Lanthanides have recently found applications in different fields of biomolecular and medical research. Luminescent lanthanide chelates have created interest mainly due to their unique luminescent properties, such as their long Stokes' shift and exceptional decay times allowing efficient temporal discrimination of background interferences in the assays, such as immunoassays. Recently, new organometallic complexes have been developed giving opportunities to novel applications, in heterogeneous and homogeneous immunoassays, DNA hybridization assays, high-throughput screening as well as in imaging. In addition, encapsulating the chelates into suitable matrix in beads enables the use of new members of lanthanides extending the emission wavelength to micrometer range and decays from a few microseconds to milliseconds. As the luminescence is derived from complicated intra-chelate energy transfer, it also gives novel opportunities to exploit these levels in different types of energy transfer based applications. This review gives a short overview of recent development of lanthanide chelate-labels and discusses in more details of energy levels and their exploitation in new assay formats.
Duchenne muscular dystrophy (DMD/Duchenne) is a progressive X-linked disease and is the most common pediatric-onset form of muscular dystrophy, affecting approximately 1:5000 live male births. DNA testing for mutations in the dystrophin gene confirms the diagnosis of this disorder. This study involves assessment of screening newborns for DMD using an immunoassay for muscle-type (MM) creatine kinase (CK) isoform—the GSP Neonatal CK-MM kit. Comparisons were made with CK activity determination by fluorescence measurement. In addition, the study evaluated the effect of gestational age, age of infant at time of sampling and how stable the CK-MM was over time. This assay discriminates well between normal, unaffected and Duchenne affected populations and is suitable for Duchenne newborn screening.
We report here a novel, time-resolved, lanthanide-based energy-transfer assay utilizing nonoverlapping acceptor fluorophores, which have their absorption energetically at a higher level than the emittive transitions of the donor. The technique was studied by comparing a series of nonoverlapping acceptors in a homogeneous DNA model assay utilizing Eu3+ chelate as a donor. The assay provides strong energy-transfer enhanced acceptor emission and enables the anti-Stokes' shift FRET measurement, in which the induced acceptor emission is at shorter wavelength than the donor emission. This results in high sensitivity, and 0.8 pM detection limit was measured for the DNA target. The acceptor signal of the assay is characterized by exceptional lifetime properties and is not strictly following the Forster's theory. The mechanism of nonoverlapping energy transfer is considered, and we propose that when nonoverlapping acceptors are utilized, the energy transfer arises from the upper 5D2 and 5D1 excited states of europium. The assumption was studied using a simplified energy level scheme of the Eu3+ donor and the acceptors, and a correlation between the acceptor emission behavior and the energy level scheme was found.
We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.
BackgroundThe Northern Finland 1966 birth cohort (NFBC 1966) is an epidemiological study where the participants have been controlled since pregnancy both in field tests and using questionnaires. This study aimed to evaluate cross-sectionally the association of self-reported oral symptoms (dental caries and bleeding of gums) with sociodemographic and health behavior factors among the subjects.MethodsOf the 11,541 original members of the cohort, 8,690 (75%) responded to the questionnaire on oral health (dental decay, gingival bleeding and self-estimated dental treatment need) and sociodemographic factors, general health and health behavior. Cross-tabulation and chi-squared tests as well as multiple logistic regression analysis were used to analyze the association between the outcome and explanatory variables.ResultsThe study group was equally distributed between the genders. One third of the subjects reported having dental decay, one fourth gingival bleeding and a half a dental treatment need. As compared to women, men reported significantly more frequently symptoms (p < 0.05). Logistic regression analysis revealed low tooth brushing frequency increasing the odds most for all oral symptoms ((OR 1.57 (1.39–1.78) for dental decay, 1.94 (1.68-2.24) for gingival bleeding and 1.42 (1.26-1.61) for dental treatment need). Frequent smoking was associated with dental decay (OR 1.63 (1.44–1.84)) and treatment need OR (1.39 (1.23–1.56)), whereas poor general health (OR 1.71 (1.48–1.96)) and high BMI (OR 1.19 (1.03–1.36)) both were associated with gingival bleeding.ConclusionsMales with single marital status, BMI over 25, poor general health and poor oral health behaviors are at risk for self-reported poor oral health and dental treatment need.
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