Method and test strip for the on-site detection of a fecal pigment in a water sample and method for manufacturing the test strip

Abstract

A method (100) for on-site detection at trace levels in a water sample of a fecal pigment, i.e. a metabolite or degradation product of a hemoglobin, such as urobilin and / or stercobilin is suggested, comprising: (i) providing a zinc salt and silane functionalized fibrous substrate (2), in particular a zinc salt and silane functionalized glass fiber substrate (2); (ii) wetting a detection region (3) of the zinc salt functionalized fibrous substrate (2) with the water sample; (iii) exposing the detection region (3) to an excitation light, the excitation light typically comprising a wavelength in a range of about 450 – 490 nm, particularly of about 475 nm, a full width half maximum of the excitation light typically being at most about 25 nm; (iv) detecting a fluorescence light emitted in response to exposing the detection region (3) to the excitation light; and at least one of: (v) detecting, based on the fluorescence light, a presence of the fecal pigment in the water sample; and (vi) correlating an intensity of the detected fluorescence light with a calibration data set for determining a concentration of the fecal pigment in the water sample.

Description

Z.-24P008Method and test strip for the on-site detection of a fecal pigment in a water sample and method for manufacturing the test stripFIELD

[0001] The present invention relates to tests for the assessment of water quality, in particular to the detection of a fecal contamination of water, based on the detection of fecal pigments, such as urobilin and stercobilin. Accordingly, it also relates to the analysis of water and wastewater as well as methods for wastewater treatment and water management.BACKGROUND

[0002] Water contamination caused by human or animal waste is considered as a worldwide issue. A fecal pollution in water can cause serious diseases due to the exposure to fecal pathogens, including bacteria, viruses, and parasites. In 2022, the World Health Organization (WHO) already estimated that globally at least 2 billion people use drinking water sources contaminated with feces (https: / / www.who.int / news-room / fact- sheets / detail / drinking-water). Another example is cited in 2023 by AQA GmbH and Fraunhofer IGB, presenting a study on drinking water quality that indicated that 3.6% of the water samples from households in Germany contain fecal germs (https: / / www.igb.fraunhofer.de / de / presse-medien / presseinformationen / 2023 / aqa-gmbh-und- fraunhofer-igb-stellen-studie-zur-trinkwasserqualitaet-vor.html). The cultivation of fecal indicator bacteria (FIB), such as E. coh. is the common technique for monitoring the quality of drinking as well as recreational waters and assessing the risk of the presence of fecal pathogens in these waters. However, these methods for detecting pathogens are very costly, require an incubation period of 18 to 24 hours before a result is available, are impractical for on-site analysis and require the use of qualified personnel.

[0003] Therefore, the need for a rapid and sensitive detection method for fecal contaminants in water remains paramount to facilitate water quality testing. As an alternative to FIB, chemical indicator compounds can play an important role for rapid and universal primary screening for water quality testing. The use of urobilin (UB) as a biomarker for fecal pollution in water is well known and urobilin is therefore considered a reliable chemical indicator compound. Urobilin is a porphyrinoid compound released into the environment via the urine and feces of mammals. It is thought to be formed from bilirubin, the main metabolite of hemoglobin from over-aged and therefore destroyed erythrocytes. Bilirubin, the metabolic precursor of fecal pigments, is excreted by the reticulo-endothelial tissues (spleen and liver) of all mammals into their intestine. There it is broken down by the intestinal flora into variousZ.-24P008 other fecal indicator pigments (FIP), with orange / yellow urobilin being the most stable. Its fluorescence in water can be enhanced by complexation with Zn2+. The nevertheless very weak fluorescence emission in aqueous media can be enhanced for urobilin and other FIPs by complexation with Zn2+ions in alcoholic media, which is known as the Schlesinger test (W. Schlesinger, Dtsch Med Wochenschr. 1903, 29, 561-563).

[0004] This Zn2+complexation-based fluorescence method has been used for both urobilin and stercobilin estimation (Miyabara et al, J Chromatogr B 1992, 574, 261-265). However, the Schlesinger test used therein has considerable limitations, as an organic solvent is required or the fluorescence intensity of the FIP-Zn2+complexes rapidly decreases in aqueous media (Prakash et al., Chemosphere 2021, 265, 129189) which makes the detection of trace concentrations of urobilin in water considerably more difficult.

[0005] Another drawback of fluorescence-based analysis of UB-Zn2+complexes, especially in natural surface waters, is the interference caused by the fluorescence of dissolved organic matter (DOM), which is omnipresent in open waters.

[0006] Other analytical methods were also used for FIP analysis like RP-HPTLC (Lam et al., Clin Chem 1998, 44, 345-346), solid-phase extraction (Piocos & de la Cruz, J Liq Chromatogr Relat Technol 2000, 23, 1281-1291) and HPLC-ES-MS (Jones-Lepp, J Enivron Monit 2006, 8, 472-478).

[0007] In addition, bacterial and genetic markers have also been used to identify and determine fecal contamination (Borrego et al, Water Res 1987, 21, 1473-1480). However, many of these methods are expensive, require complex sample preparation techniques in a laboratory, require highly specialized personnel and are therefore hardly suitable for an onsite analysis.

[0008] Currently, a method for detecting fecal pigments in water using solid-state green fluorescence on metal salts (Prakash & Mishra, Anal Methods 2022, 14, 2907-2912; IITM IDF Ref. 2246, Patent No: IN 405277, Patent Appl. No: 202141059590 A) is proposed to detect the urobilin specifically in the micromolar concentration range (e.g., around 100 pM).

[0009] However, this sensitivity is still insufficient because in most cases of FIP contamination, trace concentrations in the range of picomolar to nanomolar are reported for environmental freshwater (Bixler et al, Proc Natl Acad Sci USA 2014, 111, 7208-7211; US2016 / 0209388 Al).Z.-24P008

[0010] Most of the established methods used in practice are based on the traditional fecal indicator bacteria (FIB) approach which takes 18 to 24 hours for confirmation of a fecal contamination of water.BRIEF SUMMARY

[0011] Against this background, a rapid and simple test with high sensitivity for urobilin detection in water is highly desirable. Ideally, such a test fulfils the so-called ASSURED principle of the WHO, i.e. it is Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end users. Thus, the object of the invention is to provide a robust and simple test for reliable detection of a fecal water pollution.

[0012] According to the invention, a substrate is suggested that is specifically coated with a functional silane and subsequently impregnated with zinc chloride to be used in a fluorometric assay for urobilin detection. The substrate is a fibrous material comprising, e.g., cellulose, another organic polymer, mineral fibers, ceramic fibers, and / or glass fibers.

[0013] A drop of an aqueous sample to be analyzed for water quality is deposited directly on the functionalized substrate. Afterwards a fluorescence of the sample application site of the substrate is evaluated. For reproducibility this can be done, for instance, by inserting the substrate into a suitable holder to be fit on a mobile computing device having or being provided with a camera, such as a smartphone or tablet. Subsequently, an excitation source (e.g., a light-emitting diode) connected through the USB connector of the mobile computing device is used, to selectively excite any fluorescence of the sought for UB-Zn2+complex at the sample application site of the substrate. The excitation light may be passed through a filter, e.g., of the type MF475-35 - FITC Excitation Filter, CWL = 475 nm, BW = 35 nm. A possible fluorescence may advantageously be recorded with the camera of the mobile device. Preferably a filter may be used to avoid interference by scattering and stray light. A suitable filter is, e.g., a MF530-43 - FITC Emission Filter, CWL = 530 nm, BW = 43 nm. The obtained fluorescence signal is analyzed, e.g. by comparison with previously recorded calibration values and / or reference spots of known intensity also applied to the strip.

[0014] Said analysis can also be performed using a suitable software application, a so- called app, installed and running directly on the mobile computing device. Alternatively, the fluorescence signal or an image taken of the substrate comprising the excited sample application site can be sent to a remote computer for analysis.Z.-24P008

[0015] Advantageously, all the components required for the suggested assay are commercially available and can be used and adapted in a facile manner without the need of a laboratory setup or hazardous chemicals.

[0016] Finally, it should be remembered that the measurement or testing of FIP fluorescence has not yet been demonstrated for on-site analysis. Also, reliable test strips with high sensitivity focusing on the detection of FIP traces are not yet known.

[0017] According to an embodiment a method is suggested for an on-site detection of a fecal pigment, in particular of a metabolite or degradation product of a hemoglobin, in particular of urobilin, and / or stercobilin, at trace levels in a water sample, the method comprising:(i) providing a fibrous substrate functionalized with a zinc salt and a silane, in particular a zinc salt and silane functionalized glass fiber substrate;(ii) wetting a detection region of the zinc salt and silane functionalized fibrous substrate with the water sample;(iii) exposing the wetted detection region to an excitation light, the excitation light typically comprising a wavelength in a range of about 450 - 490 nm, particularly of about 475 nm, a full width half maximum of the excitation light typically being at most about 25 nm;(iv) detecting a fluorescence light emitted in response to exposing the detection region to the excitation light; and at least one of:(v) detecting, based on the fluorescence light, a presence of the fecal pigment in the water sample; and(vi) correlating an intensity of the detected fluorescence light with a calibration data set for determining a concentration of the fecal pigment in the water sample.

[0018] Advantageously, a fluorescence emitted at the detection region indicates the presence of a fecal pigment in the water sample and optionally, depending on its intensity, may be used to determine a concentration of the fecal pigment in the water sample. In contrast to known methods for the detection of fecal water contamination, the proposed method is quick and easy to perform on-site, i.e. it does not require sophisticated equipment or speciallyZ.-24P008 trained personnel. The ‘calibration data set’ may comprise a set of fluorescence intensity data obtained from known concentrations of the sought fecal pigment during an earlier calibration, or the ‘calibration data set’ may comprise a specific area on the fibrous substrate whose fluorescence is generated in parallel with the current measurement by a previously known amount of the sought fecal pigment, its Zn(II)-complex or a spectroscopically similar fluorescent dye such as a suitably substituted boron-dipyrromethene (BODIPY) dye like, for example, l,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene ((CH3)4BDP-H). (CH3)4BDP-H, or other R4BDP-H with R being a short alkyl group, is a suitable reference dye because it is well soluble in water, hydrophilic and hydrophobic solvents and its fluorescence is insensitive to the nature of a solvent and / or its pH.

[0019] According to an embodiment a test strip for on-site determination of a trace amount of a fecal pigment, in particular a metabolite and / or a degradation product of hemoglobin in a water sample, particularly of urobilin and stercobilin in a water sample according to any of the preceding claims, the test strip comprising: an amphiphilic detection region comprising a zinc salt and an amphiphilic silane functionalized fibrous material, in particular the zinc salt and the amphiphilic silane functionalized glass fiber material, the amphiphilic silane functionalized fibrous material typically being provided by a fibrous substrate.

[0020] Advantageously, the test strip can be easily stored and used on site by simply wetting the detection region or even the entire strip with a suspect water sample. After measuring the fluorescence of the strip at the detection region, a decision can be made as to whether water contamination is present, whether it is necessary to perform a bacteriological test, or whether, for example, a particular water treatment was effective.

[0021] In particular, a test strip for on-site determination of a trace amount of a fecal pigment in a water sample is suggested which comprises a hydrophobic surface with a detection region which is made amphiphilic in nature, the detection region comprising a fibrous material which is modified with a zinc salt and with an amphiphilic silane, in particular a silane and zinc salt functionalized glass fiber material.

[0022] According to an embodiment, a method for manufacturing a test strip, the method comprises: a) functionalizing at least a detection area of a fibrous substrate, in particular a glass fiber substrate, with an amphiphilic silane such as BHEAPTES; andZ.-24P008 b) supplementing the detection area of the fibrous substrate with a zinc salt to form a detection region of the test strip for on-site detection at trace levels in a water sample of a fecal pigment, in particular of a metabolite or degradation product of a hemoglobin, in particular of urobilin, and / or stercobilin.

[0023] Advantageously, the method does not require expensive substrates, chemicals or procedures, is easy to perform and reproducibly yields suitable test strips, which can easily be stored and used for on-site determination of fecal pigments in water samples.

[0024] In particular, according to an embodiment a method for manufacturing a test strip, suitable for detecting a fecal pigment at trace levels in a water sample is suggested. The method comprises: inserting, for a predetermine time of typically at least several hours, a fibrous substrate, in particular a glass fiber substrate (2), into a solution comprising water, an alcohol, in particular ethanol, a tetraethyl orthosilicate (TEOS) and a silane such as trimethoxy(octyl)silane (TMOS);- washing the fibrous substrate (2) with alcohol, e.g., with ethanol; drying the washed fibrous substrate (2), preferably under reduced pressure; drop casting an amphiphilic silane such as 3-(bis(2 -hydroxy ethyl))aminopropyl-triethoxysilane (BHEAPTES) onto the dried fibrous substrate to form the detection region; impregnating the dried fibrous substrate after modification with the drop cast amphiphilic silane with an aqueous zinc salt solution; and drying the impregnated fibrous substrate.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] A full and enabling disclosure of the present invention, including the best mode thereof, to one of ordinary skill in the art, is set forth more particularly in the remainder of the description, including reference to the accompanying figures.

[0026] Fig. 1 illustrates the fluorescence of different urobilin-Zn11complexes on a glassZ.-24P008 fiber substrate modified with a mixture of tetraethyl orthosilicate (TEOS) and the silanes 1 through 8. Fig. 1A shows their spectral fluorescence intensity in arbitrary units at different wavelengths at excitation of 480 nm as measured with a conventional fluorometer by drop casting of (UB (lpM)-ZnNO3(20 mM) in aqueous media,). Fig. IB shows digital images as acquired with a smartphone-based optical setup. Therein “Z” indicates the relative luminance, i.e., fluorescence intensity calculated by "Image J’ software using the formula: L = 0.2126 x Run+ 0.7152 x Gun + 0.0722 x Bun(Sugawara et al., IEEE Signal Process Mag, 2014, 31, 170- 174.). Fig. 1C shows the silanes 1 through 8 used.Fig- 2 shows (A) fluorescence emission spectra of UB-Zn on BHEAPTES modified glass fiber substrate as a function of the zinc salt used; and (B) corresponding digital images acquired with the used smartphone-based setup.

[0027] Fig. 3 shows fluorescence signals generated by the UB-Zn complex, in aqueous solution (Fig. 3A) depending on its concentration and on glass fiber substrate modified with TMOS (drop cast of 10 pL of aqueous UB-Zn(II) complex) as captured by the suggested smartphone-based set up (Fig. 3B). Please note, that UB-Zn complexes may have a broken stoichiometry, because many different conformers can form. For the sake of simplicity, UB-Zn complexes in the following are often denoted as UB-Zn11complexes.

[0028] Fig. 4 shows time dependent change in fluorescence response of drop casting of 10 pL of Teltow canal water spiked with 50 nM urobilin on BHEAPTES / ZnCh functionalized glass fiber substrate.

[0029] Fig. 5 shows measurement results obtained with a natural water spiked with UB. Fig. 5 A represents a fluorescence excitation emission matrix (EEM) of Teltow canal water spiked with 50 nM urobilin in presence and absence of BHEAPTES test strip and Fig. 5B shows digital images of fluorescence response of drop casting of 10 pL of Teltow canal water spiked with 50 nM urobilin on BHEAPTES / ZnCh functionalized test strip.

[0030] Fig. 6 shows digital images of the different silane-coated strips acquired with the smartphone-based setup under the same settings as used for the assay with Milli-Q water which was spiked with 1 pM UB. “Blank” corresponds to a strip not coated with a silane.

[0031] Fig. 7 shows excitation spectra of UB-Zn11complexes from different Zn11salts on glass fibre substrates modified with 3-(bis(2-hydroxyethyl))aminopropyl triethoxysilane (BHEAPTES / silane 3). It indicates the excitation maxima of UB-Zn11complexes at ~ 505 nm except for UB / Zn(BF4)2. For UB-Zn The change in excitation maxima from 490 nm to 505 nm indicates the complex formation on the BHEAPTES / silane 3 test strip.Z.-24P008

[0032] Fig. 8 illustrates the influence of the ZnCl2concentration used with the TMOS / BHEAPTES modification of glass fiber substrates on the fluorescence of 1 pM urobilin in Milli-Q water.

[0033] Fig. 9 shows digital images with fluorescence signals of two different urobilin concentrations (30 nM and 120 nM) in a natural water sample taken from Han River, Da Nang, in Vietnam.

[0034] Fig. 10A, B show exemplary embodiments of the suggested method of test strip manufacturing.

[0035] Fig. 11 illustrates three ‘architectural’ routes suitable to produce test strips according to the invention.

[0036] Fig. 12 illustrates schematically the sequence of coating and spotting steps according to the routes depicted in Fig. 11.

[0037] Fig. 13 shows schematically the suggested mobile phone-based setup for the onsite fecal pigment assay. It illustrates the implementation of the smartphone-based method proposed here for the detection of water contamination by fecal pigments. The test strip, which is wetted with a few drops of the water sample to be analyzed (typical sample volume 10 pl), is inserted with a carrier into a lateral opening 3a of a measuring chamber 3, for example an additively manufactured one. The light emitted by an LED 3e, which is powered by the mobile computing device 4 via an electric connection, such as an USB cable, is directed via a bandpass filter 3d and / or an edge filter 3d onto the detection region of the test strip 2 via a converging lens 3b. Emitted from the detection and / or calibration region(s) fluorescent light is directed via a condenser lens 3c and an emission filter 3g onto the camera 4.1 of the mobile telecommunication device 4, 4.2, 4.3, 4.5 used.

[0038] Fig. 14 shows measurement results obtained with a Milli-Q water spiked with UB (1 pM). Fig. 14 A represents emission intensity with variation of time (min) for urobilin 1 pM in presence of TMOS / BHEAPTES / ZnCl2test strip by benchtop spectrometer and Fig. 14B shows fluorescence response with time at 516 nm on TMOS / BHEAPTES / ZnCl2functionalized test strip.

[0039] Fig. 15 shows time dependent change in fluorescence response of drop casting of 10 pL of Milli-Q water spiked with (1 pM) urobilin on TMOS / BHEAPTES / ZnCl2functionalized glass fiber substrate.Z.-24P008

[0040] Fig. 16 shows the morphology of test strip using FESEM images.

[0041] Fig. 17 shows the morphology of test strip using FESEM images.

[0042] Fig. 18 shows the elemental analysis of Glass fiber / TMOS / BHEAPTES / ZnCl2test strip and TMOS coated glass fiber test strip.

[0043] Fig. 19 shows the TGA analysis of Glass fiber / TMOS / BHEAPTES / ZnCl2test strip and Glass fiber / BHEAPTES / ZnCl2test strip. Glass fiber / TMOS / BHEAPTES / ZnCl2test strip shows higher stability than Glass fiber / BHEAPTES / ZnCl2test strip.

[0044] Fig. 20 shows higher luminance value for Glass fiber / TMOS / BHEAPTES / ZnCl2test strip than Glass fiber / BHEAPTES / ZnCl2test strip.

[0045] Fig. 21 shows spectrophotometrically, cuvette based results of absorbance and fluorescence measurements of different Zn11salt (20 mM) complexation with urobilin (IpM) in Milli-Q water.

[0046] Fig. 22 shows the fluorescence intensity enhancement for urobilin (1 pM) and corresponding fluorescence of the detection region obtained by ZnCl2complexation of urobilin. The enhancement was calculated after background subtraction. In A, a blank spectrum, which is virtually identical to the x axis, was subtracted from both spectra before calculating the quotient. In B, the luminance L of the blank was subtracted from both luminance values of the samples before calculating the quotient.

[0047] In the following detailed description, reference is made to the accompanying figures, which form a part hereof, and which show by way of illustration specific embodiments and features of the invention. It is to be understood that other embodiments may be utilized, and structural or logical changes may be made without departing from the scope of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.Z.-24P008DETAILED DESCRIPTION

[0048] As used in this description (above and below) and claims, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0049] As used in this description (above and below) and claims, the use of the expression “n = 1 to 20” means that “n” is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0050] As used in this description (above and below) and claims, the use of the word "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or".

[0051] As used in this description (above and below) and claims, the used word “about” before a numerical value indicates a range of numerical values encompassing, i.e. including, a deviation from the indicated numerical value by ±5%.

[0052] As used in this description (above and below) and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), "containing" (and any form of containing, such as "contains" and "contain") or “encompassing” (and any form of encompassing, such as "encompass" and " encompasses") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0053] As used in this description (above and below) and claims, i.e. herein, the terms "fluorescence", "fluorescent", “fluorescence measurement”, “fluorescent dye”, “fluorescent particle”, “fluorescent ion”, “fluorescent probe”, “fluorescent indicator” and any related thereto term is to be understood as comprising an optical property or its detection, e.g., an excitation wavelength, an emission wavelength, a fluorescence intensity, a fluorescence quantum yield, a fluorescence lifetime or decay, a fluorescence quenching or bleaching and / or a ratio of any of their values and its(their) detection.Z.-24P008

[0054] As used in this description (above and below) and claims, i.e. herein, the terms "Zn2+", "Zn(II)", “Zn11”, “Zn-ion” and any related thereto term is to be understood as comprising a doubly positively charged zinc ion.

[0055] Against the above background according to an embodiment a method is suggested for an on-site detection of a fecal pigment, in particular of a metabolite or a degradation product of a hemoglobin, in particular for the detection of urobilin, and / or stercobilin, at trace levels in a water sample, the method comprising:(i) providing a fibrous substrate functionalized with a zinc salt and a silane, in particular a zinc salt and silane functionalized glass fiber substrate;(ii) wetting a detection region of the zinc salt functionalized fibrous substrate with the water sample;(iii) exposing the wetted detection region to an excitation light, the excitation light typically comprising a wavelength in a range of about 450 - 490 nm, particularly of about 475 nm, a full width half maximum of the excitation light typically being at most about 25 nm;(iv) detecting a fluorescence light emitted in response to exposing the detection region to the excitation light; and at least one of:(v) detecting, based on the fluorescence light, a presence of the fecal pigment in the water sample; and(vi) correlating an intensity of the detected fluorescence light with a calibration data set for determining a concentration of the fecal pigment in the water sample.

[0056] The advantage of the method is that it enables simple and rapid detection of fecal contamination in the water sample. Thus, the lengthy and expensive incubation of the sample for a microbiological test can be omitted as soon as no fecal contamination is detected. On the other hand, the method makes it possible to check whether a particular water needs to be treated before being discharged into natural surface waters.

[0057] According to an embodiment the above processes (iii) to (v) are performed after a wetting time of typically about 10 to 20 sec has allowed for wetting of the entire detectionZ.-24P008 region by the deposited sample droplet. Typically, the wetting time is about a minute, within a time interval of about 20 s to 50 s after performing process (ii), more particular within a time interval of about 25 s to 45 s after performing process (ii), and even more particular within a time interval of about 30 s to 40 s after performing process (ii).

[0058] Advantageously, such short time is sufficient for a fluorescent complex to be formed between the zinc provided by the substrate and the fecal pigment. As the complex is amphiphilic in nature it is attracted / held at the detection region and can thus be focused on the fibrous substrate of the test strip.

[0059] According to an embodiment the zinc salt used during preparation of the test strip is a Zn(II)-salt, in particular a salt selected from the list consisting of: ZnCl2, Zn(NO3)2, Zn(CH3COO)2, ZnSO4, Zn(C104)2and Zn(BF4)2.

[0060] Advantageously, these zinc salts are generally recognized as ‘safe’ and not considered hazardous. These salts are also used in food as dietary supplements and as nutrients.

[0061] According to an embodiment the silane the test strip is treated with is selected from the list consisting of: 3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane (BHEAPTES), triethoxy(3-cyanobutyl)silane (TE3CBS), triethoxy(3-cyanopropyl)silane (TE3CPS), triethoxy(2-cyanoethyl)silane (TE2CES), triethoxy(acetoxypropyl)silane (TEAPS), triethoxy(acetoxyethyl)silane (TEAS), triethoxy(acetoxymethyl)silane (TEAMS), trimethoxy(benzoyloxypropyl)silane (TMBOPS), trimethoxy[(2-carbomethoxy)ethyl]silane (TECMES), trimethoxy(3-methoxypropyl)silane (TM3MPS), triethoxy(tetrahydrofurylpropyl)silane (TETHFPS),3 [2-(3 -tri ethoxy silylpropoxy)ethoxy] sulfonate (TEOSPES), triethoxy(hydroxymethyl)silane (TEHMS), trimethoxy(N-[hydroxyethyl]-N-methylaminopropyl)silane (TMHEMAPS), trimethoxy(3-acetamidopropyl)silane (TMAAPS), trimethoxy(N-(acetylglycyl)-3- aminopropyl)silane (TMAGAPS), triethoxy(4-hydroxybutyramido)propyl)silane (TEHBAPS), triethoxy((gluconamido)propyl)silane (TEGAPS), trimethoxy(3-[2-pyridylethyl]thiopropyl)silane (TM2PETPS), trimethoxy(3-[4- pyridylethyl]thiopropyl)silane (TM4PETPS), trimethoxy(2-[4-pyridylethyl])silane (TM4PES), trimethoxy(2-[2-pyridylethyl])silane (TM2PES), trimethoxy(N-[4,5- dihydroimidazolyl]propyl)silane (TMDHIES), trimethoxy {(N-3 [amino(propylenoxy)]aminopropyl)silane (TMAPPAPS), trimethoxy(3- [methoxy(polyethylenoxy)6.9]propyl)silane (TMMPEOPS), trimethoxy(methoxytriethylenoxypropyl)silane (TMMTEOPS).Z.-24P008

[0062] Optionally, the amphiphilic silane is deposited on a hydrophobic fibrous substrate, typically comprising a glass fiber substrate functionalized with a hydrophobic silane selected from the list consisting of a trimethoxy(octyl)silane (TMOS), trimethoxy(methyl)silane (TMMS), triethoxy(methyl)silane (TEMS), trimethoxy(propyl)silane (TMPS), triethoxy(decyl)silane (TEDS), ethynyltrimethylsilane (ETMS), methoxytrimethylsilane (MTMS), any linear alkyltrialkoxysilane with alkyl chains of a length from 1 to 9 carbon atoms; any branched alkyltrialkoxysilane having a carbon chain length from 2 through 9; and linear dialkyldialkoxysilanes with alkyl chains of a length from 1 to 7 carbon atoms.

[0063] Advantageously, the residues of indicated silanes, optionally together with the functional groups of the substrate, are selected to provide at the detection region an amphiphilicity which allows to attract the complex formed by the fecal pigment and the zinc salt. The detection spot helps to limit, fix or confine the diameter of the drop-casted water sample potentially contaminated with UB / feces. The outer hydrophobic surface is not allowing the uncontrolled spreading out of the drop casted water sample of the detection region. Layering the amphiphilic BHEAPTES over the hydrophobic surface of TMOS inhibits the adsorption / diffusion of analyte across the test strip's thickness, allowing to capture the fluorescence signal from the top surface (Fig 20). Hence a signal gain can be attained while minimizing scattering.

[0064] According to an embodiment the suggested zinc chloride functionalized fibrous substrate (2) is provided as a test strip or stick.

[0065] Advantageously, the test strip is planar and can easily be placed in the measurement chamber of the proposed device.

[0066] According to an embodiment the fluorescence light is a green fluorescence light, wherein the fluorescence light is detected at an expected fluorescence peak wavelength of the formed zinc-complex of the fecal pigment, wherein the expected fluorescence peak wavelength is typically in a range from about 510 nm to about 525 nm.

[0067] Advantageously the sensor chip of the suggested mobile device used for performing the test is sufficiently sensitive towards a green light in the indicated wavelength range.

[0068] According to an embodiment wetting the detection region (3) comprises a dropwise application of a predefined amount of the water sample, typically of 5-20 pl, onto the zinc chloride functionalized fibrous substrate.Z.-24P008

[0069] Advantageously, small volumes of about 10 pl can reproducibly applied using a micro-pipette even by unlearned personnel.

[0070] According to an embodiment an amount of the zinc salt in the detection region and the amount of the water sample used for wetting is chosen such that a Zn-ion concentration in the wetted detection region is in a range from about 10 mM to about 30 mM, more typically in a range from about 10 mM to about 30 mM, such as about 30 mM.

[0071] Advantageously the optimum fluorescence signal could be obtained in this ZnCl2concentration. Higher Zn-ion concentration may prevent the interaction of UB-Zn(II) with silane surfaces.

[0072] According to an embodiment the above described method is performed using a mobile computing device which comprises a camera, a mass storage, a computing unit connected with the mass storage, a display, and an electrical energy storage. The mobile telecommunication device may preferably be selected from a smartphone. The method comprises at least one of, typically all of:- electrical and mechanical connecting the mobile computing device with a measurement chamber comprising a condenser lens, an emission filter, an excitation filter, at least one convex lens and a light source to be energized from the mobile computing device, the light source typically being implemented as an LED, in particular an LED, emitting a light within a range of about 350 to 600 nm, particularly in a range from 450 to 500 nm, preferably emitting at 475 nm;- arranging the mobile computing device on a housing of the measurement chamber;- connecting the mobile computing device and the measurement chamber via an electronic component connection, in particular via an USB connection;- arranging, after wetting the detection region of the zinc salt functionalized fibrous substrate with the water sample, the wetted zinc chloride functionalized fibrous substrate in the measurement chamber at a defined distance to the camera of the mobile computing device;Z.-24P008- energizing the light source from the mobile computing device to expose the detection region, via the excitation filter and the at least one convex lens, to the excitation light;- detecting with the camera, via the condenser lens and the emission filter, the fluorescence light;- performing process (v) by the computing unit; and- indicating the presence of the fecal pigment and / or the concentration of the fecal pigment in the water sample on the display.

[0073] Advantageously described mobile telecommunication or mobile computing devices are widely distributed and use more or less standardized software. The use of a corresponding software or an installation of a corresponding application (vulgo - app), i.e., a software which is configured to allow - together with a suitable measurement chamber and light source, fluorescence measurements in the indicated wavelength range and to perform required calculations / estimations, can easily be installed and run, even by unexperienced users.

[0074] According to an embodiment the concentration of the fecal pigment in the water sample which can reproducibly be detected using the disclosed method is in a range of nanomolar or even picomolar concentrations. For example, a LOD of 1.08 nM was achieved.

[0075] Advantageously, these concentrations are orders of magnitude lower than anything previously detected. Thus, the sensitivity of the proposed method allows to reach particularly high sensitivity.

[0076] According to an embodiment the calibration data set is a predetermined data set or the calibration data set is determined based on a fluorescence measurement performed at a calibration region of the fibrous substrate functionalized with the zinc salt and the silane and comprising a known concentration of an organic dye. Advantageously a known quantity of a fluorescent dye deposited on a spot the size of the detection region, in particular a BODIPY dye such as R4BDP-H can be used for a quantification of the fluorescence signal caused by the water sample, i.e. the fecal pigment. Alternatively, a known quantity of a zinc salt - complex of the metabolite or of the degradation product of hemoglobin can be used for calibration. The measurements can be calibrated beforehand or in parallel with a current sample applied to the test strip. Thus, according to embodiments, the test strip comprises oneZ.-24P008 or more calibration spots of known concentration or corresponding to defined concentrations of the fecal pigment(s) sought for.

[0077] Advantageously, a measurement result obtained using a calibrated set-up can indicate the requirement to dilute the sample under investigation in order to verify a concentration or a presence of a certain fecal pigment in the sample.

[0078] According to an embodiment a test strip for on-site determination of a trace amount of a fecal pigment in a water sample is suggested using the method described according to any of the preceding embodiments.

[0079] According to an embodiment of a test strip for on-site determination of a trace amount of a fecal pigment, in particular a metabolite and / or a degradation product of hemoglobin in a water sample, particularly of urobilin and stercobilin in a water sample according to any of the preceding claims, the test strip comprises: an amphiphilic detection region comprising a zinc salt and an amphiphilic silane functionalized fibrous material, in particular the zinc salt and the amphiphilic silane functionalized glass fiber material, the amphiphilic silane functionalized fibrous material typically being provided by a fibrous substrate.

[0080] The suggested test strip may comprise: a hydrophobic surface surrounding an amphiphilic detection region, wherein the amphiphilic in nature detection region comprises a fibrous material, in particular a glass fiber substrate which is functionalized with a zinc salt and made amphiphilic by a silane functionalization. The amphiphilic detection spot in a hydrophobic strip could help in maintain or fix the diameter of sample drop cast. As a consequence, more uniform results could be obtained. The layer-by-layer coating of amphiphilic spot on hydrophobic strip further help to prevent diffusion of sample analyte and the fluorescence signal can be captured from its surface. Similar hydrophobic polymers or amphiphilic substrate can be used for strip design.

[0081] Advantageously, such test strip allows performing a test for fecal pigments according to the embodiments described above.

[0082] According to an embodiment the test strip comprises a calibration region comprising a known concentration of a calibration substance. The calibration substance and its quantity on the calibration region is selected such as to comprise a fluorescence having an intensity at the same excitation wavelength (e.g. 475 nm) which allows to conclude about the unknown concentration of the sought for fecal pigment in the detection region. TheZ.-24P008 calibration substance is selected from a fluorescent dye, e.g. a BODIPY dye, such as R4-BDP- H according to the formula:

[0083] The calibrating substance can also be selected from a zinc-complex of the fecal pigment searched for, e.g., an urobilin-zinc salt - complex, wherein the zinc salt is selected from the following list: ZnCl2, Zn(NO3)2, Zn(CH3COO)2, ZnSO4, Zn(C104)2and Zn(BF4)2, wherein the zinc salt is non-chemically bound to the glass fiber substrate, and wherein the silane is selected from the list consisting of: 3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane (BHEAPTES), TE3CBS, TE3CPS, TE2CES, TEAPS, TEAS, TEAMS, TMBOPS, TECMES, TM3MPS, TETHFPS, TEOSPS, TEHMS, TMHEMAPS, BHEAPTES, TMAAPS, TMAGAPS, TEHBAPS, TEGAPS, TM2PES, TM2PETPS, TM4PETPS, TM4PES, TMDHIES, TMAPPAPS, TMMPEOPS, and TMMTEOPS. Further, the amphiphilic silane may be deposited on a hydrophobic fibrous substrate, typically comprising a glass fiber substrate functionalized with a hydrophobic silane selected from the list consisting of: a trimethoxy(octyl)silane (TMOS), trimethoxy(methyl)silane (TMMS), triethoxy(methyl)silane (TEMS), trimethoxy(propyl)silane (TMPS), triethoxy(decyl)silane (TEDS), ethynyltrimethylsilane (ETMS), methoxytrimethyl silane (MTMS), any linear alkyltrialkoxy silane with alkyl chains of a length from 1 to 9 carbon atoms; any branched alkyltrialkoxysilane having a carbon chain length from 2 through 9; and linear dialkyldialkoxysilanes with alkyl chains of a length from 1 to 7 carbon atoms.

[0084] Advantageously, the calibration region which can also comprise some spots of different quantities but having the same or a similar size (i.e. diameter in case of a circular spot) allows for safe determination of a concentration of the fecal pigment, since the quantity of applied sample is known.

[0085] According to an embodiment a method for manufacturing a test strip is suggested, the method comprising: inserting, for a predetermine time of typically at least several hours (e.g., 24 hours), a fibrous substrate, in particular a glass fiber substrate, into a solution comprising water, an alcohol, in particular ethanol, tetraethyl orthosilicate (TEOS) and a hydrophobic silane such as a trimethoxy(octyl)silane (TMOS),Z.-24P008 trimethoxy(methyl)silane (TMMS), triethoxy(methyl)silane (TEMS), trimethoxy(propyl)silane (TMPS), triethoxy(decyl)silane (TEDS), ethynyltrimethylsilane (ETMS), methoxytrimethylsilane (MTMS), any linear alkyltrialkoxysilane with alkyl chains of a length from 1 to 9 carbon atoms; any branched alkyltrialkoxysilane having a carbon chain length from 2 through 9; and linear dialkyldialkoxysilanes with alkyl chains of a length from 1 to 7 carbon atoms;- washing the fibrous substrate with an alcohol, preferably ethanol; drying the washed fibrous substrate, preferably under a reduced pressure and / or above room temperature; drop casting an amphiphilic silane onto the dried fibrous substrate locally in order to form the detection region, the silane being selected from the list consisting of:3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane (BHEAPTES), TE3CBS, TE3CPS, TE2CES, TEAPS, TEAS, TEAMS, TMBOPS, TECMES, TM3MPS, TETHFPS, TEOSPS, TEHMS, TMHEMAPS, BHEAPTES, TMAAPS, TMAGAPS, TEHBAPS, TEGAPS, TM2PES, TM2PETPS, TM4PETPS, TM4PES, TMDHIES, TMAPPAPS, TMMPEOPS, and TMMTEOPS; impregnating the dried fibrous substrate (2) with an aqueous zinc salt solution; and drying the impregnated fibrous substrate.

[0086] The detection area of the test strip is formed by selecting a small spot on the substrate surface and applying the amphiphilic silane drop by drop to the same spot, interrupted by drying. This dropwise application corresponds approximately to the application of a sample in thin-layer chromatography. The advantage of this method is that it is easy to carry out and does not require any expensive chemicals.

[0087] According to an embodiment the zinc salt in the aqueous zinc salt solution used for impregnating is a Znn-salt. Cd11and Hgnsalts are not considered due to the (i) toxicity and (ii) the fluorescence intensity was quenched after complexation (Prakash & Mishra, Anal Methods 2021, 13, 5573-5588). In particular it is selected from the list consisting of: ZnCl2, Zn(NO3)2, Zn(CH3COO)2, ZnSO4, Zn(C104)2and Zn(BF4)2, wherein the zinc salt is non-Z.-24P008 chemically bound to the glass fiber substrate (2), and / or wherein the amphiphilic silane is selected from the list consisting of 3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane (BHEAPTES), TE3CBS, TE3CPS, TE2CES, TEAPS, TEAS, TEAMS, TMBOPS, TECMES, TM3MPS, TETHFPS, TEOSPS, TEHMS, TMHEMAPS, TMAAPS, TMAGAPS, TEHBAPS, TEGAPS, TM2PES, TM2PETPS, TM4PETPS, TM4PES, TMDHIES, TMAPPAPS, TMMPEOPS, and TMMTEOPS.

[0088] Optionally, the amphiphilic silane is deposited on a hydrophobic fibrous substrate, typically comprising a glass fiber substrate functionalized with a hydrophobic silane selected from the list consisting of a trimethoxy(octyl)silane (TMOS), trimethoxy(methyl)silane (TMMS), triethoxy(methyl)silane (TEMS), trimethoxy(propyl)silane (TMPS), triethoxy(decyl)silane (TEDS), ethynyltrimethylsilane (ETMS), methoxytrimethylsilane (MTMS), any linear alkyltrialkoxysilane with alkyl chains of a length from 1 to 9 carbon atoms; any branched alkyltrialkoxysilane having a carbon chain length from 2 through 9; and linear dialkyldialkoxysilanes with alkyl chains of a length from 1 to 7 carbon atoms.

[0089] For screening of different silanes, Zn(NO3)2of 20 mM were used for complexation with UB (1 pM). UB- Zn(NO3)2complexes show maximum fluorescence intensity in water than other Zn11complexes (Fig. 1).

[0090] Advantageously, the indicated substances effectively modify the fibrous substrate to impart the required amphiphilic properties to the substrate surface.

[0091] Each embodiment described above may be combined with any other embodiment or embodiments disclosed herein unless clearly indicated to the contrary.

[0092] Further, several aspects of the disclosed embodiments will be discussed.

[0093] As disclosed by the examples given below and the fluorescence intensity data provided, the proposed test strip and method of its use are applicable for on-site detection of fecal pigments in water.

[0094] The suggested photoluminescence detection methods can be an easy-to-operate, portable, cost-effective and hence, a user-friendly alternative for the non-specialist user when compared with other conventional methods of FIP detection such as laboratory -based high- performance liquid chromatography and mass spectrometry. In addition, photoluminescence is well-recognized by its low detection limits (high sensitivity) and real-time responses.Z.-24P008DETAILED DESCRIPTION OF THE DRAWINGS

[0095] In particular, Fig. 1 shows that glass fiber strips co-coated with BHEAPTES yield the highest fluorescence signal with an emission maximum Xem= 518 nm. In contrast, the TMMS strips show an emission maximum at 500 nm. Based on the spectroscopic properties of urobilin and its Zn11complexes, the different fluorescence features suggest that whereas the complex is the major species on BHEAPTES strips, unbound urobilin is dominating on TMMS strips. The broad emission spectrum of the TMPS strips indicate the presence of both, free urobilin and UB-Zn11complexes. Fig. 1 further shows that the response pattern obtained with a conventional fluorometer and the miniaturized setup agree well, i.e., the relative luminance ( / .) of urobilin on BHEAPTES strips is the highest. Importantly, the silane functionalized glass fiber strips showed negligible auto- and background fluorescence when the assay was conducted in the absence of UB, contributing to robustness and reliability of the assay and facilitating its quantitation (Figs. IB, 14).

[0096] Thus, the amphiphilic silane for the detection region may be selected from the following polar amphiphilic silanes:Z.-24P008triethoxy(3-cyanobutyl)silane triethoxy(3-cyanopropyl)silane triethoxy(2-cyanoethyl)silaneTE3CBS TE3CPS TE2CEStriethoxy(acetoxypropyl)silane triethoxy(acetoxyethyl)silane triethoxy(acetoxymethyl)silaneTEAPS TEAES TEAMStrimethoxy(benzoyloxypropyl)silane triethoxy[(2- trimethoxy(3-methoxypropyl)silane carbomethoxyjethyllsilaneTMBOPS TM3MPS TECMESr e oxy( y roxymethyl)silane triethoxy(tetrahydrofurfuryl propyljsilane 3-[2-(3-triethoxysilylpropoxy)ethoxy]sulfolaneTEHMSTETHFPS TEOSPEStrimethoxy(N-[hydroxyethyl]-N- 3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane methylaminopropyljsilaneTMHEMAPS BHEAPTESZ.-24P008trimethoxy(3- trimethoxy(N-(acetylglycyl)-3- acetamidopropyljsilane aminopropyljsilaneTMAAPS TMAGAPStriethoxy(gluconamido)propyl)silane triethoxy(4- hydroxybutyramido)propyl)silaneTEGAPSTEHBAPStrimethoxy(3-[4- trimethoxy(2-[4- trimethoxy(3-[2- pyridylethyl]thiopropyl)silane pyridylethyl])silane pyridylethyl]thiopropyl)silaneTM4PETPS TM4PESTM2PETPStrimethoxy(2-[2- trimethoxy(N-[4,5- pyridylethyl])silane dihydroimidazolyl]propyl) silaneTM2PES TMDHIESTMMTEOPSZ.-24P008

[0098] For the hydrophobization of hydrophilic substrates such as glass fiber paper, cellulose, mineral and ceramic fibers and hydrophilic polymers, the following hydrophobic silanes may be used:linear alkyltrialkoxysilanes branched alkyltrialkoxysilanes linear dialkyldialkoxysilanesmethoxytrimethylsilane ethynyltrimethylsilaneMTMS ETMS

[0099] The arrows on branched alkyltriethoxysilanes above indicated that many different branching options are possible. In fact, many differently branched alkyltriethoxysilanes as commercially available can be used.

[0100] As can be seen in Fig. 2, the strongest fluorescence enhancement was observed for ZnCl2. Importantly, UB-Zn complexes showed weak fluorescence intensity if measured in aqueous solution due to the low solubility in the polar environment. Hence, the amphiphilic surface of the BHEAPTES modified substrate supposedly provides the best matrix to anchor UB-Zn complexes, the UB-Zn11is known to be singly negatively charged, producing maximum fluorescence enhancement. Interestingly, the tetrafluoroborate salt does not seem to promote complexation under the present conditions, the emission maximum remaining at 500 nm as for free urobilin (cf. Fig. 2A). All the other counterions support complex formation, yet the enhancement is dramatically lower for acetate and still distinctly less for sulfate, perchlorate and nitrate. The observed response pattern suggests that the coordination ability and the geometry of the counterion are more important for this tuning mode than the hydrophobicity. BF4, C1O4and AcO are all well soluble in hydrophobic media, yet an outer F atom of BF4coordinates much less to Zn11than an outer O atom of C1O4, and for AcO the preferred coordination mode through the Y-shaped carboxylate group is most likely sterically more demanding. Of the ‘hard’ counterions, in terms of Pearson’s concept of hard and soft acids and bases [R. G. Pearson, J Am Chem Soc 1963, 85, 3533-3539], chloride is the smallest one and of spherical shape so that it is most likely best fulfilling the coordinativeZ.-24P008 need of octahedral Zn11, while not interfering with urobilin binding. Again, like above, the results obtained with the fluorometer and the miniaturized setup agree well.

[0101] Fig. 3 illustrates the functional relationship of UB concentration with relative luminance calculated from digital image using the Eqn 1. Fig 3A indicates the average of three independent experiments with variation in UB concentration in Milli-Q water. Image analysis of the strips was referenced against Image . / by extracting the standard relative linear luminance of the strips, which is directly proportional to the measured fluorescence intensity. From the calibration plot, an LOD of 1.08 nM was determined from linear regression plot (insert)).

[0102] Fig. 4 illustrates measurement results obtained with real water samples which can contain other fluorescent material from dissolved organic matter (DOM) or competing ionic species. Accordingly, water samples were taken from a canal near Berlin [Teltow Canal, Germany; slow-flowing water, neighboring residential and industrial areas and commercial shipping; sampling in 07 / 2024] and spiked with 50 nM UB. A drop of this sample was deposited on a BHEAPTES strip impregnated with ZnCl2and left to dry for 30 s before 3- dimensional fluorescence excitation-emission matrices were recorded with a fluorometer to assess the spectral signature and potential interference of fluorescent DOM. A comparison of the spiked vs. a native sample in Fig. 5B shows that the signal generated by urobilin is approx. 3.6 -times higher than the background of the sample.

[0103] Fig. 5 shows measurement data obtained for a natural water sample spiked with UB. The sensitivity of the camera as in all images shown was set to ISO 500. Fig. 5A illustrates impressively reveals the importance of the silane functionalization, because the signal of the spiked sample is 10-times higher on a BHEAPTES strip than on an uncoated strip. Similar results obtained with the proposed smartphone-based setup, i.e., negligible interference by sample autofluorescence and a higher relative luminance for the silane-coated strip illustrates Fig. 5B.

[0104] Fig. 6 confirms that no background fluorescence / autofluorescence is present in blank glass fiber strips and in glass fiber strips coated only with different silanes under excitation of 470 nm. The images were captured with the smartphone integrated miniaturized optical setup

[0105] Fig. 7 shows the fluorescence excitation spectra of UB-Zn11complexes generated by different Zn11salts on BHEAPTES strips as measured with a fluorometer).Z.-24P008

[0106] Fig. 8 shows the influence of the variation of the ZnCl2concentration used with the TMOS / BHEAPTES-modified glass fiber substrate on the fluorescence of drop casted 10 pl samples containing 1 pM urobilin in Milli-Q water. The ZnCl2concentration was varied from 10 mM to 30 mM. As can be seen in both Fig. 8A and 8B, the relative luminance L increases linearly with the concentration of ZnCl2. In the graph in Fig. 8B, the luminance L = 13.64 of the blank value was less than the luminance L actually measured for UB-Zn complex.

[0107] Relative luminance ( / .) is calculated by L = 0.2126 x Rlin+ 0.7152 x G[jn+ 0.0722 x Biin(Sugawara et al., IEEE Signal Process Mag 2014, 31, 170-174: Eqn 1). Image analysis of the strips was referenced against Image J by extracting the standard linear luminance of the strips, which is directly proportional to the measured fluorescence intensity.

[0108] Fig. 9 shows digital images of fluorescing detection zones after application of two different urobilin concentrations (30 nM and 120 nM) in a natural water sample taken from a river of Vietnam (Han River, Da Nang) in comparison to a reference spot (BODIPY dye). The reference spots were generated by applying 5 pl of a reference dye having a concentration of 40 nM. The reference spot of the fluorescent dye is located close to the detection region and can be thus used for quantification of the analyte, if calibrated accordingly. As evident, its intensity (L) remains more or less constant, while the luminescence intensity of the UB-doped 5 pl sample changes with the concentration of UB. In the embodiment shown, a BODIPY dye, in particular (CH3)4BDP-H, was used. Any other R4BDP-H with R = short alkyl group could also be used. Both the detection points were designed in similar methodology (TMOS- BHEAPTES-ZnCl2), as described earlier.

[0109] Fig. 10 illustrates exemplary embodiments of the suggested method of test strip manufacturing. Particularly, Fig. 10A shows the two principal steps: a) functionalizing at least a detection area of a fibrous substrate, in particular a glass fiber substrate (2), with an amphiphilic silane such as BHEAPTES; and b) supplementing the detection area of the fibrous substrate (2) with a zinc salt to form a detection region of the test strip for on-site detection at trace levels in a water sample of a fecal pigment, in particular of a metabolite or degradation product of a hemoglobin, in particular of urobilin, and / or stercobilin.

[0110] Fig. 10B illustrates a preferred embodiment of the manufacture method. In particular, step al) designates inserting, for a predetermined time of typically at least severalZ.-24P008 hours, a fibrous substrate, in particular a glass fiber substrate (2), into a solution comprising water, an alcohol, in particular ethanol, tetraethyl orthosilicate (TEOS) and a silane such as trimethoxy(octyl)silane (TMOS). This step is followed by steps a2) and a3) (not shown).

[0111] Step a2) comprises washing the fibrous substrate (2) with an alcohol, and step a3) comprises drying the washed fibrous substrate (2) under reduced pressure. Subsequently, on the washed and dried substrate (2) 10 pl of BHEAPTES are drop cast (step a4), e.g. with a pipet tool, e.g. a piezoelectric or acoustophoretic drop on demand device.

[0112] Afterwards, the dried spot is modified through impregnation by dropwise addition of ZnCl2solution (step bl). The dried (step b2) substrate strip comprising the detection region is ready for use. The strip can be stored in a dry state to be used for on-site detection of the fecal pigment sought for. For this purpose, the detection region is wetted with the water sample (step (ii)). As can be seen in the last picture, the substrate surrounding the amphiphilic detection region is hydrophobic.

[0113] In the shown exemplary embodiment, part “aO” is a neat glass fiber substrate as commercially available (cut to a strip of 0.5 cm x 2 cm). After incubation in TMOS for 24 h at room temperature, the substrate strip is functionalized through drop casting with 10 pl BHEAPTES for 1 min at room temperature (step a4). Afterwards the strip is immersed into an aqueous ZnCl2solution of a defined concentration, e.g., for 1 minute at room temperature. The thus impregnated strip is dried, e.g. at 50 °C for 10 minutes, optionally under reduced pressure. The thus dried strip if wetted with an aqueous sample proves to be mainly hydrophobic but has, at the position where BHEAPTES and ZnCl2were applied on it (by immersion in ZnC12 solution for 1 min), an amphiphilic region (indicated by the circular dotted line). This amphiphilic region can be used as detection region

[0114] Fig. 11 depicts three ‘architectural’ routes suitable to produce test strips according to the invention. Depending on the initial polarity of the neat fibrous substrate used, different coating procedures (a*), (b*) and (c*) are suitable to generate a test strip with the described detection region. The most sensitive strips thereof have been obtained following the steps of route (a*).

[0115] Fig. 12 illustrates schematically the sequence of immersion / spotting with corresponding substances according to the routes depicted in Fig. 11. Therein, Fig. 12 (a) shows the hybrid strip obtained according to route (a*) of Fig. 11. Accordingly, Figs. 12 (b) and 12(c) show the hybrid strips as obtained according to routes (b*) and (c*) of Fig. 11. The strip according to route (a*) was the most suitable, i.e. most sensitive one.Z.-24P008

[0116] As evident from Figs. 11 and 12, the preparation of the test strips comprising a suitable detection region can be started from a fibrous substrate of any polarity. Advantageously, the substrates are fibrous for the surface enlargement in comparison to a solid substrate. As shown in Fig. 12(a) the polar glass fiber substrate is immersed at first in TMOS. The strong binding of the silanol groups and the octyl chain of TMOS modifies the substrate. On such TMOS- modified substrate BHEAPTES and ZnCl2are locally spotted and thus, the detection region is formed. As illustrated by Fig. 12(b), alternatively, a fibrous hydrophilic substrate can be modified directly with BHEAPTES. Such can be reached by immersion into BHEAPTES, i.e., impregnating the substrate with BHEAPTES. However, in order to define the detection region, a hydrophobic barrier such as a wax barrier has to be printed or applied to the substrate. Fig. 12(c), depicts the production of a test strip starting from a fibrous hydrophobic substrate that is locally modified (functionalized) with BHEAPTES. Afterwards, the chosen Zn(II) salt is applied.

[0117] Fig. 13 illustrates the implementation of the smartphone-based method proposed here for the detection of water contamination by fecal pigments. The test strip, which is wetted with a few drops of the water sample to be analyzed (typical sample volume 10 pl), is inserted with a carrier into a lateral opening 3a of a measuring chamber 3, for example an additively manufactured one. The light emitted by an LED 3e is directed via a bandpass filter 3d and / or an edge filter 3d onto the detection region of the test strip 2 via a converging lens 3b. Emitted fluorescent light is directed via a condenser lens 3c and an emission filter 3g onto the camera 4.1 of the mobile telecommunication device 4 , 4.2, 4.3, 4.5 used. The camera 4.1 is used for detecting via the condenser lens 3c and the emission filter 3g a fluorescence light emitted by the fecal pigment (e.g. urobilin or stercobilin) after a reaction thereof with the zinc chloride of the functionalized glass fiber substrate 2. The computing unit 4.3 of the mobile computing device (mobile telecommunication device) is used for determining a presence and / or a concentration of the corresponding analyte by correlating an intensity of the fluorescence light with a calibration data set stored by the mass storage of the smartphone 4 and the presence and / or concentration of the corresponding analyte in the water sample is optionally indicated on the display / screen of the mobile computing unit.

[0118] Fig. 14 shows measurement results obtained on a benchtop fluorometer with a Milli-Q water spiked with LTB (1 pM). Fig. 14 A represents emission spectra with variation of time (min) for urobilin 1 pM in presence ofGlass fiber / TMOS / BHEAPTES / ZnCl2test strip and Fig. 14B shows fluorescence intensity response with time at 516 nm on Glass fiber / TMOS / BHEAPTES / ZnCl2test strip.Z.-24P008

[0119] Fig. 15 shows the time dependent change in fluorescence response measured with the smartphone-integrated optical setup of drop casted 10 pL of Milli-Q water spiked with (1 pM) urobilin on Glass fiber / TMOS / BHEAPTES / ZnCl2functionalized glass fiber substrate. In particular, Fig. 15A represents the relative luminance with variation of time (min) for urobilin 1 pM in presence of Glass fiber / TMOS / BHEAPTES / ZnCl2test strip by smartphone digital images and Fig. 15B shows the digital images with time for urobilin (1 pM) in presence of Glass fiber / TMOS / BHEAPTES / ZnCl2functionalized test strip.

[0120] Fig. 16 shows field emission scanning electron microscopy (FESEM) images of the test strip. Fig. 16A indicates the FESEM image of glass fiber strip and Fig. 16B indicates the modification of the glass fibers by functionalization with TMOS.

[0121] Fig. 17 shows the morphology of test strip using FESEM images. Fig. 17A indicates the FESEM image of glass fiber / BHEAPTES strip and Fig. 17B indicates the modification of surface after functionalization of Glass fiber / TMOS / BHEAPTES / ZnCl2.

[0122] Fig. 18 shows a comparison of the elemental composition of Glass fiber / TMOS / BHEAPTES / ZnCl2and Glass fiber / BHEAPTES / ZnCl2test strip. As obvious, the TMOS coated glass fiber test strip shows an increase in C% and H% due to its additional N-octyltrimethoxy silane coating.

[0123] Fig. 19 illustrates the thermogravimetric analysis (TGA) of Glass fiber / TMOS / BHEAPTES / ZnCl2test strip and Glass fiber / BHEAPTES / ZnCl2test strip. As evident, the initial TMOS modification allows for Glass fiber / TMOS / BHEAPTES / ZnCl2test strip according to route (a*) a higher stability than for BHEAPTES / ZnCl2test strip.

[0124] Fig. 20 shows higher luminance value for Glass fiber / TMOS / BHEAPTES / ZnCl2test strip than Glass fiber / BHEAPTES / ZnCl2test strip. The 10 pl sample applied to the spot was 50 nM urobilin in Milli-Q water

[0125] Fig. 21 shows cuvette based measurement data obtained with a spectrometer of absorbance and fluorescence for complexes formed of urobilin (IpM) in Milli-Q water with different Zn11salts (each 20 mM).

[0126] Fig. 22 shows the fluorescence intensity enhancement for urobilin and corresponding urobilin-Zn complexes on Glass fiber / TMOS / BHEAPTES / ZnCl2. Fig. 22 A and 22B compare the enhancement factor obtained with a standard fluorescence spectrometer and a mobile smartphone-integrated optical setup. The enhancement was calculated afterZ.-24P008 background subtraction. In A, a blank spectrum, which is virtually identical to the x axis, was subtracted from both spectra before calculating the quotient. In B, the luminance L of the blank was subtracted from both luminance values of the samples before calculating the quotient

[0127] As can be seen, these results confirm the excellent sensitivity achieved with detection limits in the nano- to picomolar range for Milli-Q water spiked with UB, even for a natural water sample the sensitivity is in similar range. Thus, the proposed assay proved to be suitable for use under real conditions.Test strip preparation

[0128] Urobilin hydrochloride was purchased from Porphyrin Systems Hombrecher e. K. The zinc salts were purchased from Sigma-Aldrich (Zn(NO3)2, Zn(C104)2), Thermo Scientific (Zn(BF4)2), Chemsolute (ZnCl2) and Merck (ZnAc2, ZnSO4) and the silanes from Sigma- Aldrich (ethynyltrimethylsilane, triethoxy(methyl)silane), Fluorochem (triethoxy(decyl)silane, 3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane), ABCR (trimethoxy(octyl)silane, methoxytrimethylsilane, trimethoxy(methyl)silane), and Alfa Aesar (trimethoxy(propyl)silane). Tetraethyl-orthosilicate (TEOS) and ammonia were purchased from Merck. Glass fiber paper was acquired from GE Healthcare lifescience. Milli-Q ultrapure water was obtained from Merck Millipore.

[0129] Although we used borosilicate glass fiber substrates, other fibrous substrates such as fibers made of cellulose or another organic polymer, mineral fibers, ceramic fibers or fibers of other glasses can also be used as fibrous substrate. The skilled person can easily select a silane that is adapted to the surface chemistry of the respective fiber material.Advantageously, hydrolysable groups present directly on the Si atom of the respective silane form silanol groups (Si - OH) in an aqueous environment, which in turn can form covalent bonds with hydroxyl groups on the surface of the corresponding substrate.PRACTICAL EXAMPLES

[0130] Practical examples illustrated by the Figs. 1 - 22 demonstrate the feasibility of the chosen substrate modification and fluorescence properties of detection regions after wetting with water samples containing minute amounts of a typical fecal pigment according to some typical embodiments. The suggested method is suitable for on-site detection of practically important fecal pigments.Z.-24P008Example 1 glass fiber substrate

[0131] Glass Microfiber Retention Filter GF / A Whatman Glass Fiber Filter Paper Membrane 1820-025 / 150 (GE Healthcare lifescience; package 100 circles of diameter 25 mm / 150 mm, having the following technical parameters: Pore size: 1.6 pm; Filtration velocity: 62 Herzberg(s); Air flow velocity: 4.3 Gurley(s); DOP retention efficiency: 99.95%; Thickness: 260 pm; Weight: 55g / m2; Operating temperature: < 500 °C.Example 2 substrate modification

[0132] According to a practical example, a mixture of 3.4 mL Milli-Q water, 7.4 mL ethanol, 2.7 mL TEOS, 300 pL of silane (TMOS) and 180 pL NH3(32 %) was placed in a 20 mL vial containing 30 glass fiber strips of 8 * 2.5 cm2. After reaction for 24 h at room temperature with orbital stirring, the TMOS silane-glass fiber strips were washed with ethanol and dried under reduced pressure.

[0133] Another BHEAPTES silane solution was prepared without glass fiber strip and waited for 24 h to complete the reaction (same protocol). 10 pL of fresh BHEAPTES solution was drop casted on TMOS silane-glass fiber strips and dried out.Example 3 substrate modification

[0134] Glass fiber / TMOS / BHEAPTES strip was impregnated with ZnCl2of 30 mM solution and dried in an oven for 10 min at 50 °C.SUMMARY

[0135] In addition to the above, aspects of the present application can be worded as follows:1. The proposed invention comprises the design or methodology of a test strip-based fluorometric assay for rapid and simple urobilin detection at trace levels directly at a point- of-need for on-site water quality analysis (see Figure 1), including- functionalizing a test strip (e.g., glass fiber or cellulose) with a silane,Z.-24P008- impregnating the functionalized strip with zinc chloride,- dropping a water sample on the strip (or an area of a strip, whether confined by e.g. wax barriers or not)- measuring the fluorescence with a mobile device (e.g., smartphone, tablet, digital camera) after insertion into a 3D-printed holder and excitation with a miniaturized excitation source (e.g., LED via USB), preferably including also miniaturized optics (lenses, filters) for optimum signal acquisition.- analyzing the data onboard or after transfer to a computer.2. A test strip described in aspect 1, in which the sensing material comprises of a glass fiber strip the fibers of which are silane-functionalized on their surface to tune optimum hydrophobicity for FlP-Zn11complexes detection. The fibrous network presents a large specific surface area which stabilizes FP-Zn11complexes in a quasi-solid-state, formed after dropping an aqueous sample on the strip and subsequent air-drying (see Figure 2).3. A test strip described in aspect 1, in which the sensing material able to interact with the urobilin molecule is a glass fiber strip modified with certain silanes and zinc chloride.4. A test strip described in aspect 1, in which urobilin detection in the pico- to nanomolar concentration range is facilitated.5. A simple drop-&-detect assay in which the total testing time is less than 1 minute.Areas of application / Advantages

[0136] As already mentioned, the detection of fecal contamination is relevant in cases where either the time for a microbiological test involving a cultivation step is too long or too costly, or where a decision has to be made as to whether such a test is necessary (promising) at all.

[0137] The functionalization of fibrous materials suitable as test strips with silanes allows to tune their hydrophobicity, facilitating the interaction of chemical species (Znn-complexes of the fecal pigments) or supporting certain physico-chemical properties of said complexes. In this case, coating of the strips with silanes enhances the selective interaction of, e.g., urobilin with zinc chloride impregnated on the substrate surface and provides a less hydrophilicZ.-24P008 environment than neat water, enhancing the fluorescence of the corresponding Zn11complex. The impregnation step further concentrates the formed UB-Zn2+complexes directly on the surfaces of the substrate fibers. If specific features are wax-printed on the strip, devising specific detection zones, an additional spatial concentration leads to improved FIP detection.

[0138] Remarkably, the fluorescence signal detected by the suggested assay / setup is only based on urobilin molecules interacting with the impregnated metal ion on the test strip. No labeling or use of secondary binding agents or any harmful chemicals are required to produce or amplify a fluorescence signal in the pre-defined detection region of the substrate strip.

[0139] Further, the proposed 3D printed measurement chamber and optical setup considerably reduces background interference through daylight, straylight or scattering, especially when convex and / or condenser lenses and optical filters for excitation and / or emission light are used.

[0140] Furthermore, background fluorescence of dissolved organic matter (DOM) is minimized by dropping only a small volume of the investigated water sample onto the strip and letting it air-dry for several seconds before measurement, confining the target analyte fecal pigment, as shown for UB, onto the Zn2+-impregnated surface of the fibers yielding a strong, quasi-solid-state fluorescence while quenching most of the DOM fluorescence.

[0141] Thus, a test strip, an analyte-sensitive layer and a method for on-site detection of typical analytes indicative of the presence of fecal matter in a sample are proposed, i.e. a rapid on-site method with all that is involved in the detection of fecal contamination in a water sample is disclosed.Z.-24P008Abbreviations and reference signsTEOS tetraethyl orthosilicate TMMS trimethoxy(methyl)silane (Silane 1) TEMS triethoxy (methyl) silane (Silane 2) BHEAPTES 3(bis(2-hydroxyethyl)aminopropyltriethoxysilane (Silane 3) TMPS trimethoxy (propyl) silane (Silane 4) TMOS trimethoxy (octyl) silane (Silane 5) TEDS triethoxy (decyl) silane (Silane 6) ETMS ethynyltrimethylsilane (Silane 7) MTMS methoxytrimethylsilane (Silane 8) PEGS 2-(methoxy(polyethyleneoxy)propyl)trimethoxy silane TE3CBS triethoxy(3 -cyanobutyl)silane TE3CPS triethoxy (3 -cyanopropyl) silane TE2CES triethoxy(2-cyanoethyl)silane TEAPS triethoxy(acetoxypropyl)silane, TEAS triethoxy(acetoxyethyl)silane TEAMS triethoxy(acetoxymethyl)silane TMBOPS trimethoxy(benzoyloxypropyl)silane TECMES trimethoxy[(2-carbomethoxy)ethyl]silane TM3MPS trimethoxy(3 -methoxypropyl)silane TETHFPS triethoxy(tetrahydrofurylpropyl)silane TEOSPES 3 [2-(3 -triethoxysilylpropoxy)ethoxy] sulfonate TEHMS triethoxy(hydroxymethyl)silane TMHEMAPS trimethoxy (N - [hydroxy ethyl] -N -methylaminopropyl) silane TMAAPS trimethoxy(3-acetamidopropyl)silane TMAGAPS trimethoxy (N -(acetylglycyl) -3 -aminopropyl) silane TEHBAPS triethoxy(4-hydroxybutyramido)propyl)silane TEGAPS triethoxy((gluconamido)propyl)silane TM2PETPS trimethoxy(3-[2-pyridylethyl]thiopropyl)silane TM4PETPS trimethoxy(3-[4-pyridylethyl]thiopropyl)silane TM4PES trimethoxy(2-[4-pyridylethyl])silane TM2PES trimethoxy(2-[2-pyridylethyl])silane TMDHIES trimethoxy (N -[4,5 -dihydroimidazolyl] propyl) silane TMAPPAPS trimethoxy {(N-3[amino(propylenoxy)]aminopropyl)silane TMMPEOPS trimethoxy(3-[methoxy(polyethylenoxy)6.9]propyl)silane TMMTEOPS trimethoxy(methoxytriethylenoxypropyl)silane DOM dissolved organic matter FIB fecal indicator bacteriaFIP fecal indicator pigment SB stercobilin UB urobilin Zn(Ac)2 zinc acetate, (Zn(CH3COO)2) ZnCl2zinc chloride, (ZnCl2) Zn(NO3)2zinc nitrate Zn(C104)2zinc perchlorate Zn(BF4)2zinc tetrafluoroborate hydrate ZnSO4zinc sulfate 1 water sample 2 fibrous substrate, test strip 3 housing, measurement chamber 3a receiving chamber 3b convex lens 3c condenser lensZ.-24P0083d excitation filter3e light source, e.g. LED3g emission filter3h lid of measurement chamber3x permanent magnets4 mobile computing device, mobile telecommunication device, smartphone4. 1 camera4.2 mass storage4.3 computing unit4.4 display, screen4.5 energy storage, battery100 manufacturing method of test strip500 hand held portable system

[0142] The present invention has been explained with reference to various illustrative embodiments and examples. These embodiments and examples are not intended to restrict the scope of the invention, which is defined by the claims. As apparent to one skilled in the art, the embodiments can be implemented in various ways without departing from the scope of what is invented. Various features, aspects, and functions described in each of the embodiments can be combined with features, aspects, and functions of other embodiments.

Claims

Z.-24P008Claims1. A method (100) for on-site detection at trace levels in a water sample of a fecal pigment, in particular of a metabolite or degradation product of a hemoglobin, in particular of urobilin, and / or stercobilin, the method comprising:(i) providing a zinc salt and silane functionalized fibrous substrate (2), in particular a zinc salt and silane functionalized glass fiber substrate (2);(ii) wetting a detection region of the zinc salt functionalized fibrous substrate (2) with the water sample;(iii) exposing the detection region to an excitation light, the excitation light typically comprising a wavelength in a range of about 450 - 490 nm, particularly of about 475 nm, a full width half maximum of the excitation light typically being at most about 25 nm;(iv) detecting a fluorescence light emitted in response to exposing the detection region to the excitation light; and at least one of:(v) detecting, based on the fluorescence light, a presence of the fecal pigment in the water sample; and(vi) correlating an intensity of the detected fluorescence light with a calibration data set for determining a concentration of the fecal pigment in the water sample.

2. The method of claim 1, wherein the processes (iii) to (v) are performed after an wetting time of typically about 10 to 20 sec is at least reached, while the wetted detection region is not dried again, within a minute and / or within a time interval of about 20 s to 50 s after performing process (ii), more particular within a time interval of about 25 s to 45 s after performing process (ii), and even more particular within a time interval of about 30 s to 40 s after performing process (ii).

3. The method of any of the preceding claims, wherein the zinc salt is a Zn(II)-salt, in particular a salt selected from the following list: ZnCl2, Zn(NO3)2, Zn(CH3COO)2, ZnSO4, Zn(C104)2and Zn(BF4)2.Z.-24P0084. The method of any of the preceding claims, wherein the silane is an amphiphilic silane, typically selected from the list consisting of: 3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane (BHEAPTES), TE3CBS, TE3CPS, TE2CES, TEAPS, TEAS, TEAMS, TMBOPS, TECMES, TM3MPS, TETHFPS, TEOSPS, TEHMS, TMHEMAPS, TMAAPS, TMAGAPS, TEHBAPS, TEGAPS, TM2PES, TM2PETPS, TM4PETPS, TM4PES, TMDHIES, TMAPPAPS, TMMPEOPS, and TMMTEOPS, wherein the amphiphilic silane is deposited on a hydrophobic fibrous substrate (2), typically comprising a glass fiber substrate functionalized with a hydrophobic silane selected from the list consisting of: a trimethoxy(octyl)silane (TMOS), trimethoxy(methyl)silane (TMMS), triethoxy(methyl)silane (TEMS), trimethoxy(propyl)silane (TMPS), triethoxy(decyl)silane (TEDS), ethynyltrimethylsilane (ETMS), methoxytrimethylsilane (MTMS), any linear alkyltrialkoxysilane with alkyl chains of a length from 1 to 9 carbon atoms; any branched alkyltrialkoxysilane having a carbon chain length from 2 through 9; and linear dialkyldialkoxysilanes with alkyl chains of a length from 1 to 7 carbon atoms.

5. The method of any of the preceding claims, wherein the zinc chloride functionalized fibrous substrate (2) is provided as a test strip.

6. The method of any of the preceding claims, wherein the fluorescence light is a green fluorescence light, wherein the fluorescence light is detected at an expected fluorescence peak wavelength of formed zinc salt - complex of the fecal pigment, the expected fluorescence peak wavelength typically being in about 510 nm to about 525 nm.

7. The method of any of the preceding claims, wherein wetting the detection region comprises a dropwise application of a predefined amount of the water sample (1), typically of 5-20 pl, onto the zinc chloride functionalized fibrous substrate (2) such as 10 pl, in particular using a pipette.

8. The method of any of the preceding claims, wherein an amount of the zinc salt in the detection region and the amount of the water sample used for wetting is chosen such that a Zn-ion concentration in the wetted detection region is in a range from about 10Z.-24P008 mM to about 30 mM, more typically in a range from about 10 mM to about 30 mM, such as about 30 mM.

9. The method of any of the preceding claims, wherein the method (100) is performed using a mobile computing device (4) comprising a camera (4.1), a mass storage (4.2), a computing unit (4.3) connected with the mass storage (4.2), a display (4.4), and an electrical energy storage (4.5), in particular a smartphone (4), and wherein the method comprises at least one of, typically all of:- electrical and mechanical connecting the mobile computing device (4) with a measurement chamber (3) comprising a condenser lens (3c), an emission filter (3g), an excitation filter (3d), at least one convex lens (3b) and a light source (3e) to be energized from the mobile computing device (4), the light source (3e) typically being implemented as an LED, in particular a 475 nm LED;- arranging the mobile computing device (4, ) on a housing of the measurement chamber (3);- connecting the mobile computing device (4, 4.1, 4.2, 4.3, 4.4, 4.5) and the measurement chamber (3) via an electronic component connection, in particular via an USB connection;- arranging, after wetting the detection region of the zinc salt functionalized fibrous substrate (2) with the water sample, the zinc chloride functionalized fibrous substrate (2) in the measurement chamber at a defined distance to a camera (4.1) of the mobile computing device (4);- energizing the light source (3e) from the mobile computing device (4) to expose the detection region, via the excitation filter (3d) and the at least one convex lens (3b), to the excitation light;- detecting with the camera (4.1), via the condenser lens (3c) and the emission filter (3 g), the fluorescence light;- performing process (v) by the computing unit (4.3); and- indicating the presence of the fecal pigment and / or the concentration of the fecal pigment in the water sample on the display.Z.-24P00810. The method (100) according to any of the preceding claims, wherein a detectable concentration of the fecal pigment in the water sample is in a nM range or even in a pM range.

11. The method (100) according to any of the preceding claims, wherein the calibration data set is a predetermined data set or wherein the calibration data set is determined based on a fluorescence measurement performed at a calibration region of the zinc salt and silane functionalized fibrous substrate (2) comprising a known concentration of an organic dye, in particular to a BODIPY dye, such as (CH3)4BDP-H or a known concentration of a zinc salt - complex of the metabolite or the degradation product of hemoglobin, and / or wherein the zinc salt and silane functionalized fibrous substrate (2) is provided by the test strip according to any of the claims 12 to 17.

12. A test strip (2) for on-site determination of a trace amount of a fecal pigment, in particular a metabolite and / or a degradation product of hemoglobin in a water sample (1), particularly of urobilin and stercobilin in a water sample according to any of the preceding claims, the test strip comprising: an amphiphilic detection region comprising a zinc salt and an amphiphilic silane functionalized fibrous material (2), in particular the zinc salt and the amphiphilic silane functionalized glass fiber material (2), the amphiphilic silane functionalized fibrous material (2) typically being provided by a fibrous substrate (2).

13. The test strip (2) of claim 12, wherein the amphiphilic detection region is bounded by a hydrophobic region, at least when seen from above.

14. The test strip (2) of claim 13, wherein the test strip (2) comprises the amphiphilic silane functionalized fibrous material (2) outside the amphiphilic detection region, and wherein the test strip (2) comprises a hydrophobic barrier formed on the amphiphilic silane functionalized fibrous material (2) and around the amphiphilic detection region.

15. The test strip (2) of claim 13, wherein the test strip (2) comprises a hydrophobic silane functionalized fibrous material (2) which is, in the amphiphilic detection region, additionally functionalized with the amphiphilic silane.

16. The test strip (2) of claim 12, wherein the amphiphilic detection region is formed on a hydrophobic fibrous material (2), in particular comprising directly spotting the amphiphilic silane onto the hydrophobic fibrous material (2).Z.-24P00817. The test strip (2) of any of claims 12 to 16, wherein the test strip (2) comprises a calibration region comprising a known concentration of urobilin - zinc salt - complexes, wherein the zinc salt is a Zn(II)-salt, in particular selected from the following list: ZnCl2, Zn(NO3)2, Zn(CH3COO)2, ZnSO4, Zn(C104)2and Zn(BF4)2, wherein the zinc salt is non-chemically bound to the amphiphilic silane functionalized fibrous material (2), in particular the glass fiber material (2), and / or wherein the amphiphile silane is selected from the list consisting of:3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane (BHEAPTES), TE3CBS, TE3CPS, TE2CES, TEAPS, TEAS, TEAMS, TMBOPS, TECMES, TM3MPS, TETHFPS, TEOSPS, TEHMS, TMHEMAPS, TMAAPS, TMAGAPS, TEHBAPS, TEGAPS, TM2PES, TM2PETPS, TM4PETPS, TM4PES, TMDHIES, TMAPPAPS, TMMPEOPS, and TMMTEOPS, and / or wherein the amphiphilic silane is deposited on a hydrophobic fibrous substrate (2), typically comprising a glass fiber substrate functionalized with a hydrophobic silane selected from the list consisting of: a trimethoxy(octyl)silane (TMOS), trimethoxy(methyl)silane (TMMS), triethoxy(methyl)silane (TEMS), trimethoxy(propyl)silane (TMPS), triethoxy(decyl)silane (TEDS), ethynyltrimethylsilane (ETMS), methoxytrimethylsilane (MTMS), any linear alkyltrialkoxysilane with alkyl chains of a length from 1 to 9 carbon atoms; any branched alkyltrialkoxysilane having a carbon chain length from 2 through 9; and linear dialkyldialkoxysilanes with alkyl chains of a length from 1 to 7 carbon atoms.

18. A method for manufacturing a test strip, the method comprising: a) functionalizing at least a detection area of a fibrous substrate, in particular a glass fiber substrate (2), with an amphiphilic silane such as BHEAPTES; and b) supplementing the detection area of the fibrous substrate (2) with a zinc salt to form a detection region of the test strip for on-site detection at trace levels in a water sample of a fecal pigment, in particular of a metabolite or degradation product of a hemoglobin, in particular of urobilin, and / or stercobilin.Z.-24P00819. The method of claim 18, wherein only the detection area of the fibrous substrate is functionalized with the amphiphilic silane.

20. The method of claim 18 or 19, wherein functionalizing at least the detection region comprises: coating a hydrophilic fibrous substrate with a hydrophobic silane such as TMOS to form a hydrophobic fibrous substrate; and spotting an amphiphilic silane onto the detection area.

21. The method of claim 18, wherein at least a major part of the fibrous substrate is functionalized with the amphiphilic silane, further comprising: printing a hydrophobic barrier around the detection area.

22. The method of any of claims 18 to 21, wherein supplementing the detection area of the fibrous substrate (2) comprises at least one of: applying an aqueous zinc salt solution onto the detection area; and a drying process.

23. The method of any of claims 18 to 21, comprising at least one of: al) inserting, for a predetermine time of typically at least several hour, a fibrous substrate, in particular a glass fiber substrate (2), into a solution comprising water, an alcohol, in particular ethanol, tetraethyl orthosilicate (TEOS) and a silane such as trimethoxy(octyl)silane (TMOS); a2) washing the fibrous substrate (2) with an alcohol; a3) drying the washed fibrous substrate (2) under reduced pressure; a4) drop cast of the amphiphilic silane onto a detection area (3) of the dried fibrous substrate;Z.-24P008 bl) impregnating at least the detection area (3) of the fibrous substrate (2) with an aqueous zinc salt solution; and b2) drying the fibrous substrate (2).

24. The method of any of claims 18 to 23, wherein the zinc salt solution is a Zn(II)-salt, in particular selected from the following list: ZnCl2, Zn(NO3)2, Zn(CH3COO)2, ZnSO4, Zn(C104)2and Zn(BF4)2, wherein the zinc salt is non-chemically bound to the glass fiber substrate (2), and / or wherein the amphiphilic silane is selected from the list consisting of:3-(bis(2-hydroxyethyl))aminopropyl-triethoxysilane (BHEAPTES), TE3CBS, TE3CPS, TE2CES, TEAPS, TEAS, TEAMS, TMBOPS, TECMES, TM3MPS, TETHFPS, TEOSPS, TEHMS, TMHEMAPS, TMAAPS, TMAGAPS, TEHBAPS, TEGAPS, TM2PES, TM2PETPS, TM4PETPS, TM4PES, TMDHIES, TMAPPAPS, TMMPEOPS, and TMMTEOPS, and / or wherein the amphiphilic silane is deposited on a hydrophobic fibrous substrate (2), typically comprising a glass fiber substrate functionalized with a hydrophobic silane selected from the list consisting of: a trimethoxy(octyl)silane (TMOS), trimethoxy(methyl)silane (TMMS), triethoxy(methyl)silane (TEMS), trimethoxy(propyl)silane (TMPS), triethoxy(decyl)silane (TEDS), ethynyltrimethylsilane (ETMS), methoxytrimethylsilane (MTMS), any linear alkyltrialkoxysilane with alkyl chains of a length from 1 to 9 carbon atoms; any branched alkyltrialkoxysilane having a carbon chain length from 2 through 9; and linear dialkyldialkoxysilanes with alkyl chains of a length from 1 to 7 carbon atoms.

25. The method of any of claims 18 to 24, further comprising: forming a calibration region on the fibrous substrate, the calibration region comprising a known concentration of an organic dye, in particular to a BODIPY dye, such as (CH3)4BDP-H or a known concentration of a zinc salt - complex of the metabolite or the degradation product of hemoglobin.

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