Oligonucleotide Array-in-Well Platform for Detection and Genotyping Human Adenoviruses by Utilizing Upconverting Phosphor Label Technology
Department of Biotechnology, University of Turku, Turku, Finland. Analytical Chemistry
(Impact Factor: 5.64).
02/2011; 83(4):1456-61. DOI: 10.1021/ac103155f
We have developed a robust array-in-well test platform based on an oligonucleotide array, combining advantages of simple instrumentation and new upconverting phosphor reporter technology. Upconverting inorganic lanthanide phosphors have a unique property of photoluminescence emission at visible wavelengths under near-infrared excitation. No autofluorescence is produced from the sample or support material, enabling a highly sensitive assay. In this study, the assay is performed in standard 96-well microtiter plates, making the technique easily adaptable to high-throughput analysis. The oligonucleotide array-in-well assay is employed to detect a selection of ten common adenovirus genotypes causing human infections. The study provides a demonstration of the advantages and potential of the upconverting phosphor-based reporter technology in multianalyte assays and anti-Stokes photoluminescence detection with an anti-Stokes photoluminescence imaging device.
Available from: Terhi Riuttamäki (née Rantanen)
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ABSTRACT: Clin Microbiol Infect
A robust oligonucleotide array-in-well hybridization assay using novel up-converting phosphor reporter technology was applied for genotyping clinically relevant human adenovirus types. A total of 231 adenovirus-positive respiratory, ocular swab, stool and other specimens from 219 patients collected between April 2010 and April 2011 were included in the study. After a real-time PCR amplification targeting the adenovirus hexon gene, the array-in-well assay identified the presence of B03 (n = 122; 57.5% of patients), E04 (29; 13.7%), C02 (21; 9.9%), D37 (14; 6.6%), C01 (12; 5.7%), C05 (5; 2.4%), D19 (4; 1.9%), C06 (2; 0.9%), D08 (1; 0.5%), A31 (1; 0.5%) and F41 (1; 0.5%) genotypes among the clinical sample panel. The typing result was obtained for all specimens that could be amplified (n = 223; 97%), and specificity of the typing was confirmed by sequencing specimens representing each of the different genotypes. No hybridization signal was obtained in adenovirus-negative specimens or specimens with other viruses (n = 30). The array-in-well hybridization assay has great potential as a rapid and multiplex platform for the typing of clinically relevant human adenovirus genotypes in different specimen types.
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ABSTRACT: Marine toxins from microscopic algae can accumulate through the food chain and cause various neurological and gastrointestinal illnesses for human health. Herein, we designed a new ultrasensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of brevetoxin B (BTX-2) and dinophysistoxin-1 (DTX-1) in seafood using distinguishable metal nanocluster-labeled molecular tags as traces on bifunctionalized magnetic capture probes. To construct such a bifunctionalized probe, monoclonal mouse anti-BTX-2 (mAb(1)) and anti-DTX-1 (mAb(2)) antibodies were co-immobilized on a magnetic bead (MB-mAb(1,2)). The distinguishable metal nanoclusters including cadmium nanoclusters (CdNC) and copper nanoclusters (CuNC) were synthesized using the artificial peptides with amino acid sequence CCCYYY, which were used as distinguishable signal tags for the label of the corresponding bovine serum albumin-BTX-2 and bovine serum albumin-DTX-1 conjugates. A competitive-type immunoassay format was adopted for the online simultaneous monitoring of BTX-2 and DTX-1 on a homemade flow-through magnetic detection cell. The assay was based on the stripping voltammetric behaviors of the labeled CdNC and CuNC at the various peak potentials in pH 2.5 HCl containing 0.01 M KCl using square wave anodic stripping voltammetry (SWASV). Under optimal conditions, the multiplexed immunoassays enabled simultaneous detection of BTX-2 and DTX-1 in a single run with wide working ranges of 0.005-5 ng mL(-1) for two marine toxins. The limit of detection (LOD) and limit of quantification (LOQ) were 1.8 and 6.0 pg mL(-1) for BTX-2, while those for DTX-1 were 2.2 and 7.3 pg mL(-1), respectively. No non-specific adsorption and electrochemical cross-talk between neighboring sites were observed during a series of procedures to detect target analytes. The covalent conjugation of biomolecules onto the nanoclusters and magnetic beads resulted in good repeatability and intermediate precision down to 9.5%. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of 12 spiked samples, including Sinonovacula constricta , Musculista senhousia , and Tegillarca granosa , between the multiplexed immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for analysis of BTX-2 and DTX-1.
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