is a major issue worldwide. The aim of this study to examine whether the serum PCa-associated α2,3-linked sialyl N -glycan-carrying PSA (S2,3PSA) ratio measured by automated micro-total immunoassay systems (μTAS system) can be applied as a diagnostic marker of PCa. The μTAS system can utilize affinity-based separation involving noncovalent interaction between the immunocomplex of S2,3PSA and Maackia amurensis lectin to simultaneously determine concentrations of free PSA and S2,3PSA. To validate quantitative performance, both recombinant S2,3PSA and benign-associated α2,6-linked sialyl N -glycan-carrying PSA (S2,6PSA) purified from culture supernatant of PSA cDNA transiently-transfected Chinese hamster ovary (CHO)-K1 cells were used as standard protein. Between 2007 and 2016, fifty patients with biopsy-proven PCa were pair-matched for age and PSA levels, with the same number of benign prostatic hyperplasia (BPH) patients used to validate the diagnostic performance of serum S2,3PSA ratio. A recombinant S2,3PSA- and S2,6PSA-spiked sample was clearly discriminated by μTAS system. Limit of detection of S2,3PSA was 0.05 ng/mL and coefficient variation was less than 3.1%. The area under the curve (AUC) for detection of PCa for the S2,3PSA ratio (%S2,3PSA) with cutoff value 43.85% (AUC; 0.8340) was much superior to total PSA (AUC; 0.5062) using validation sample set. Although the present results are preliminary, the newly developed μTAS platform for measuring %S2,3PSA can achieve the required assay performance specifications for use in the practical and clinical setting and may improve the accuracy of PCa diagnosis. Additional validation studies are warranted.

A digital microfluidic biochip (DMFB) is an emerging technology that enables miniaturized analysis systems for point-of-care clinical diagnostics, DNA sequencing, and environmental monitoring. A DMFB reduces the rate of sample and reagent consumption, and automates the analysis of assays. In this paper, we provide the first assessment of the security vulnerabilities of DMFBs. We identify result-manipulation attacks on a DMFB that maliciously alter the assay outcomes. Two practical result-manipulation attacks are shown on a DMFB platform performing enzymatic glucose assay on serum. In the first attack, the attacker adjusts the concentration of the glucose sample and thereby modifies the final result. In the second attack, the attacker tampers with the calibration curve of the assay operation. We then identify denial-of-service attacks, where the attacker can disrupt the assay operation by tampering either with the droplet-routing algorithm or with the actuation sequence. We demonstrate these attacks using a digital microfluidic synthesis simulator. The results show that the attacks are easy to implement and hard to detect. Therefore, this work highlights the need for effective protections against malicious modifications in DMFBs.

A microfluidic platform or "microfluidic mapper" is demonstrated, which in a single experiment performs 36 parallel biochemical reactions with 36 different combinations of two reagents in stepwise concentration gradients. The volume used in each individual reaction was 36 nl. With the microfluidic mapper, we obtained a 3D enzyme reaction plot of horseradish peroxidase (HRP) with Amplex Red (AR) and hydrogen peroxide (H2O2), for concentration ranges of 11.7 μM to 100.0 μM and 11.1 μM to 66.7 μM for AR and H2O2, respectively. This system and methodology could be used as a fast analytical tool to evaluate various chemical and biochemical reactions especially where two or more reagents interact with each other. The generation of dual concentration gradients in the present format has many advantages such as parallelization of reactions in a nanoliter-scale volume and the real-time monitoring of processes leading to quick concentration gradients. The microfluidic mapper could be applied to various problems in analytical chemistry such as revealing of binding kinetics, and optimization of reaction kinetics.

Echogenic particles, such as microbubbles and volatile liquid micro/nano droplets, have shown considerable potential in a variety of clinical diagnostic and therapeutic applications. The accurate prediction of their response to ultrasound excitation is however extremely challenging, and this has hindered the optimisation of techniques such as quantitative ultrasound imaging and targeted drug delivery. Existing characterisation techniques, such as ultra-high speed microscopy provide important insights, but suffer from a number of limitations; most significantly difficulty in obtaining large data sets suitable for statistical analysis and the need to physically constrain the particles, thereby altering their dynamics. Here a microfluidic system is presented that overcomes these challenges to enable the measurement of single echogenic particle response to ultrasound excitation. A co-axial flow focusing device is used to direct a continuous stream of unconstrained particles through the combined focal region of an ultrasound transducer and a laser. Both the optical and acoustic scatter from individual particles are then simultaneously recorded. Calibration of the device and example results for different types of echogenic particle are presented, demonstrating a high throughput of up to 20 particles per second and the ability to resolve changes in particle radius down to 0.1 μm with an uncertainty of less than 3%.

The objective of the study has been the development of a new sensing platform, called Real-time Electrochemical Profiling (REP) that relies on real-time electrochemical immunoassay detection. The proposed REP platform consists of new electrode arrays that are easy to fabricate, has a small imprint allowing microfluidic system integration, enables multiplexed amperometric measurements and performs well in terms of electrochemical immunoassay detection as shown through the deoxynivalenol detection assays. The deoxynivalenol detection has been conducted according to an optimised REP assay protocol using deoxynivalenol standards at varying concentrations and a standard curve was obtained (y=-20.33ln(x)+124.06; R(2)=0.97) with a limit of detection of 6.25 ng/ml. As both ELISA and REP detection methods use horse radish peroxidase as the label and 3.3',5.5'-Tetramethylbenzidine as the substrate, the performance of the REP platform as an ELISA reader has also been investigated and a perfect correlation between the deoxynivalenol concentration and the current response was obtained (y=-14.56ln(x)+101.02; R(2)=0.99). The calibration curves of both assays have been compared to conventional ELISA tests for confirmation. After assay optimisation using toxin spiked buffer, the deoxynivalenol detection assay has also been performed to detect toxins in wheat grain., (Copyright © 2014 Elsevier B.V. All rights reserved.)

An extremely simple, self-standing microfluidic cell culture system is reported. The whole system is confined in a 35 mm culture dish, and requires only a standard CO2 incubator. The culture medium is perfused by gravity. We successfully cultured NIH3T3-derived cells up to 10 days with a viability of ∼90%., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.

Acoustofluidic devices for manipulating microparticles in fluids are appealing for biological sample processing due to their gentle and high-speed capability of sorting cell-scale objects. Such devices are generally limited to moving particles toward locations at integer fractions of the fluid channel width (1/2, 1/4, 1/6, etc.). In this work, we introduce a unique approach to acoustophoretic device design that overcomes this constraint, allowing us to design the particle focusing location anywhere within the microchannel. This is achieved by fabricating a second fluid channel in parallel with the sample channel, separated from it by a thin silicon wall. The fluids in both channels participate to create the ultrasound resonance, while only one channel processes the sample, thus de-coupling the fluidic and acoustic boundaries. The wall placement and the relative widths of the adjacent channels define the particle focusing location. We investigate the operating characteristics of a range of these devices to determine the configurations that enable effective particle focusing and separation. The results show that a sufficiently thin wall negligibly affects focusing efficiency and location compared to a single channel without a wall, validating the success of this design approach without compromising separation performance. Using these principles to design and fabricate an optimized device configuration, we demonstrate high-efficiency focusing of microspheres, as well as separation of cell-free viruses from mammalian cells. These "transparent wall" acoustic devices are capable of over 90% extraction efficiency with 10 μm microspheres at 450 μL min(-1), and of separating cells (98% purity), from viral particles (70% purity) at 100 μL min(-1).

Mesoporous silica nanoparticles are promising drug delivery agents; however, their interaction with various in vivo biological components is still under investigation. In this work, the impact of sub-50 nm diameter mesoporous silica nanoparticles on platelet function is investigated using a microfluidic platform to model blood vessel characteristics. Platelet adhesion and aggregation in the presence of mesoporous silica nanoparticles is investigated, controlling whether or not platelets are activated ahead of nanoparticle exposure. The results indicate that nanoparticles slightly compromise platelet adhesion to endothelial cells at low nanoparticle doses, but that high nanoparticle doses significantly increase the number of platelet adhesion events, leading to higher probability for uncontrolled platelet actions (e.g. clot formation in vivo). High nanoparticle doses also induced platelet aggregation. While platelet activation and aggregation occurred, in no case did nanoparticle exposure result in significant loss of platelet viability; as such, this work clearly demonstrates that aspects besides viability, such as cellular adhesion and interaction with other cell types, have to be considered in the context of nanotoxicology. This simple and highly adaptable analytical platform will be useful for further nanotoxicity studies involving other nanoparticle and cell types.

The human signal-molecule-profiling database (HSMPD) is designed as a prospective medical database for translational bioinformatics (TBI). To explore the feasibility of low-cost database construction, we studied the roadmap of HSMPD. A HSMPD-oriented tool, called "signal-molecule-profiling (SMP) chip" was developed for data acquisition, which can be employed in the routine blood tests in hospitals; the results will be stored in the HSMPD system automatically. HSMPD system can provide data services for the TBI community, which generates a stable income to support the data acquisition. The small-scale experimental test was performed in the hospital to verify SMP chips and the demo HSMPD software. One hundred and eighty nine complete SMP records were collected, and the demo HSMPD system was also evaluated in the survey study on patients and doctors. The function of SMP chip was verified, whereas the demo HSMPD software needed to be improved. The survey study showed that patients would only accept free tests of SMP chips when they originally needed blood examinations. The study indicated that the construction of HSMPD relies on the self-motivated cooperation of the TBI community and the traditional healthcare system. The proposed roadmap potentially provides an executable solution to build the HSMPD without high costs.

We report a high performance autonomous analytical system based on the vanadomolybdate method for the determination of soluble reactive phosphorus in seawater. The system combines a microfluidic chip manufactured from tinted poly (methyl methacrylate) (PMMA), a custom made syringe pump, embedded control electronics and on-board calibration standards. This "lab-on-a-chip" analytical system was successfully deployed and cross-compared with reference analytical methods in coastal (south west England) and open ocean waters (tropical North Atlantic). The results of the miniaturized system compared well with a reference bench-operated phosphate auto-analyser and showed no significant differences in the analytical results (student's t-test at 95% confidence level). The optical technology used, comprising of tinted PMMA and polished fluidic channels, has allowed an improvement of two orders of magnitude of the limit of detection (52 nM) compared to currently available portable systems based on this method. The system has a wide linear dynamic range 0.1-60 μM, and a good precision (13.6% at 0.4 μM, n=4). The analytical results were corrected for silicate interferences at 0.7 μM, and the measurement frequency was configurable with a sampling throughput of up to 20 samples per hour. This portable micro-analytical system has a low reagent requirement (340 μL per sample) and power consumption (756 J per sample), and has allowed accurate high resolution measurements of soluble reactive phosphorus in seawater., (© 2013 Elsevier B.V. All rights reserved.)

Lipidic mesophases are a class of highly ordered soft materials that form when certain lipids are mixed with water. Understanding the relationship between the composition and the microstructure of mesophases is necessary for fundamental studies of self-assembly in amphiphilic systems and for applications, such as the crystallization of membrane proteins. However, the laborious formulation protocol for highly viscous mesophases and the large amounts of material required for sample formulation are significant obstacles in such studies. Here we report a microfluidic platform that facilitates investigations of the phase behavior of mesophases by reducing sample consumption 300-fold, and automating and parallelizing sample formulation. The mesophases were formulated on-chip using less than 80 nL of material per sample and their microstructure was analyzed in situ using small-angle X-ray scattering (SAXS). The 220 μm-thick X-ray compatible platform was comprised of thin polydimethylsiloxane (PDMS) layers sandwiched between cyclic olefin copolymer (COC) sheets. Uniform mesophases were prepared using an active on-chip mixing strategy coupled with periodic cooling of the sample to reduce viscosity. We validated the platform by preparing and analyzing mesophases of the lipid monoolein (MO) mixed with aqueous solutions of different concentrations of β-octylglucoside (βOG), a detergent frequently used in membrane protein crystallization. Four samples were prepared in parallel on chip, by first metering and automatically diluting βOG to obtain detergent solutions of different concentration, then metering MO, and finally mixing by actuation of pneumatic valves. Integration of detergent dilution and subsequent mixing significantly reduced the number of manual steps needed for sample preparation. Three different types of mesophases typical for MO were successfully identified in SAXS data from on-chip samples. Microstructural parameters of identical samples formulated in different chips showed excellent agreement. Phase behavior of samples on-chip (~80 nL per sample) corresponded well with that of samples prepared via the traditional coupled-syringe method using at least two orders of magnitude more material ("off-chip", 35-40 μL per sample), further validating the applicability of the microfluidic platform for on-chip characterization of mesophase microstructure.

We have applied self-assembled DNA tetrahedral nanostructures for the precise and tunable control of the gain in an optofluidic fluorescence resonance energy transfer (FRET) laser. By adjusting the ratio of the donor and the acceptor attached to the tetrahedral vertices, 3.8 times reduction in the lasing threshold and 28-fold enhancement in the lasing efficiency were demonstrated. This work takes advantage of the self-recognition and self-assembly capabilities of biomolecules with well-defined structures and addressability, enabling nano-engineering of the laser down to the molecular level.

Measurement of cell oxygenation and oxygen consumption is useful for studies of cell bioenergetics, metabolism, mitochondrial function, drug toxicity and common pathophysiological conditions. Here we present a new platform for such applications which uses commercial multichannel biochips (μ-slides, Ibidi) and phosphorescent O2 sensitive probes. This platform was evaluated with both extracellular and intracellular O2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca(2+) and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the μ-slide platform provides facile O2 measurements with both suspension and adherent cells, higher sensitivity and reproducibility, and faster measurement time. It also allows re-perfusion and multiple treatments of cells and multi-parametric analyses in conjunction with other probes. Optical measurements are conducted on standard fluorescence readers and microscopes.

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z' factor, which incorporates both standard deviation and signal window. A Z' factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z' factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z' factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Here, we employ a model electrochemical DNA sensor to demonstrate that the detection limit and specificity of surface-based sensors often are not dependent on the true affinity of the probe for its target but are simply dependent on the effective probe concentration. Under these circumstances, the observed affinity (and thus the sensor's detection limit and specificity) will depend on the density with which the probes are packed on the surface of the sensor, the surface area, and even the volume of sample employed.

Standardized, affordable, user-friendly world-to-chip interfaces represent one of the major barriers to the adoption of microfluidics. We present a connector system for plug-and-play interfacing of microfluidic devices to multiple input and output lines. The male connectors are based on existing standardized housings from electronics that are inexpensive and widely available. The female connectors are fabricated using familiar replica molding techniques that can easily be adopted by microfluidic developers.

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.

Solid-state nanopores have emerged as versatile single-molecule sensors for applications including DNA sequencing, protein unfolding, micro-RNA detection, label-free detection of single nucleotide polymorphisms, and mapping of DNA-binding proteins involved in homologous recombination. While machining nanopores in dielectric membranes provides nanometer-scale precision, the rigid silicon support for the membrane contributes capacitive noise and limits integration with microfluidic networks for sample preprocessing. Herein, we demonstrate a technique to directly transfer solid-state nanopores machined in dielectric membranes from a silicon support into a microfluidic network. The resulting microfluidic-addressable nanopores can sense single DNA molecules at high bandwidths and with low noise, owing to significant reductions in membrane capacitance. This strategy will enable large-scale integration of solid-state nanopores with microfluidic upstream and downstream processing and permit new functions with nanopores such as complex manipulations for multidimensional analysis and parallel sensing in two and three-dimensional architectures.

In this work, a photoelectrochemical (PEC) method was introduced into a microfluidic paper-based analytical device (μ-PAD), and thus, a truly low-cost, simple, portable, and disposable microfluidic PEC origami device (μ-PECOD) with an internal chemiluminescence light source and external digital multimeter (DMM) was demonstrated. The PEC responses of this μ-PECOD were investigated, and the enhancements of photocurrents in μ-PECOD were observed under both external and internal light sources compared with that on a traditional flat electrode counterpart. As a further amplification of the generated photocurrents, an all-solid-state paper supercapacitor was constructed and integrated into the μ-PECOD to collect and store the generated photocurrents. The stored electrical energy could be released instantaneously through the DMM to obtain an amplified (∼13-fold) and DMM-detectable current as well as a higher sensitivity than the direct photocurrent measurement, allowing the expensive and sophisticated electrochemical workstation or lock-in amplifier to be abandoned. As a model, sandwich adenosine triphosphate (ATP)-binding aptamers were taken as molecular reorganization elements on this μ-PECOD for the sensitive determination of ATP in human serum samples in the linear range from 1.0 pM to 1.0 nM with a detection limit of 0.2 pM. The specificity, reproducibility, and stability of this μ-PECOD were also investigated.

An all-fibre based Raman-on-chip setup is introduced which enables analysis of solutions and trapped particles without microscopes or objectives. Beside the novel quartz microfluidic chip, innovative multi-core single-mode fibres with integrated fibre Bragg gratings are used for detection. The limit of quantitation is 7.5 mM for urea and 2.5 mM for nicotine with linear Raman spectroscopy. This is an improvement of more than two orders of magnitude compared with previous fibre-based microfluidic Raman detection schemes. Furthermore, our device was combined with optical traps to collect Raman-on-chip spectra of spherical polymer beads.

Nanotechnology provides the tools to develop novel biosensors with improved performance, including sensitivity and response time that can be readily integrated into diagnostic devices. We have developed a miniaturized cell analysis platform to advance microfluidic cell cultures by combining two complementary, label-free and non-invasive cell analysis methods for the long-term monitoring of dynamic cell behavior. The novel dual-parameter cell-on-a-chip detects light scattering from adherent cells to provide information on cell numbers and intracellular granularity, while simultaneously performing impedance spectroscopy to monitor cell adhesion and cell-cell interaction. In the present work we have integrated spray-coated organic photodiode arrays with a lab-on-a-chip containing embedded interdigitated electrode structures to improve assay reproducibility, reliability and accuracy. We successfully demonstrate that the complementary cell chip technology can accurately detect cell numbers, clarify misleading results during cell-substance interaction assays, as well as the cytotoxicity screening of drug substances. The ability to precisely determine cell numbers within minutes constitutes a major step towards standardization.

This study reports on dual on-column Fabry-Pérot (FP) cavity sensor-based gas chromatography (GC) of mixtures of volatile organic compounds (VOCs) utilizing an on-chip device, the so called "microfabricated passive preconcentrator/injector (μPPI)". Comprehensive analysis of the sampling, desorption/injection, and compound separation performance of the μPPI-based optofluidic GC system is described. Here, the combined use of the μPPI and on-column FP cavity sensors in a common GC platform enabled diffusion-based passive sampling, rapid (<7 min) chromatographic separation, and optical detection for the quaternary VOC mixtures of benzene, TCE, toluene, and m-xylene at sub-ppm concentrations with a simpler fluidic setup than conventional GC systems. The FP cavity sensor arrangement provided the means to study the dynamics of the thermal desorption/injection of VOCs by the μPPI and its effect on the GC separation resolution. Our analysis of obtained chromatograms revealed a presence of the competitive adsorptions of VOC mixtures onto the adsorption sites of trapping materials in the μPPI, which decreased the effective sampling rate by ~50% for compounds with high volatility. The validated performance of the optofluidic GC system promises future development of a field deployable GC microsystem incorporating the μPPI and the FP cavity sensors.

Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ∼2% to ∼23%, by (i) using a more efficient reverse transcriptase, (ii) introducing RNase H to break up the DNA:RNA hybrid, and (iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from four patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient's HIV RNA. We learned the following three lessons from this work: (i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful validation is essential before a method is considered to provide absolute quantification; (ii) the sensitivity of dLAMP to the sequence of the target nucleic acid necessitates additional validation with patient samples carrying the full spectrum of mutations; (iii) for multistep digital amplification chemistries, such as a combination of reverse transcription with amplification, microfluidic devices may be used to decouple these steps from one another and to perform them under different, individually optimized conditions for improved efficiency.

A common application for microfluidics can be found in medical devices where the advantages of small volume measuring equipment can be exploited for In Vitro Diagnostics. This chapter focuses on the US and the EU regulations, explaining the broad landscape and regulatory pathways of each market.

Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T)), the rate of platelet accumulation (V(PLT)), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT) and SC at all wall shear rates. A longer Lag(T) for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

Ribonucleic acid (RNA) is gaining utility as a key component of immunotherapeutics to transiently express antigens or to modulate endogenous gene expression for clinical applications. As a key ancillary component of clinical grade products, RNA requires a robust method for quality control. Here we evaluated the microfluidics based platform and slab electrophoresis for determination of integrity, concentration and size of four in vitro-transcribed RNA products with sizes of 1611, 808, 475 and 290 nucleotides (nts). Our data demonstrate that the Bioanalyzer can determine both size and integrity of the RNA, but the analysis suffers from a strong well position effect. For the RNAs tested, the integrity values obtained by the Bioanalyzer demonstrate a reverse correlation with the size of the molecule and are lower than those obtained using slab electrophoresis. Agarose gel electrophoresis produced the information on size of the RNA molecule with good precision, accuracy and reproducibility. We highlight observations which need to be taken into account when developing and qualifying a method of choice for assessment of in vitro-transcribed RNA using either approach., (Copyright © 2012. Published by Elsevier B.V.)

A novel concept for electroosmotic flow (EOF) control in a microfluidic chip is presented by using a self-assembled monolayer as the insulator of a flow field-effect transistor. Bidirectional EOF control with mobility values of 3.4 × 10(-4) and -3.1 × 10(-4) cm(2)/V s can be attained, corresponding to the applied gate voltage at -0.8 and 0.8 V, respectively, without the addition of buffer additives. A relatively high control factor (approximately 400 × 10(-6) cm(2)/V(2) s) can be obtained. The method presented in this study offers a simple strategy to control the EOF.

During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

Biofouling or adsorption of biomolecules onto surfaces in microfluidic devices limits the type of samples which can be handled. In this paper, we take advantage of the high adsorption capacity of graphene oxide (GO) for proteins as a strategy to limit biofouling, while preserving their activity for droplet-based lab-on-chip applications.

We develop a versatile, self-referenced composite Fabry-Pérot (FP) sensor and the corresponding detection scheme for rapid and precise measurement of vapors. The composite FP vapor sensor is formed by etching two juxtaposed micron-deep wells, with a precisely controlled offset in depth, on a silicon wafer. The wells are then coated with a vapor sensitive polymer and the reflected light from each well is detected by a CMOS imager. Due to its self-referenced nature, the composite FP sensor is able to extract the change in thickness and refractive index of the polymer layer upon exposure to analyte vapors, thus allowing for accurate vapor quantitation regardless of the polymer thickness, refractive index, and light incident angle and wavelength. Theoretical analysis is first performed to elucidate the underlying detection principle, followed by experimental demonstration at two different incident angles showing rapid and consistent measurement of the polymer changes when the polymer is exposed to three different analytes at various concentrations. The vapor detection limit is found to be on the order of a few pico-grams (~100 ppb).

The success of a shotgun proteomic experiment relies heavily on the performance and optimization of both the LC and the MS systems. Despite this, little consideration has, so far, been given to the importance of evaluating and optimizing the MS instrument settings during data-dependent acquisition mode. Moreover, during data-dependent acquisition, the users have to decide and choose among various MS parameters and settings, making a successful analysis even more challenging. We have systematically investigated and evaluated the effect of enabling and disabling the preview mode for FTMS scan, the number of microscans per MS/MS scan, the number of MS/MS events, the maximum ion injection time for MS/MS, and the automatic gain control target value for MS and MS/MS events on protein and peptide identification rates on an LTQ-Orbitrap using the Saccharomyces cerevisiae proteome. Our investigations aimed to assess the significance of each MS parameter to improve proteome analysis and coverage. We observed that higher identification rates were obtained at lower ion injection times i.e. 50-150 ms, by performing one microscan and 12-15 MS/MS events. In terms of ion population, optimal automatic gain control target values were at 5×10(5) -1×10(6) ions for MS and 3×10(3) -1×10(4) ions for MS/MS. The preview mode scan had a minimal effect on identification rates. Using optimized MS settings, we identified 1038 (±2.3%) protein groups with a minimum of two peptide identifications and an estimated false discovery rate of ∼1% at both peptide and protein level in a 160-min LC-MS/MS analysis., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many sensors have to be used simultaneously for multipoint carbon dioxide (CO(2)) observation. All the sensors should be calibrated in advance, but this is a time-consuming process. To seek a simplified calibration method, we used four commercial CO(2) sensor models and characterized their output tendencies against ambient temperature and length of use, in addition to offset characteristics. We used four samples of standard gas with different CO(2) concentrations (0, 407, 1,110, and 1,810 ppm). The outputs of K30 and AN100 models showed linear relationships with temperature and length of use. Calibration coefficients for sensor models were determined using the data from three individual sensors of the same model to minimize the relative RMS error. When the correction was applied to the sensors, the accuracy of measurements improved significantly in the case of the K30 and AN100 units. In particular, in the case of K30 the relative RMS error decreased from 24% to 4%. Hence, we have chosen K30 for developing a portable CO(2) measurement device (10 × 10 × 15 cm, 900 g). Data of CO(2) concentration, measurement time and location, temperature, humidity, and atmospheric pressure can be recorded onto a Secure Digital (SD) memory card. The CO(2) concentration in a high-school lecture room was monitored with this device. The CO(2) data, when corrected for simultaneously measured temperature, water vapor partial pressure, and atmospheric pressure, showed a good agreement with the data measured by a highly accurate CO(2) analyzer, LI-6262. This indicates that acceptable accuracy can be realized using the calibration method developed in this study.

The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.

This paper reports the feasibility of optical coherence tomography (OCT) technology for inspection of bonding quality of microfluidic devices in manufacturing environments. A compact optical-fiber-based OCT is developed and its measurement performance is characterized. A series of microfluidic devices respectively bonded by adhesive tape, thermal method, and oxygen plasma, are inspected. The defects of geometry deformation and sealing completeness are emphasized during measurements. Based on the inspection results, some discoveries related to the production of microfluidic devices are discussed.

The current need for high-throughput genotyping platforms for targeted validation of disease-associated single nucleotide polymorphisms (SNPs) motivated us to evaluate a novel nanofluidics platform for genotyping DNA extracted from peripheral blood and buccal wash samples. SNP genotyping was performed using a Fluidigm 48.48 Dynamic Array biochip on the BioMark polymerase chain reaction platform and results were compared against standard TaqMan assays and DNA sequencing. Pilot runs using these dynamic arrays on 90 samples against 20 SNP assays had an average call rate of 99.7%, with 100% call rates for 16 of the assays. Manual TaqMan genotyping of these samples against three SNPs demonstrated 100% correlation between the two platforms. To understand the influence of DNA template variability, three sources of blood samples (CH-1, n = 20; CH-2, n = 47; KK, n = 47) and buccal washes (n = 37) were genotyped for 24 SNPs. Although both CH-1 and CH-2 batches showed good base calling (≥98.8%), the KK batch and buccal wash samples exhibited lower call rates (82.1% and 94.0%). Importantly, repurification of the KK and buccal wash samples resulted in significant improvements in their call rates (to ≥97.9%). Scale-up for genotyping 1698 cases and controls for 24 SNPs had overall call rates of 97.6% for KK and 99.2% for CH samples. The Dynamic Array approach demonstrated accuracy similar to that of TaqMan genotyping, while offering significant savings in DNA, effort, time, and costs., (Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

In recent years, the interest in small-scale bio-reactors has increased dramatically. To ensure homogeneous conditions within the complete area of perfused microfluidic bio-reactors, we develop a general design of a continually feed bio-reactor with uniform perfusion flow. This is achieved by introducing a specific type of perfusion inlet to the reaction area. The geometry of these inlets are found using the methods of topology optimization and shape optimization. The results are compared with two different analytic models, from which a general parametric description of the design is obtained and tested numerically. Such a parametric description will generally be beneficial for the design of a broad range of microfluidic bioreactors used for, e.g., cell culturing and analysis and in feeding bio-arrays.

Detection and accurate quantitation of biomarkers such as alpha-fetoprotein (AFP) can be a key aspect of early stage cancer diagnosis. Microfluidic devices provide attractive analysis capabilities, including low sample and reagent consumption, as well as short assay times. However, to date microfluidic analyzers have relied almost exclusively on calibration curves for sample quantitation, which can be problematic for complex mixtures such as human serum. We have fabricated integrated polymer microfluidic systems that can quantitatively determine fluorescently labeled AFP in human serum using either the method of standard addition or a calibration curve. Our microdevices couple an immunoaffinity purification step with rapid microchip electrophoresis separation in a laser-induced fluorescence detection system, all under automated voltage control in a miniaturized polymer microchip. In conjunction with laser-induced fluorescence detection, these systems can quantify AFP at approximately 1 ng/mL levels in approximately 10 microL of human serum in a few tens of minutes. Our polymer microdevices have been applied in determining AFP in spiked serum samples. These integrated microsystems offer excellent potential for rapid, simple, and accurate biomarker quantitation in a point-of-care setting.