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The thesis presents a study of time-resolved ﬂuorescence system designs based on novel light sources for Radiometers next generation immunoassay analyzer for in vitro diagnostics. The general concept of time-resolved ﬂuoroimmunoassays is given. The main parts constituting the basis of time-resolved ﬂuoroimmunoassays, such as lanthanide based labels, time-resolved ﬂuorimetry, and all-in-one dry cup concept, are explained. Critical analytical parameters of immunoassays are discussed. The design, development and investigation of an optical system based on UV LED excitation at 340 nm for time-resolved ﬂuorescence measurement of immunoassays are presented. For the ﬁrst time to the authors knowledge, a LED based time-resolved ﬂuorescence measurement system is successfully employed for immunoassays detection. The system is tested to measure cardiac marker troponin I in immunoassays. The signal-to-noise ratio is comparable to a state-of-the-art Xenon ﬂash lamp based unit with equal excitation energy and without overdriving the LED. A comparative study of the ﬂash lamp and the LED based system is performed and temporal, spatial, and spectral features of LED excitation for time-resolved ﬂuorimetry are discussed. Optimization of the suggested key parameters of the LED promises signiﬁcant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems. Preliminary tests of an immunoassay analyzer employing an optimized LED excitation are reported, measuring on a standard troponin I and a novel research high-sensitivity troponin I assay. High-sensitivity cardiac troponin assay development enables determination of biological variation in healthy populations, more accurate interpretation of clinical results and points towards earlier diagnosis and rule-out of acute myocardial infarction. With the optimized LED based system, the limit of detection is improved by a factor of 5 for the standard troponin I and by a factor of 3 for the research high-sensitivity troponin I assay, compared to that of the ﬂash lamp excitation. The obtained limit of detection was 0.22 ng/L measured on plasma with the research high-sensitivity troponin I assay and 1.9 ng/L measured on tris-saline-azide buffer containing bovine serum albumin with the standard troponin I assay. The main highlight of the thesis is the ﬁrst demonstration of an optimized LED excitation based point-of-care immunoassay analyzer. We have preliminarily fulﬁlled the high-sensitivity criteria for troponin measurements in immunoassays. The demonstrated results are an important step in the development of high-sensitivity troponin I assays for point-of-care testing, and will ultimately lead to more accurate interpretation of clinical results and earlier diagnosis and rule-out of acute myocardial infarction. A general model of background noise sources in heterogeneous timeresolved ﬂuoroimmunoassays is suggested. The suggested model can be generally applied to ﬂuoroimmunoassays employing the dry-cup concept. The optimization of timing parameters in the time-resolved measurement of lanthanide ﬂuorescence is discussed. Impact of the light sources on main immunoassay parameters is discussed, and potentials and limitations of immunoassay measurements are investigated. For the ﬁrst time to the authors knowledge, a passively Q-switched, solidstate UV laser at 343 nm is demonstrated. The novel cost-effective passively Q-switched 343 nm solid-state laser delivers up to 20 µJ per pulse, with a pulse width of 2.3 ns at a repetition rate of 100 Hz. The 343 nm is obtained through third harmonic generation of a passively Q-switched 1030 nm Yb:YAG laser with pulse energy of 190 µJ at 100 Hz and a pulse width of 5.4 ns. The IR-UV conversion efﬁciency is 10.4%, comparable to that achieved with mode locked IR lasers. The light source is electronically controlled for easy synchronization with a detection circuit. The controllable low repetition rate speciﬁcally targets applications exploiting the millisecond scale lifetime of lanthanides employed in ﬂuoroimmunoassay measurements for time-resolved ﬂuorescence spectroscopy. Low repetition rate and even pulse-on-demand operation is demonstrated. The main challenges of the design of a high-power passively Q-switched solid state laser at 343 nm are discussed. Finally, the results of laser induced time-resolved ﬂuorescence measurements in immunoassays are presented. The impact of laser illumination area, narrow emission spectrum, and signiﬁcantly higher peak power compared to the LED are discussed.
|Number of pages||129|
|Publication status||Published - 2018|