A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.     

Virtual microwells for digital microfluidic reagent dispensing and cell culture

Digital microfluidic (DMF) liquid handling includes active (electrostatic) and passive (surface tension) mechanisms for reagent dispensing. Here we implement a simple and straightforward Teflon-AF liftoff protocol for patterning hydrophilic sites on a two-plate device for precise passive dispensing of reagents forming virtual microwells--an analogy to the wells found on a microtitre plate. We demonstrate here that devices formed using these methods are capable of reproducible dispensing of volumes ranging from ~80 to ~800 nL, with CVs of 0.7% to 13.8% CV. We demonstrate that passive dispensing is compatible with DMF operation in both air and oil, and provides for improved control of dispensed nano- and micro- litre volumes when compared to active electrostatic dispensing. Further, the technique is advantageous for cell culture and we report the first example of reagent dispensing on a single-plate DMF device. We anticipate this method will be useful for a wide range of applications--particularly those involving adherent cell culture and analysis.     

Immunoassays in microfluidic systems

Immunoassays have greatly benefited from miniaturization in microfluidic systems. This review, which summarizes developments in microfluidics-based immunoassays since 2000, includes four sections, focusing on the configurations of immunoassays that have been implemented in microfluidics, the main fluid handling modalities that have been used for microfluidic immunoassays, multiplexed immunoassays in microfluidic platforms, and the emergence of label-free detection techniques. The field of microfluidic immunoassays is continuously improving and has great promise for the future.     

Analytical performance of molecular beacons on surface immobilized gold nanoparticles of varying size and density

The high quenching efficiency of metal nanoparticles has facilitated its use as quenchers in molecular beacons. To optimize this system, a good understanding of the many factors that influence molecular beacon performance is required. In this study, molecular beacon performance was evaluated as a function of gold nanoparticle size and its immobilization characteristics. Gold nanoparticles of 4 nm, 15 nm and 87 nm diameter, were immobilized onto glass slides. Each size regime offered distinctive optical properties for fluorescence quenching of molecular dyes that were conjugated to oligonucleotides that were immobilized to the gold nanoparticles. Rigid double stranded DNA was used as a model to place fluorophores at different distances from the gold nanoparticles. The effect of particle size and also the immobilization density of nanoparticles was evaluated. The 4 nm and 87 nm gold nanoparticles offered the highest sensitivity in terms of the change in fluorescence intensity as a function of distance (3-fold improvement for Cy5). The optical properties of the molecular fluorophore was of significance, with Cy5 offering higher contrast ratios than Cy3 due to the red-shifted emission spectrum relative to the plasmon peak. A high density of gold nanoparticles reduced contrast ratios, indicating preference for a monolayer of immobilized nanoparticles when considering analytical performance. Molecular beacon probes were then used in place of the double stranded oligonucleotides. There was a strong dependence of molecular beacon performance on the length of a linker used for attachment to the nanoparticle surface. The optimal optical performance was obtained with 4 nm gold nanoparticles that were immobilized as monolayers of low density (5.7 × 10¹¹ particles cm⁻²) on glass surfaces. These nanoparticle surfaces offered a 2-fold improvement in analytical performance of the molecular beacons when compared to other nanoparticle sizes investigated. The principles developed in this study would assist in the design of solid phase molecular beacons using gold nanoparticles.     

A digital microfluidic approach to heterogeneous immunoassays

A digital microfluidic (DMF) device was applied to a heterogeneous sandwich immunoassay. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in microchannels. Since droplets are manipulated on relatively generic 2-D arrays of electrodes, DMF devices are straightforward to use, and are reconfigurable for any desired combination of droplet operations. This flexibility makes them suitable for a wide range of applications, especially those requiring long, multistep protocols such as immunoassays. Here, we developed an immunoassay on a DMF device using Human IgG as a model analyte. To capture the analyte, an anti-IgG antibody was physisorbed on the hydrophobic surface of a DMF device, and DMF actuation was used for all washing and incubation steps. The bound analyte was detected using FITC-labeled anti-IgG, and fluorescence after the final wash was measured in a fluorescence plate reader. A non-ionic polymer surfactant, Pluronic F-127, was added to sample and detection antibody solutions to control non-specific binding and aid in movement via DMF. Sample and reagent volumes were reduced by nearly three orders of magnitude relative to conventional multiwell plate methods. Since droplets are in constant motion, the antibody–antigen binding kinetics is not limited by diffusion, and total analysis times were reduced to less than 2.5 h per assay. A multiplexed device comprising several DMF platforms wired in series further increased the throughput of the technique. A dynamic range of approximately one order of magnitude was achieved, with reproducibility similar to the assay when performed in a 96-well plate. In bovine serum samples spiked with human IgG, the target molecule was successfully detected in the presence of a 100-fold excess of bovine IgG. It was concluded that the digital microfluidic format is capable of carrying out qualitative and quantitative sandwich immunoassays with a dramatic reduction in reagent usage and analysis time compared to macroscale methods.