Surface-selective Luminescence Methods: Enabling technologies for rapid immunoassays and chemical sensing in cell cultures

Luminescence is the most widely used technology in chemical and biochemical assays and sensor applications. It is used extensively in the fields of biotechnology, medical diagnostics, DNA sequencing, genetic research, forensic analysis as well as in various on-site diagnostic, screening and safety applications. Luminescence technology is attractive, since it provides high sensitivity, a wide variety of sensing schemes and several measurable parameters. The technology is based on the use of luminescent labels or indicator molecules, whose radiation in the detector is modulated by the analyte in terms of emission intensity, lifetime, anisotropy, energy transfer or spectroscopic properties.

Typically, the luminescent molecules to be detected are located at interfaces. There is a myriad of fluorescent immunoassays, where the fluorescently labelled biomolecules bind on transparent surfaces. Similarly, a typical sensing element a luminescence-based chemical sensor is composed of a thin dielectric layer embedded with analyte-sensitive indicators. In these common sensing geometries, the dielectric interfaces create discontinuities in the refractive index, which has substantial effects on the local electric field and the spatial radiation distribution of the adjacent molecular emitters. With appropriate optical arrangements, these effects can be exploited in the luminescence detection. Indeed, both the luminescence excitation and emission collection can be transformed into surface-selective or layer-selective modes, which significantly enhances the detected emission and suppresses the background signals.

The surface-selective fluorescence detection is an excellent technological choice for rapid fluorescent immunoassays. The technique gives highly sensitive means to monitor biochemical binding processes in real time and thus generate estimates for the analyte concentrations as soon as the assay begins. The technique is also relatively insensitive to many bulk matrix effects, which makes it especially well-suited for rapid on-site applications with challenging sample matrices, such as point-of-care diagnostics and rapid on-site screening of many small but important molecules, including steroids, hormones, toxins and abused drugs.

In this thesis, a fluorescent measurement platform with surface-selective techniques is developed for rapid immunoassays. For maximal surface-selectivity, the platform incorporates both the technique of total internal reflection excitation (TIR) and supercritical angle fluorescence (SAF). The key element of the platform is a parabolic lens, designed in the thesis and custom-manufactured from polystyrene, which facilitates the simultaneous application of TIR and SAF. The relatively high refractive index of polystyrene is advantageous in terms of the SAF collection efficiency and, in addition, polystyrene lens makes the platform compatible with standard flat-bottom microtiter plates. The thesis reports on the design, construction and characterization of the platform, as well as on its application to rapid immunoassays of two abused drug molecules: morphine and cocaine.

The morphine assay is based on a noncompetitive immunoassay utilizing two antibodies. The fluorescent detection antibodies bind on the sensing surface, which is monitored with the developed platform in real time. As a result, morphine concentrations below one tenth of the typical screening cutoff concentration of the authorities (40 ng/mL) can be detected in 30 sec. The cocaine assay is based on a displacement-type immunoassay. The developed platform makes it possible to follow the displacement process in real time. The results show that cocaine concentrations down to 10 ng/mL, comparable to the common cutoff values of the authorities (8 - 20 ng/mL), can be detected in untreated saliva samples in 30 sec.

Chemical sensing in in vitro cell cultures represents another key application for the surface-selective luminescence technology. In a widely applied chemical sensing geometry, analyte-specific indicators are embedded in an analyte-permeable thin film on the culturing substrate, and the analyte molecules modulate the luminescence properties of the indicator. To this geometry, the surface-selective (or layer-selective) detection techniques bring certain advantages, especially in terms of minimizing the invasiveness and the disruptions the cells experience. Firstly, compared to conventional luminescence collection set-ups, the surface-selective methods enhance the signal multiple times, thus allowing the use of low indicator concentrations and excitation power. Secondly, the surface-selective excitation protects the cells from a direct illumination. Especially in sensitive cell cultures, such as based on human induced pluripotent stem cell-derived (hiPSC-derived) cells, these features can be significant.

In the thesis, luminescence-based oxygen sensors with surface-selective TIR and SAF techniques are developed for in vitro cell cultures. The key factors in the designs are the minimal invasiveness and the integrability to the modular cell culture platform with numerous other functionalities, including temperature and gas control, microelectrode arrays and microscopy. The optical read-outs are based on custommanufactured parabolic lenses. The developed oxygen sensors are characterized and applied in experiments, where the functionality of hiPSC-derived cardiomyocytes is monitored under varying oxygen conditions. Finally, the thesis demonstrates how the luminescent sensing material itself can be made significantly more biocompatible by linking of the indicators covalently into the supporting matrix, that is, by preventing the leaching of the indicators.