Correction: Virtual microwells for digital microfluidic reagent dispensing and cell culture.

Correction for 'Virtual microwells for digital microfluidic reagent dispensing and cell culture' by Irwin A. Eydelnant et al., Lab Chip, 2012, 12, 750-757, https://doi.org/10.1039/C2LC21004E.

Canadian Society of Clinical Chemists (CSCC) consensus guidance for testing, selection and quality management of SARS-CoV-2 point-of-care tests.

OBJECTIVES: A consensus guidance is provided for testing, utility and verification of SARS-CoV-2 point-of-care test (POCT) performance and implementation of a quality management program, focusing on nucleic acid and antigen targeted technologies. DESIGN AND METHODS: The recommendations are based on current literature and expert opinion from the members of Canadian Society of Clinical Chemists (CSCC), and are intended for use inside or outside of healthcare settings that have varied levels of expertise and experience with POCT. RESULTS AND CONCLUSIONS: Here we discuss sampling requirements, biosafety, SARS-CoV-2 point-of-care testing methodologies (with focus on Health Canada approved tests), test performance and limitations, test selection, testing utility, development and implementation of quality management systems, quality improvement, and medical and scientific oversight.

The Pharmacokinetics and Pharmacodynamics of Carfentanil After Recreational Exposure: A Case Report.

Carfentanil and related fentanyl analogs have been linked to a number of overdose deaths from drug users in several cities across North America. Diagnosis of carfentanil exposure requires a very high index of clinical suspicion, especially because available laboratory narcotic screens do not detect this agent. We describe a 34-year-old man admitted with depressed level of consciousness and in respiratory failure after recreational exposure to a white powder later inferred to contain carfentanil. Urine and whole blood samples were obtained for conventional preliminary drug screen immunoassays for unknown exposures, in addition to utilizing a high-pressure liquid chromatography-tandem mass spectrometry assay for quantification of carfentanil and its metabolite. The patient was intubated and required mechanically assisted ventilation for 31 hours until he was able to breathe safely on his own. Pharmacokinetic modeling of three timed blood samples identified the elimination half-life as 5.7 hours for carfentanil and 11.8 hours for the norcarfentanil metabolite. Awakening and breathing spontaneously corresponded to an interpolated blood carfentanil concentration of 0.52 ng/ml. This is the first pharmacokinetic and pharmacodynamic case report on the recreational use of carfentanil. Critical care clinicians should anticipate long periods of ventilatory support in the care of patients exposed to carfentanil.

Inorganic Nanoparticles as Donors in Resonance Energy Transfer for Solid-Phase Bioassays and Biosensors.

Bioassays for the rapid detection and quantification of specific nucleic acids, proteins, and peptides are fundamental tools in many clinical settings. Traditional optical emission methods have focused on the use of molecular dyes as labels to track selective binding interactions and as probes that are sensitive to environmental changes. Such dyes can offer good detection limits based on brightness but typically have broad emission bands and suffer from time-dependent photobleaching. Inorganic nanoparticles such as quantum dots and upconversion nanoparticles are photostable over prolonged exposure to excitation radiation and tend to offer narrow emission bands, providing a greater opportunity for multiwavelength multiplexing. Importantly, in contrast to molecular dyes, nanoparticles offer substantial surface area and can serve as platforms to carry a large number of conjugated molecules. The surface chemistry of inorganic nanoparticles offers both challenges and opportunities for the control of solubility and functionality for selective molecular interactions by the assembly of coatings through coordination chemistry. This report reviews advances in the compositional design and methods of conjugation of inorganic quantum dots and upconversion nanoparticles and the assembly of combinations of nanoparticles to achieve energy exchange. Our interest is the exploration of configurations where the modified nanoparticles can be immobilized to solid substrates for the development of bioassays and biosensors that operate by resonance energy transfer (RET).

Fast, Sensitive, and Quantitative Point-of-Care Platform for the Assessment of Drugs of Abuse in Urine, Serum, and Whole Blood.

Drug abuse is a major public health problem in many countries in Europe and North America. Currently available platforms for drug abuse assessment are facing technical challenges of nonquantitation, inaccuracy, low throughput, incompatibility with diverse complex specimens, long assay times, and requirement of instrument and/or expertise for readout. Here, we report an integrated competitive volumetric-bar-chart chip (CV-Chip) to assay multiple drug targets at the point-of-care (POC). To the best of our knowledge, it is the first time that a POC platform has been demonstrated to fully address the above-mentioned limitations. We applied this integrated CV-chip platform to assay multiple drugs in 38 patient urine and serum samples and validated the on-chip results with an LC-MS/MS method, indicating a clinical sensitivity and specificity of 0.94 and 1.00, respectively. We further demonstrated that the combination of an on-chip blood separator with the CV-Chip enabled the platform to directly assay finger-prick whole blood samples, which have always been recognized as an ideal biospecimen for POC detections. In summary, this integrated CV-Chip is able to serve as a sensitive, accurate, fast, portable, readout visible, and minimally invasive platform for drug abuse assessment.

A paper-based multiplexed resonance energy transfer nucleic acid hybridization assay using a single form of upconversion nanoparticle as donor and three quantum dots as acceptors.

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reductive amination to evaluate the multiplexing capacity of luminescence resonance energy transfer (LRET) between UCNPs and quantum dots (QDs). This is the first account of a multiplexed bioassay strategy that demonstrates the principle of use of a single form of UCNP as donor and three different color emitting QDs as acceptors to concurrently determine three analytes. Broad absorbance profiles of green, orange and red QDs that spanned from the first exciton absorption peak to the UV region were in overlap with a blue emission band from UCNPs composed of NaYF4 that was doped with 30% Yb3+, 0.5% Tm3+, allowing for LRET that was stimulated using 980 nm near-infrared radiation. The characteristic narrow and well-defined emission peaks of UCNPs and QDs allowed for the collection of luminescence from each nanoparticle using a band-pass optical filter and an epi-fluorescence microscope. The LRET system was used for the concurrent detection of uidA, Stx1A and tetA gene fragments with selectivity even in serum samples, and reached limits of detection of 26 fmol, 56 fmol and 76 fmol, respectively.

Energy Transfer Assays Using Quantum Dot-Gold Nanoparticle Complexes: Optimizing Oligonucleotide Assay Configuration Using Monovalently Conjugated Quantum Dots.

The energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) represents a popular transduction scheme in analytical assays that use nanomaterials. The impact of the spatial arrangement of the two types of nanoparticles on analytical performance has now been evaluated using a nucleic acid strand displacement assay. The first spatial arrangement (configuration 1) involved the assembly of a number of monovalently functionalized QD-oligonucleotide conjugates around a single central AuNP that was functionalized with complementary oligonucleotide sequences. The assembly of these complexes, and subsequent disassembly via target oligonucleotide-mediated displacement, were used to evaluate energy transfer efficiencies. Furthermore, the inner filter effect of AuNPs on the fluorescence intensity of the QD was studied. AuNPs of three different diameters (6, 13, and 30 nm) were used in these studies. Configuration 2 was based on the placement of monovalently functionalized AuNP-oligonucleotide conjugates around a single QD that was functionalized with a complementary oligonucleotide. The optimal assay configuration, established by evaluating energy transfer efficiencies and inner filter effects, was obtained by arranging at most 15 QDs around the 13 nm AuNP (configuration 1). These assays provided a 2.5-fold change in fluorescence intensity in the presence of target oligonucleotides. To obtain the same response with configuration 2 required the placement of three 6 nm AuNPs around the QD. This resulted in configuration 2 having a 5-fold lower fluorescence intensity when compared to configuration 1. The use of low-cost detection systems (digital camera) further emphasized the higher analytical performance of configuration 1. Response curves obtained using these detection systems demonstrated that configuration 1 had a 10-fold higher sensitivity when compared to configuration 2. This study provides an important framework for the development of sensitive assays using gold nanoparticles and quantum dots.

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.

Evaluation of nanoparticle-ligand distributions to determine nanoparticle concentration.

The concentration of nanoparticles in solution is an important, yet challenging, parameter to quantify. In this work, a facile strategy for the determination of nanoparticle concentration is presented. The method relies on the quantitative analysis of the inherent distribution of nanoparticle-ligand conjugates that are generated when nanoparticles are functionalized with ligands. Validation of the method was accomplished by applying it to gold nanoparticles and semiconductor nanoparticles (CdSe/ZnS; core/shell). Poly(ethylene glycol) based ligands, with functional groups that quantitatively react with the nanoparticles, were incubated with the nanoparticles at varying equivalences. Agarose gel electrophoresis was subsequently used to separate and quantify the nanoparticle-ligand conjugates of varying valences. The distribution in the nanoparticle-ligand conjugates agreed well with that predicted by the Poisson model. A protocol was then developed, where a series of only eight different ligand amounts could provide an estimate of the nanoparticle concentration that spans 3 orders of magnitude (1 µM to 1 mM). For the gold nanoparticles and semiconductor nanoparticles, the measured concentrations were found to deviate by only 7% and 2%, respectively, from those determined by UV-vis spectroscopy. The precision of the assay was evaluated, resulting in a coefficient of variation of 5-7%. Finally, the protocol was used to determine the extinction coefficient of alloyed semiconductor nanoparticles (CdSxSe1-x/ZnS), for which a reliable estimate is currently unavailable, of three different emission wavelengths (525, 575, and 630 nm). The extinction coefficient of the nanoparticles of all emission wavelengths was similar and was found to be 2.1 × 10(5) M(-1)cm(-1).

Solid-phase supports for the in situ assembly of quantum dot-FRET hybridization assays in channel microfluidics.

Semiconductor quantum dots (QDs) have long served as integral components in signal transduction modalities such as Förster resonance energy transfer (FRET). The majority of bioanalytical methods using QDs for FRET-based techniques simply monitor binding-induced conformational changes. In more recent work, QDs have been incorporated into solid-phase support systems, such as microfluidic chips, to serve as physical platforms in the development of functional biosensors and bioprobes. Herein, we describe a simple strategy for the transduction of nucleic acid hybridization that combines a novel design method based on FRET with an electrokinetically controlled microfluidic technology, and that offers further potential for amelioration of sample-handling issues and for simplification of dynamic stringency control.

Solid-phase covalent immobilization of upconverting nanoparticles for biosensing by luminescence resonance energy transfer.

Monodisperse water-soluble upconverting nanoparticles (UCNPs) were immobilized onto modified glass substrates for development of biosensing surfaces that operated using luminescence resonance energy transfer (LRET). Amine modified UCNPs were prepared from oleic acid capped UCNPs by ligand exchange using o-phosphorylethanolamine (PEA). PEA-UCNPs were covalently immobilized on aldehyde functionalized coverslips. Environmental scanning electron microscopy (ESEM) images indicated a homogeneous distribution of UCNPs on surfaces with a high immobilization density of approximately 1.3 × 10(11) UCNP cm(-2). This is the first account of covalent immobilization of UCNPs for bioassay and biosensor development where the density is on par with the high immobilization densities reported for other types of nanoparticles. The functionality and stability of the immobilized NPs were demonstrated by examining an LRET-based bioassay. The well-known sandwich assay for the detection of thrombin was selected as a model in which UCNPs were used as donors and quantum dots (QDs) as acceptors. The closely packed UCNPs on the glass surface showed a 2.5-fold enhancement in assay sensitivity compared to less-densely packed surfaces. In addition, a 1.5-fold enhancement in energy transfer efficiency was shown for solid-phase compared to solution-phase LRET.

Isolation of monovalent quantum dot-nucleic acid conjugates using magnetic beads.

Control of the valency that is achieved in the decoration of quantum dots (QDs) remains a challenge due to the high surface area of nanoparticles. A population distribution of conjugates is formed even when reactions involve use of one-to-one molar equivalents of the ligand and QD. Monovalent conjugates are of particular interest to enable the preparation of multinanoparticle constructs that afford improved analytical functionality. Herein, a facile method for the formation and purification of QD-DNA monoconjugates (i.e., 1 DNA per QD) is described. Using diethylaminoethyl (DEAE) functionalized magnetic beads, a protocol was developed and optimized to selectively isolate QD-DNA monoconjugates from a mixture. Monoconjugates prepared with oligonucleotides as short as 19 bases and as long as 36 bases were successfully isolated. The monoconjugates were isolated in less than 5 min with isolation efficiencies between 68% and 93%, depending on the length of oligonucleotide that was used. The versatility of the method was demonstrated by purifying monoconjugates prepared from commercially available, water-soluble QDs. The isolation of monoconjugates was confirmed using agarose gel electrophoresis and single molecule fluorescence spectroscopy. Examples are provided comparing the analytical performance of monoconjugates to collections of nanoparticles of mixed valencies, indicating the significance of this separation method to prepare nanomaterials for bioassay design.

Paper-based DNA detection using lanthanide-doped LiYF4 upconversion nanocrystals as bioprobe.

A novel sensitive DNA bioassay using lanthanide-doped LiYF4 upconversion nanocrystals as luminescent marker and oligonucleotide hybridization as the selective reaction is developed in a paper-based platform, providing a detection limit of 3.6 fmol.

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 (5.7×10(11)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.

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.

Biosensing with quantum dots: a microfluidic approach.

Semiconductor quantum dots (QDs) have served as the basis for signal development in a variety of biosensing technologies and in applications using bioprobes. The use of QDs as physical platforms to develop biosensors and bioprobes has attracted considerable interest. This is largely due to the unique optical properties of QDs that make them excellent choices as donors in fluorescence resonance energy transfer (FRET) and well suited for optical multiplexing. The large majority of QD-based bioprobe and biosensing technologies that have been described operate in bulk solution environments, where selective binding events at the surface of QDs are often associated with relatively long periods to reach a steady-state signal. An alternative approach to the design of biosensor architectures may be provided by a microfluidic system (MFS). A MFS is able to integrate chemical and biological processes into a single platform and allows for manipulation of flow conditions to achieve, by sample transport and mixing, reaction rates that are not entirely diffusion controlled. Integrating assays in a MFS provides numerous additional advantages, which include the use of very small amounts of reagents and samples, possible sample processing before detection, ultra-high sensitivity, high throughput, short analysis time, and in situ monitoring. Herein, a comprehensive review is provided that addresses the key concepts and applications of QD-based microfluidic biosensors with an added emphasis on how this combination of technologies provides for innovations in bioassay designs. Examples from the literature are used to highlight the many advantages of biosensing in a MFS and illustrate the versatility that such a platform offers in the design strategy.

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.

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.