A 365 nm UV LED-excitable antenna ligand for switchable lanthanide luminescence.

The time-resolved luminescence of lanthanide complexes is a highly sensitive and widely used bioassay technology for clinical diagnostics. With the time-resolved luminescence detection the naturally occurring autofluorescence of biological matrices, solid supports and plastics can be avoided. A major drawback of the current technique is that the luminescent lanthanide labels require ultraviolet (UV) excitation, typically shorter than 360 nm, which is strongly absorbed and can damage living biological systems. The lack of cost-efficient high power solid state excitation light sources for UV excitation further limits the development of low-cost and more compact measurement instruments for time-resolved luminescence and the potential use of lanthanide luminescence in different applications. Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in a separation-free detection of targets. The intrinsically luminescent lanthanide chelate is split into two nonluminescent moieties, a lanthanide ion carrier chelate and a light harvesting antenna ligand, each of which can be attached to a separate molecular probe. A luminescent lanthanide complex is formed only when the two probes bind adjacently to the target molecule. Herein we describe a new 365 nm excitable antenna ligand (AL360) for switchable lanthanide luminescence of europium(iii) (EuIII) that would enable the use of 365 nm light emitting diodes (LEDs) as an excitation light source for time-resolved fluorescence imaging and detection. With the acquired subpicomolar assay sensitivity it would be applicable for solution or surface arrays and UV LED microscopy.

Array-in-well serodiagnostic assay utilizing upconverting phosphor label technology.

In this study, a multiplex serological array-in-well assay was constructed for simultaneous detection of serum IgG antibodies against parvovirus B19 and human adenovirus. The array was prepared in streptavidin-coated 96-well microtiter plates by spotting biotinylated parvovirus B19 virus-like-particles, adenovirus type 2 and 5 hexon antigens, negative control of human serum albumin and positive controls of human IgG and anti-human IgG antibodies on the bottom of each well in an array format with a printable area of 2 mm × 2 mm. The array-in-well assay was evaluated with serum samples (n=89) of different antibody status as determined by commercial enzyme immunoassay for parvovirus IgG, and by in-house enzyme immunoassay for adenovirus IgG. The bound serum anti-parvovirus IgG, anti-adenovirus IgG, and total IgG antibodies were detected with anti-human IgG antibody coated photon upconverting nanoparticles and the assay was measured with an anti-Stokes photoluminescence imager. Detection of specific antibodies by the multiplex array-in-well assay was in good agreement (100% for parvovirus B19 and 96% for adenovirus) with the reference results. In conclusion, the array-in-well with upconverting phosphor reporter technology was able to detect antiviral antibodies in human sera, and represents an efficient serodiagnostic concept that is a promising new tool for multiplex serology.

An integrated closed-tube 2-plex PCR amplification and hybridization assay with switchable lanthanide luminescence based spatial detection.

Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in separation-free detection of DNA targets. A luminescent lanthanide complex is formed only when the two probes hybridize adjacently to their target DNA. We have now further adapted this technology for the first time in the integration of a 2-plex polymerase chain reaction (PCR) amplification and hybridization-based solid-phase detection of the amplification products of the Staphylococcus aureus gyrB gene and an internal amplification control (IAC). The assay was performed in a sealed polypropylene PCR chip containing a flat-bottom reaction chamber with two immobilized capture probe spots. The surface of the reaction chamber was functionalized with NHS-PEG-azide and alkyne-modified capture probes for each amplicon, labeled with a light harvesting antenna ligand, and covalently attached as spots to the azide-modified reaction chamber using a copper(i)-catalyzed azide-alkyne cycloaddition. Asymmetric duplex-PCR was then performed with no template, one template or both templates present and with a europium ion carrier chelate labeled probe for each amplicon in the reaction. After amplification europium fluorescence was measured by scanning the reaction chamber as a 10 × 10 raster with 0.6 mm resolution in time-resolved mode. With this assay we were able to co-amplify and detect the amplification products of the gyrB target from 100, 1000 and 10,000 copies of isolated S. aureus DNA together with the amplification products from the initial 5000 copies of the synthetic IAC template in the same sealed reaction chamber. The addition of 10,000 copies of isolated non-target Escherichia coli DNA in the same reaction with 5000 copies of the synthetic IAC template did not interfere with the amplification or detection of the IAC. The dynamic range of the assay for the synthetic S. aureus gyrB target was three orders of magnitude and the limit of detection of 8 pM was obtained. This proof-of-concept study shows that the switchable lanthanide luminescent probes enable separation-free array-based multiplexed detection of the amplification products in a closed-tube PCR which can enable a higher degree of multiplexing than is currently feasible by using different spectrally separated fluorescent probes.

Quantitative detection of well-based DNA array using switchable lanthanide luminescence.

In this report a novel wash-free method for multiplexed DNA detection is demonstrated employing target specific probe pairs and switchable lanthanide luminescence technology on a solid-phase array. Four oligonucleotide capture probes, conjugated at 3' to non-luminescent lanthanide ion carrier chelate, were immobilized as a small array on the bottom of a microtiter plate well onto which a mix of corresponding detection probes, conjugated at 5' to a light absorbing antenna ligand, were added. In the presence of complementary target nucleic acid both the spotted capture probe and the liquid-phase detection probe hybridize adjacently on the target. Consequently the two non-luminescent label molecules self-assemble and form a luminescent mixed lanthanide chelate complex. Lanthanide luminescence is thereafter measured without a wash step from the spots by scanning in time-resolved mode. The homogeneous solid-phase array-based method resulted in quantitative detection of synthetic target oligonucleotides with 0.32 nM and 0.60 nM detection limits in a single target and multiplexed assay, respectively, corresponding to 3× SD of the background. Also qualitative detection of PCR-amplified target from Escherichia coli is described.

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle.

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.