In situ non-contact sensing of microbiological contamination by fluorescence spectroscopy
Date
2022-07-13
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Abstract
Käesoleva tööstusdoktorantuuri programmi käigus töötati välja mitmeid uudseid fotoonika sensoreid ja nende rakendusi, alates lidarist kuni spektrofluoromeetrite ja fluoromeetriteni. Nende seadmete peamine eesmärk on monitoorida ja kontrollida pindadel esinevaid mikrobioloogilisi patogeene. Välja töötatud lidar on mõeldud patogeenide kaugtuvastamiseks ja klassifitseerimiseks välihaigla pindadel, samas kui spektrofluoromeetrid võimaldavad fiiberoptilise kaabli abil tuvastada patogeene reaktiivivabalt lähedalt ning multispektriline fluoromeeter võimaldab käeshoitavalt kiiresti hinnata pinna puhtust. Veel töötati doktoriõppe käigus välja täiesti uudne fotoonika sensoril põhinev lahendus vesinikperoksiidi aurude saastatusest puhastamise protseduuride jälgimiseks. See lähenemine kasutab käimasoleva H2O2 aurude saastatusest puhastamise protseduuri tõhususe reaalajas hindamiseks tavapäraselt kasutatavate bioloogiliste indikaatorite, näiteks bakterispooride, fluorestsentsi. Töös leitakse, et avastatud fluorestsentsi langusefekt on H2O2 aurude kontsentratsiooni suhtes äärmiselt tundlik, nii et praktiline rakendus kaubandusliku toote kujul vesinikperoksiidi dekontaminatsiooniprotseduuride jälgimiseks on täiesti elujõuline valik. Avastatud efekti uuriti edasi, võrreldes fluorestsentsi emissioonispektreid, aeglahutus-spektreid, UV-kiirguse mõju ja skaneeriva elektronmikroskoobi pilte ühelt poolt võrreldes H2O2 auruga hukatud patogeenide (bakterispooride) vastavaid omadusi veel elujõuliste patogeenide omadustega. Töö tulemused tõestavad, et fluorestsentsi abil saab eristada patogeenide klasse erinevatel taustmaterjalidel. Tulemused näitavad ka, et vesinikperoksiidi auruga ülitõhusa saastest puhastamise ajal peetakse patogeenide surmapõhjusteks nii võtmevalkude denatureerimist kui ka valkude sisemiste osade oksüdeerumist. Samas näitasid skaneeriva elektronmikroskoobi kujutised, et oksüdatsioonist põhjustatud mehaanilised kahjustused ei ole patogeeni surma peamine põhjus. Muu hulgas leidsime ka, kuidas UV-doos mõjutab mõõtmistulemusi ja et seda tuleb selliste fotoonikasensorite projekteerimisel arvesse võtta.
In the course of the current industrial PhD program several novel photonic sensors and their applications were developed, ranging from a lidar to spectrofluorometers and fluorometers. The main goal of these devices is to monitor and control microbiological pathogens on surfaces. The lidar was designed for remote detection and classification of pathogens on field hospital surfaces, while the spectrofluorometers enable close-range reagentless detection of pathogens with fibre-optic cable and the multi-spectral fluorometer enables handheld quick estimation of surface cleanliness. A completely novel photonic sensor solution to the monitoring of hydrogen peroxide vapour decontamination procedures was developed in the course of the PhD program as well. This approach uses the fluorescence of commonly used biological decontamination indicators, such as bacterial spores, to estimate the efficiency of an ongoing H2O2 vapour decontamination procedure in real time. The fluorescence intensity fall-off effect discovered in this work is found to be extremely sensitive to H2O2 vapour concentration, so that the practical application in the form of a commercial product for monitoring VHP decontamination procedures is a completely viable option. The discovered effect was studied further by comparing the fluorescence emission spectra, the time-resolved spectra, the effect of UV radiation and the scanning electron micrographs of H2O2-vapour-killed pathogens (bacterial spores) on one side to similarly prepared but still viable pathogens on the other side. The results of the work prove that fluorescence can be used to distinguish classes of pathogens on various background materials. The results also indicate that denaturation of key proteins as well as oxidation of the inner parts of the proteins are both considered causes of death for pathogens during the highly efficient decontamination with hydrogen peroxide vapour. At the same time, the scanning electron microscopy images indicated that the mechanical damage caused by oxidation is not the main cause of pathogen death. Finally found how UV dose affects measurement results and has to be taken into account in the design of such photonic devices.
In the course of the current industrial PhD program several novel photonic sensors and their applications were developed, ranging from a lidar to spectrofluorometers and fluorometers. The main goal of these devices is to monitor and control microbiological pathogens on surfaces. The lidar was designed for remote detection and classification of pathogens on field hospital surfaces, while the spectrofluorometers enable close-range reagentless detection of pathogens with fibre-optic cable and the multi-spectral fluorometer enables handheld quick estimation of surface cleanliness. A completely novel photonic sensor solution to the monitoring of hydrogen peroxide vapour decontamination procedures was developed in the course of the PhD program as well. This approach uses the fluorescence of commonly used biological decontamination indicators, such as bacterial spores, to estimate the efficiency of an ongoing H2O2 vapour decontamination procedure in real time. The fluorescence intensity fall-off effect discovered in this work is found to be extremely sensitive to H2O2 vapour concentration, so that the practical application in the form of a commercial product for monitoring VHP decontamination procedures is a completely viable option. The discovered effect was studied further by comparing the fluorescence emission spectra, the time-resolved spectra, the effect of UV radiation and the scanning electron micrographs of H2O2-vapour-killed pathogens (bacterial spores) on one side to similarly prepared but still viable pathogens on the other side. The results of the work prove that fluorescence can be used to distinguish classes of pathogens on various background materials. The results also indicate that denaturation of key proteins as well as oxidation of the inner parts of the proteins are both considered causes of death for pathogens during the highly efficient decontamination with hydrogen peroxide vapour. At the same time, the scanning electron microscopy images indicated that the mechanical damage caused by oxidation is not the main cause of pathogen death. Finally found how UV dose affects measurement results and has to be taken into account in the design of such photonic devices.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
biofouling, pathogenic microbes, fluorescense spectroscopy, sensors