Next generation microfluidics: fulfilling the promise of lab-on-a-chip technologies.

Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise.

Emerging Approaches for Fluorescence-Based Newborn Screening of Mucopolysaccharidoses.

Interest in newborn screening for mucopolysaccharidoses (MPS) is growing, due in part to ongoing efforts to develop new therapies for these disorders and new screening assays to identify increased risk for the individual MPSs on the basis of deficiency in the cognate enzyme. Existing tests for MPSs utilize either fluorescence or mass spectrometry detection methods to measure biomarkers of disease (e.g.

Digital Microfluidic Platform to Maximize Diagnostic Tests with Low Sample Volumes from Newborns and Pediatric Patients.

"Children are not tiny adults" is an adage commonly used in pediatrics to emphasize the fact that children often have different physiological responses to sickness and trauma compared to adults. However, despite widespread acceptance of this concept, diagnostic blood testing is an excellent example of clinical care that is not yet customized to the needs of children, especially newborns. Cumulative blood loss resulting from clinical testing does not typically impact critically ill adult patients, but can quickly escalate in children, leading to iatrogenic anemia and related comorbidities.

Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.

Background: Decreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens.

Methods: GALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone.

The dynamics and stability of lubricating oil films during droplet transport by electrowetting in microfluidic devices.

The operation of digital microfluidic devices with water droplets manipulated by electrowetting is critically dependent on the static and dynamic stability and lubrication properties of the oil films that separate the droplets from the solid surfaces. The factors determining the stability of the films and preventing surface fouling in such systems are not yet thoroughly understood and were experimentally investigated in this study. The experiments were performed using a standard digital microfluidic cartridge in which water droplets enclosed in a thin, oil-filled gap were transported over an array of electrodes.

Novel application of digital microfluidics for the detection of biotinidase deficiency in newborns.

Objective: Newborn screening for biotinidase deficiency can be performed using a fluorometric enzyme assay on dried blood spot specimens. As a pre-requisite to the consolidation of different enzymatic assays onto a single platform, we describe here a novel analytical method for detecting biotinidase deficiency using the same digital microfluidic cartridge that has already been demonstrated to screen for five lysosomal storage diseases (Pompe, Fabry, Gaucher, Hurler and Hunter) in a multiplex format.

Methods: A novel assay to quantify biotinidase concentration in dried blood spots (DBS) was developed and optimized on the digital microfluidic platform using proficiency testing samples from the Centers for Disease Control and Prevention.

Multiplex newborn screening for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases using a digital microfluidic platform.

Purpose: New therapies for lysosomal storage diseases (LSDs) have generated interest in screening newborns for these conditions. We present performance validation data on a digital microfluidic platform that performs multiplex enzymatic assays for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases.

Methods: We developed an investigational disposable digital microfluidic cartridge that uses a single dried blood spot (DBS) punch for performing a 5-plex fluorometric enzymatic assay on up to 44 DBS samples.

Rapid assays for Gaucher and Hurler diseases in dried blood spots using digital microfluidics.

Objective: Easy tool for newborn screening of Gaucher and Hurler diseases.

Methods: Method comparison between fluorometric enzymatic activity assay on a digital microfluidic platform and micro-titer plate bench assay was performed on normal (n = 100), Gaucher (n = 10) and Hurler (n = 7) dried blood spot samples.

Results: Enzymatic activity analysis of glucocerebrosidase (Gaucher) and α-l-iduronidase (Hurler) revealed similar discrimination between normal and affected samples on both platforms.

Novel microfluidic platform for automated lab-on-chip testing of hypercoagulability panel.

Current methods for hypercoagulability panel testing require large blood volumes and long turn-around testing times. A novel microfluidic platform has been designed to perform automated multiplexed hypercoagulability panel testing at near patient, utilizing only a single droplet of blood sample. We test the hypothesis that this novel platform could be utilized to perform specific multiplexed ELISA-based hypercoagulability panel testing for antithrombin III, protein C, protein S and factor VIII antigens, as well as anticardiolipin/human anti-β2-glycoprotein-1 IgG antibodies--on blood samples.

A novel fluorometric enzyme analysis method for Hunter syndrome using dried blood spots.

Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS). A convenient single-step fluorometric microplate enzyme assay has been developed and validated for clinical diagnosis of MPS II using dried blood spots (DBS). The assay compared well with a recently reported digital microfluidic method, from which it was adapted.

Droplet-based pyrosequencing using digital microfluidics.

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates.

Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns.

Background: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods.

Applications of electrowetting-based digital microfluidics in clinical diagnostics.

Digital microfluidics based on electrowetting is a type of microfluidic platform in which liquids are processed as individual unit-sized droplets that are dispensed from a source, merged together, split apart or transported between locations on demand. These devices are implemented using arrays of surface electrodes to control the shape and position of droplets through the electrowetting effect. A major thrust of digital microfluidics research has been the development of integrated lab-on-a-chip devices to perform clinical in vitro diagnostic assays.

Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens.

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens.

Multiplexed real-time polymerase chain reaction on a digital microfluidic platform.

This paper details the development of a digital microfluidic platform for multiplexed real-time polymerase chain reactions (PCR). Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting. Rapid PCR thermocycling is performed in a closed-loop flow-through format where for each cycle the reaction droplets are cyclically transported between different temperature zones within an oil-filled cartridge.

Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.

A digital microfluidic platform for performing heterogeneous sandwich immunoassays based on efficient handling of magnetic beads is presented in this paper. This approach is based on manipulation of discrete droplets of samples and reagents using electrowetting without the need for channels where the droplets are free to move laterally. Droplet-based manipulation of magnetic beads therefore does not suffer from clogging of channels.

An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids.

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip systems, especially in a point-of-care setting. Conventional microfluidic devices are usually based on continuous-flow in microchannels, and offer little flexibility in terms of reconfigurability and scalability. Handling of real physiological samples has also been a major challenge in these devices.

Electrowetting-based droplet mixers for microfluidic systems.

Mixing of analytes and reagents is a critical step in realizing a lab-on-a-chip. However, mixing of liquids is very difficult in continuous flow microfluidics due to laminar flow conditions. An alternative mixing strategy is presented based on the discretization of liquids into droplets and further manipulation of those droplets by electrowetting.

Rapid droplet mixers for digital microfluidic systems.

The mixing of analytes and reagents for a biological or chemical lab-on-a-chip is an important, yet difficult, microfluidic operation. As volumes approach the sub-nanoliter regime, the mixing of liquids is hindered by laminar flow conditions. An electrowetting-based linear-array droplet mixer has previously been reported.