Spacer length, label moiety interchange and probe pair orientation in a homogeneous solid-phase hybridization assay utilizing lanthanide chelate complementation

Lahdenperä, Susanne; Manninen, Julius; Joki, Laura; Karhunen, Ulla; Soukka, Tero

doi:10.1039/c4ay00466c

Cited by 2 publications

(5 citation statements)

References 13 publications

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“…Probe oligonucleotides (Table 1) for gyrB and IAC 61 Thermal bond laminate foil (4titude, http://www.4ti.co.uk, Surrey, UK) was used for chip sealing. Polypropylene chips were designed for liquid phase PCR applications, but offered a suitable heat-resistant platform to be utilized also with immobilized probes and spatial detection on the surface.…”

Section: Methodsmentioning

confidence: 99%

“…2.3.3 Hybridization assay. The switchable lanthanide luminescence based solid-phase hybridization assay 61 was implemented into a new closed-tube assay platform with covalently immobilized antenna probes and surface detection through the plastic bottom in a closed-tube polypropylene PCR chip with aluminium foil backing. To compare the new assay platform with the conventional microtiter well based assay all parameters other than the antenna probe immobilization chemistry and assay platform were kept as similar as possible.…”

Section: Instrumentationmentioning

confidence: 99%

“…The LOD was calculated from the mean of the blank samples (0 nM target, n = 4) plus 3 standard deviations and compared with the LOD of the microtiter well based hybridization assay. 61 2.3.4 Integrated amplification and detection. Each asymmetric amplification and hybridization reaction (30 μL) was conducted in a sealed reaction chamber on the PCR chip containing gyrB and IAC antenna probe spots (1.67 μM spotting concentration), 50 nM gyrB and 50 nM IAC Eu-carrier probes, optimized concentrations (results not shown) of primers (0.2 μM gyrB forward/2 μM gyrB reverse, 1 μM IAC forward/ 0.1 μM IAC reverse) and 1 × 10 2 , 1 × 10 3 or 1 × 10 4 copies of S. aureus genome together with 5000 copies of the synthetic IAC template diluted in the amplification and hybridization buffer containing 0.4 mM dNTP (LAROVA GmbH http://www.larova.…”

Section: Instrumentationmentioning

confidence: 99%

“…2) and the dynamic range was three orders of magnitude which equaled well the LOD (18 pM) of the previously developed solid-phase hybridization assay in the conventional microtiter well format utilizing biotin-streptavidin antenna probe immobilization chemistry. 61 With high target concentrations (>10 nM) the spot signal started to satu-rate and the concentration of the target approached the concentration of the Eu-carrier probe.…”

Section: Hybridization Assaymentioning

confidence: 99%

“…55 Light harvesting antenna ligand labeled alkyne-modified capture probes (antenna probes) were covalently attached as spots to the reaction chamber of the azide-functionalized PCR chip using fast and site-specific copper(I)-catalyzed azide-alkyne cyclo-addition (CuAAC), the so called click-chemistry. [56][57][58][59][60] To improve the hybridization efficiency of the targets into the surface bound capture probes the target specific hybridization sequence of the capture probe was separated from the solid support with a 25 thymidine spacer 61 and the polyethylene glycol linker (NHS-PEG 4 -azide) that was used in the azide-functionalization of the surface. Covalent attachment of the capture probes and the high thermal stability of the polypropylene substrate enabled the usage of high temperatures required in PCR.…”

Section: Introductionmentioning

confidence: 99%

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

Lahdenperä

Spangar

Lempainen

et al. 2015

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Instrumentationmentioning

confidence: 99%

Section: Instrumentationmentioning

confidence: 99%

Section: Hybridization Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Lahdenperä

Spangar

Lempainen

et al. 2015

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

Lahdenperä

Wang

Vainio

et al. 2017

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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) (Eu) 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.

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Spacer length, label moiety interchange and probe pair orientation in a homogeneous solid-phase hybridization assay utilizing lanthanide chelate complementation

Abstract: We have studied parameters affecting DNA hybridization and lanthanide chelate complementation based signal formation in a separation-free solid-phase assay suitable for spatial multiplexing.

Cited by 2 publications

References 13 publications

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

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

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

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