Open Access

Introduction. W.H.O estimated that globally at least 2 billion people use drinking water sources contaminated with faeces and according to UNICEF, most of these faecal detection methods are expensive, time-consuming (18–24 h time to result), and, with few exceptions, not suited for on-site analysis. Hence, there is an urgent need for the development of analytical methods that allow to unequivocally test for drinking water quality directly on-site. Aims. Development of rapid and sensitive fluorescence based analytical methods for faecal pigments (FPs) detection towards on-site water quality testing. Methods. Silane functionalized glass fiber paper test strips were developed for the sensitive drop-&-detect analysis of FPs. Drop casting of water samples containing faecal pigment on specifically functionalized test strips allowed the sensitive detection of FPs with a smartphone coupled to a 3D printed optical setup. Results. A series of silanes were used to functionalize glass fiber paper and tune its hydrophobicity, exploiting the influence of matrix tailoring to enhance binding of the Zn salt used as co-reagent to urobilin for optimal fluorescence response. Combination of bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane with zinc chloride-impregnated test strips demonstrated optimum fluorescence response for sensitive (nano- and sub-nanomolar concentration) smartphone-based faecal pigments detection. The obtained fluorescence sensing results were validated with a benchtop fluorometer. Furthermore, the developed analytical method was successfully applied to the analysis of real water samples. Conclusions. The possibilities of matrix tailoring and co-reagent nature on the development of a rapid, sensitive, and embedded fluorescence-based on-site strip test are presented and discussed. This technique has potential application for faecal biomarker detection/primary screening for water quality in developing countries, where sophisticated instruments may not be available.

Faecal contaminants in water are considered serious threats for human health, due to the presence of viruses, bacteria and other harmful microorganisms.1 Urobilin (UB) is a well-known faecal pigment and can be used as a marker for faecal matter in water.2 UB is commonly present in the urine of all mammals as the catabolic end product of bilirubin degradation.2 As the only simple chemical approach to its detection, Schlesinger’s test is usually used to enhance the weak fluorescence of UB in alcoholic media by complexation with Zinc.2, 3 The major limitation of this method is the only weak enhancement of the intrinsically weak UB fluorescence in aqueous media.3 This work presents an approach to introduce different Zn salts for improved fluorescence response, where we found a clear dependence of the fluorescence yield of UB-Zn(II) complexes on the counterion of the salt in water. By employing a combination of fluorescence parameters like transition energy, fluorescence intensity, and fluorescence lifetime, a photophysical understanding of the structure and conformation of the UB-Zn(II) complexes responsible for the fluorescence enhancement in water could be gained. The possibilities of developing a sensitive analytical method based on the acquired understanding are also discussed.

Fecal pollution in water poses significant health risks, especially when contaminated sources are used for drinking and food production. Traditional water quality testing methods are expensive, slow, and require skilled personnel, limiting their accessibility. This work addresses these issues by developing a portable fluorometric assay for the detection of the fecal indicator pigment urobilin (UB). The assay uses silane-functionalized glass fiber strips impregnated with zinc chloride, providing a ‘drop-&-detect’ approach with enhanced fluorescence response mediated by the unique complexation properties of ZnCl2 and UB. This approach allows for the detection of UB at sub-nanomolar concentrations in less than 1 min using a 3D-printed setup with miniaturized optical components powered by a smartphone with its camera as a detector. The results validated with a benchtop fluorometer show the effectiveness of this method. The successful application of this userfriendly, rapid, and sensitive assay to real water samples from three rivers and the influx and efflux of a wastewater treatment plant advances field-based water quality monitoring, meets the WHO’s ASSURED criteria, and supports progress toward the global clean water and sanitation goals.

According to the World Health Organization (WHO), at least two billion people worldwide rely on drinking water sources contaminated with faeces.1 UNICEF reports that current methods for detecting faecal contamination are often expensive, time-consuming (typically requiring 18–24 hours), and generally unsuitable for on-site analysis.2 Hence, there is an urgent need for rapid analytical methods that can reliably assess drinking water quality directly in the field. Our approach utilizes the weak intrinsic fluorescence of faecal pigments such as urobilin (UB), whose emission is enhanced through complexation with Zn²⁺ in alcoholic media, known as Schlesinger’s test. However, this classical method for faecal contaminant detection has significant limitations: UB–Zn²⁺ complexes exhibit only weak fluorescence in water, their fluorescence decays over time, and is interfered by fluorescence from humic substances naturally present in surface waters. These limitations in detection in aqueous media motivated us to explore an organic–aqueous extraction system using hexanol to improve fluorescence response and signal stability. To address this, we present a dual strategy involving the use of different Zn²⁺ salts and hexanol as an extraction and fluorescence-enhancing medium. We observed a clear dependence of the fluorescence yield of UB–Zn²⁺ complexes on the counter anions of different Zn²⁺ salts. Among them, UB–Zn(NO₃)₂ complex was showing the highest fluorescence intensity in hexanol. The extraction of UB–Zn²⁺ complexes into hexanol provided enhanced and temporally stable fluorescence emission for a reliable sensing approach.3 The photophysics of the UB–Zn²⁺ complex in hexanol and Job’s plots confirmed non-trivial complex stoichiometries. Nonetheless, the developed analytical method showed sensitive (nano- and sub-nanomolar concentration) response for UB detection with negligible fluorescence interference from humic substances commonly present in environmental water. Furthermore, a 3D-printed optofluidic platform was developed to perform in-channel extraction and real-time fluorescence detection. This platform was printed all at once using a commercial SLA printer with high resolution and transparent resins. The fluidic part allowed for droplet-based extraction by chaotic advection, by means of a miniaturized pump. The optical detection combined an LED for excitation and a USB photomultiplier tube module for low fluorescence signal digital acquisition, potentially on an embedded device such as a tablet. This configuration enabled rapid, sensitive, and interference-minimized detection of faecal pigments in water, demonstrating the potential of optofluidic sensing for on-site water quality monitoring.