This invention relates to the detection and measurement of redox parameters like free chlorine based upon a lateral flow method and device which enables sensitivity down to 0.05 ppm.

Background of the invention

Test strips are mobile laboratories, which allow the semiquantitative analysis of ions, organic and inorganic substances anywhere by just dipping them for a few seconds in the sample solution. In their simplest form, these devices consist of a plastic handle with a reaction zone composed of a few square millimeters of paper impregnated with specific test reagents. In contact with an analyte containing solution, a color change of the test reagent occurs, whereby the intensity of the color can be compared to a color card to estimate the analyte concentration. The color intensity can further vary with contact time of the test in the solution, which requires precise timing for quantitative results. The sampled volume is fixed by the pore volume of the paper used for the reaction zone. This makes test strips very robust analytical systems but leaves little room to adjust their detection range, which is hence mainly determined by the choice of the test reagents.

Due to limited sensitivity of the used reagents, certain analyses, which require the detection of very low analyte amounts, are not conducted with test strips but with more complicated photometric methods. Test strips for redox parameters such as free chlorine (chlorine C , hypochlorous acid HOCI or hypochlorite ion CIO depending on pH value), peracetic acid and hydrogen peroxide have typically a detection limit between 0.5 and 5 ppm This sensitivity is not sufficient to analyze for example rinsing solutions after the disinfection of food and beverage production facilities to be disinfectant free. The disinfection of drinking water with chlorine requires also the determination of very low residual free chlorine amounts after disinfection.

The World Health Organization recommends a residual amount of 0.2 to 0.5 ppm at the point of delivery. According to the German Ordinance on

Potable Water, the free chlorine content in drinking water should be even adjusted between 0.1 and 0.3 ppm after the disinfection process.

The most common redox dyes for the analysis of redox parameters are syringaldazine (SA) and tetramethylbenzidine (TMB).

Several attempts have been made in order to increase the sensitivity of test strips. One approach was to use an absorbent wick for increased analyte volume instead of a simple SA test patch. But the respective test nevertheless reached only a detection limit of 0.5 ppm free chlorine (US3811840).

Another approach was to increase the contact time of the dye with the analyte by prolonged stirring of the test strip in the analyte solution (US 5491094). A detection limit of 0.05 ppm is reached with tetramethylbenzidine, but only of 0.2 ppm with a mixture of SA and vanillinazine. Disadvantage of the prolonged stirring method is the need for precise timing and reproducible stirring as well as bleeding of reagents in the sample solution.

An improvement over existing test strip based determinations of redox parameters such as free chlorine would give significant benefits if it were to give a reliable determination with an increased sensitivity. Brief description of the Invention It has been found that a lateral flow test strip comprising a certain format is suitable for the determination of redox parameters such as free chlorine. It has been found that the sensitivity can be increased if especially the start zone of the water wettable, i.e. bibulous and porous test strip is oxidation stable. Preferably the test strip is impregnated with a buffer providing a pH of between 3 and 7 and has a wick at the end of the flow path.

The present invention relates to a lateral flow test device comprising a test strip comprising a dry porous, matrix material capable of transporting an aqueous liquid therealong by capillarity and having at least a start zone for receiving a sample and a reaction zone, which is not identical with the start zone but is preferably spatially distant from the start zone, having at least one kind of redox dye immobilised therein, whereby at least the dry porous matrix of the start zone comprises an oxidation stable material like oxidation stable glass fibers or an oxidation stable hydrophilic polyester. In any case the start zone comprises oxidation stable glass fibers or an oxidation stable hydrophilic polyester or is preferably made of said materials. The reaction zone can have the same dry porous matrix but it can also be made of another dry porous matrix material.

In a preferred embodiment, at least the start zone, more preferred the whole porous matrix of the lateral flow test device is made of a material that is bibulous and that is not oxidized by the redox agent to be detected with the lateral flow device during the detection time.

In another preferred embodiment the dry porous matrix material of at least the start zone is made of glass fibers or a hydrophilic polyester and the reaction zone comprises cellulose fibers and/or cellulose particles and/or cellulose derivatives like cellulose acetate or hydroxyalkylcellulose and a redox dye. In one embodiment, the hydrophilic polyester is polyester impregnated with a surfactant. In a preferred embodiment the polyester is PET.

In another preferred embodiment, the dry matrix material of at least the reaction zone is impregnated with a buffer of a pH between 3 and 7, preferably 4 and 6.

In a preferred embodiment the reaction zone comprises syringaldazine ( 4- hydroxy-3,5-dimethoxybenzaldehyde azine) or tetramethylbenzidine (TMB) immobilised on to it, preferably syringaldazine.

In another embodiment the reaction zone comprises two or more separate zones which comprise at least one kind of redox dye immobilised therein, in a preferred embodiment, the zones are equidistant, parallel bands which are located perpendicular to the flow of the liquid.

In another embodiment, a wick is positioned after the reaction zone, which means on the side of the reaction zone that is distant to the start zone. The present invention further relates to a method for detecting the presence of redox agents in a sample characterised in that a lateral flow test device according to the present invention is contacted with the sample and the development of a colour is detected in the reaction zone of the lateral flow device.

In a preferred embodiment the presence of redox agents is not only detected but the level of redox agents is also measured by measuring the intensity of the colour and/or the width of the colour band and/or the number of coloured bands.

In a preferred embodiment between 0.02 and 5 ppm, preferably between 0.05 and 1 ppm, of the redox agent in a sample is detected. Description of the drawings

Figures 1 shows two schemes of suitable test strips according to the present invention.

Figures 2 and 3 show results of the analysis of free chlorine with test strips according to the present invention and test strips according to prior art. Details can be found in Example 5.

Redox parameters or redox agents are agents which have a high positive standard electrode potential and which might be present in environmental samples or water samples e.g. due to disinfection. Examples of redox agents are free chlorine, peracetic acid and hydrogen peroxide, favorably free chlorine.

Free chlorine is defined as comprising one or more of the following components: chlorine C , hypochlorous acid HOCI or hypochlorite ion CIO , depending on pH value. It should be noted that the redox dyes used in the method of the present invention are not suitable for interacting with bound chlorine. Therefore for also detecting the bound available chlorine it needs to be converted to free chlorine with a suitable catalyst such as iodide.

A dry porous matrix is a material that is suitable for transporting an aqueous solution through said matrix by capillarity. Dry in this case means that it is not already wetted with a liquid like water so that transporting of an aqueous solution through said matrix is not possible anymore. The pore structure is variable. It might comprise pores in the walls of the material but preferably it also has pore structures reaching through the material - from one side to the other. Those pore can also be called flow-through pores.

The porous material might also be a network of fibers. A wick is any absorbent pad made of a material that can absorb and transport aqueous solutions. For the application of a wick in the present invention it is not necessary for the wick to be oxidation stable. Any bibulous material is suitable. It can for example be a fibrous material or a porous material or a membrane.

An aqueous liquid or an aqueous solution is any liquid comprising more than 75% water. Typically the aqueous liquid comprises more than 90% of water. Other liquids comprised in the aqueous liquid might be water miscible organic solvents like ethanol. Typically the aqueous liquid also comprises components dissolved therein like inorganic salts, surfactants or redox agents.

A hydrophilic polyester is any polyester which fulfils the following requirements: 0.5 cm of a strip of the polyester with a length of 9 cm is put in a reservoir of water. The time needed until the whole strip is wetted is measured. A hydrophilic polyester is any material that is completely wetted within 30 minutes, preferably within 15 minutes.

In case the polyester is not sufficiently hydrophilic it can be hydrophilized by the following methods to make it sufficiently wettable:

1. Impregnation - The pores of the polyester matrix are filled with a solution containing a wetting agent, such as a surfactant. The surfactant is dried onto the surface of the matrix. The surfactant is not covalently attached to the matrix and slowly washes out of the matrix when water or an aqueous solution is applied.

2. Hydrophilization - A hydrophilic coating is polymerized around the polymer that forms the matrix. There is no covalent attachment of the hydrophilic coating to the polymer network of the matrix. The polymer network simply serves as a scaffold. During the hydrophilization process, conditions are manipulated so that the coating extends through the full depth of the matrix but is not so extensive that it alters the porous structure of the matrix to any significant degree.

3. Grafting - The hydrophobic matrix is exposed to an energy source that causes formation of free radicals on the polymer surface. This is done in the presence of low molecular weight, hydrophilic, organic molecules that react with the free radicals to form covalent bonds. The membrane now takes on the wetting properties of the organic molecules. The choice of the method may depend for example on the properties of the porous matrix. Typically, impregnation with a surfactant is preferred.

Oxidation stability of a matrix is determined as described in the following by determining the amount of chlorine consumed when the matrix is contacted with a chlorine solution:

In order to determine the amount of chlorine consumed by a matrix, 8 ml of 2 ppm chlorine solution is added to 96 mg of cut matrix pieces of approximately 0.3 cm ² . After 10 min the supernatant is filtered with a 0.2 pm PP syringe filter (VWR) and stained using the liquid reagents from the Merck Test Kit for free and total chlorine determination (1.14801, Merck KGaA). For this 6 ml of the filtrate is added to 3 drops of solution 1 (DPD dye) and 1 drop of solution 2 (sulphuric acid) of the chlorine test kit. Sample spectra are collected in PMMA macro cuvettes with a Cary 60 UVA/is spectrometer (Agilent) in a wavelength range from 400 - 600 nm with 600 nm/min scan rate. Milli-Q water is used to determine the baseline. The chlorine concentration is determined by the Beer-Lambert law using the absorbance at 551 nm (e = 21000 I mol ^-1 cm ^-1 ). The loss of chlorine should be below 10% after 10 minutes contact time. That means less than 0.2 ppm chlorine should be consumed by the reaction with the matrix within the 10 minutes contact time.

As a consequence, any material that is oxidation stable or not oxidized by the redox reagent according to the definition of the present invention is a material that leads to a consumption of less than 0.2 ppm chlorine when being incubated with a 2 ppm chlorine solution for 10 minutes. This can be analyzed according to the test described above. The present invention relates to a lateral flow test device for detecting the presence of redox agents in a sample according to the method of the present invention. The test device comprises a test strip with a dry porous matrix material capable of transporting a liquid therealong by capillarity and having at least a start zone for receiving said sample and a reaction zone having at least one kind of redox dye immobilized therein. A redox dye is any component that when being contacted with a redox agent shows a colour change due to a redox reaction taking place between the redox agent and the redox dye. Examples of redox dyes are syringaldazine ( 4- hydroxy-3, 5-dimethoxybenzaldehyde azine) or tetramethylbenzidine, preferred is syringaldazine.

The start zone is preferably situated at one end of the test strip. The sample to be analysed is applied to the start zone. It has been found that the sensitivity of a lateral flow test format for the detection of redox agents can be further improved if at least the start zone is made of a material that does not react with a redox agent and consequently does not reduce the amount of redox agent present in the sample solution. It has further been found that cellulose materials such as paper which are commonly used for test strips are not completely stable to oxidation. The inventors have found that it is very favourable, if the start zone does not comprise any cellulose material, i.e. if it is cellulose-free. The start zone according to the present invention is made of oxidation-stable, wettable materials, especially glass fibers or polyester fibers, especially the polyester 6613H by Ahlstrom-Munksjo. The polyester materials may be impregnated with a surfactant. Suitable surfactants are for example anionic surfactants like SDS (Sodium Dodecylsulfate) or SDBS (Sodium Dodecylbenzene Sulfonate).

The materials might be woven, non-woven or in the format of a membrane. Preferably such materials of the start zone should be in a single sheet or layer. The volume capacity of the start zone defines the initial sample uptake of the device. The start zone sample pad also acts as a filter to help to prevent unwanted particulate materials from reaching the reaction zone. Liquid samples can be added to the start zone as single drops e.g. by using a pipette or by dipping the start zone in the liquid sample. The start zone can comprise components which are dried onto it such as components of the assay and/or chemicals to prevent (“block”) non-specific binding effects at the reaction zone and/or to chemicals to enhance hydrophilic properties, influence the pH, enhance rehydration of assay components and lateral flow characteristics and/or analyte release agents and/or extractants, preferably detergent extractants.

The reaction zone might be located directly adjacent to the start zone or spatially distant further down the test strip. It contains one or more redox dyes, in particular at least one kind of redox dye immobilised to the reaction zone. Additional components might be additionally immobilised dyes, reagents, buffers etc. In a preferred embodiment the reaction zone is a pad or a membrane chosen to be suitable for procedures to non-covalently or covalently immobilise redox dyes and also for the lateral flow characteristics of the material. In a very preferred embodiment, the reaction zone is composed of a material that is stable to oxidation and suitable for non- covalent immobilisation of the redox dye. It has been found that this combination is hard to achieve. Cellulose based materials which show a high binding capacity for the redox dyes are typically also not fully stable to oxidation. Consequently, in a preferred embodiment, for obtaining optimal results and a very sensitive test, the reaction zone is composed of at least two materials. One base material, preferably in form of a dry porous matrix that is stable to oxidation and allows transport of a liquid therealong by capillarity, and an additive with which the base material is preferably at least partially coated or impregnated which allows for stable non-covalent attachment of the redox dye. It has been found that the base material is preferably cellulose-free. It might comprise glass fibers or a polymer such as polyesters. The coating material preferably comprises cellulose fibers and/or cellulose particles and/or cellulose derivatives like hydroxyalkylcellulose, e.g. hydroxypropylcellulose, hydroxyethylcellulose, hydroxybutylcellulose, or sodium carboxymethylcellulose or most preferred cellulose acetate. The coating might be applied to the base material by any suitable mechanical or chemical method like pressing, glueing or impregnation. In a preferred embodiment the coating is applied to the base material by printing or dispensing a liquid, e.g. an ink onto it that comprises the cellulose material, e.g. cellulose fibers and/or cellulose powders and/or cellulose acetate. Preferably, the liquid additionally comprises also the redox dye. A suitable ink comprises 2-10 mM syringaldazine in a solvent like e.g., ethanol, acetone, dioxane. The ink might also comprise further reagents, e.g. reagent which support the printability of the ink.

The coating can be applied to the whole base material in the reaction zone or preferably in a defined pattern. The pattern might have any format like a band, a ring, a dot etc. In one embodiment, one or more, preferably 3 to 6, parallel bands are applied to the base material. The bands preferably have the same size (length and width) and are equidistant and perpendicular to the flow of the liquid. It has been found that by using a reaction zone on which the redox dye is present in the form of two or more bands, a more reproducible measurement is possible. If only very few analyte is present in the sample, and only a limited amount of sample is applied, only the first or maybe the second band in the row of bands show colour formation. If the concentration of the analyte is higher, more bands show colour formation. Consequently, in addition to measuring the intensity of the colour formation to measure the amount of analyte, it is possible to just count the number of coloured bands to draw a conclusion on the amount of analyte. Typically the width of the bands is between 0.5 and 5 mm, preferably between 1 and 2 mm. The distance between the bands is typically between 2 and 10 mm, preferably between 4 and 6 mm. The length of the bands is typically equal or nearly equal to the width of the test strip and the reaction zone. The application of the one or more redox dyes can be done in parallel with the application of the cellulose containing coating material to provide for better immobilization of the dye as described above or separately by contacting the reaction zone with a solution comprising the dye and subsequent drying. In a preferred embodiment, the dye is printed or dispensed on the reaction zone in a defined pattern, e.g. one or more bands as described above in those areas of the reaction zone which are suitable for non-covalent attachment. In case the whole reaction zone is homogenously and equally suitable for covalent attachment of the redox dye, the dye can be printed or dispensed over the whole zone or only in certain areas of the reaction zone to generate a certain pattern, e.g. several bands, as discussed above.

At least the reaction zone is preferably impregnated with a buffer having a pH between 3 and 7, preferably between 4 and 6. It was found that the pH is preferably set to this range to increase both color response and sensitivity. The buffer might be for example a phosphate buffer or a citrate buffer. It is typically applied to the zones by impregnating them with a buffer solution and subsequent drying.

The sensitivity of the lateral flow device can be further increased by processing higher sample volumes, which can for example be achieved by longer test strips, a wider sample pad or a fan-shaped wicking pad. An even simpler way to increase the amount of sample passing the reaction zone, consists in the addition of a wick after the reaction zone, e.g. as part of the test strip after the reaction zone or glued in between the test strip and the backing card.

Thus, in another preferred embodiment the test strip incorporates a wick which draws liquid sample from the matrix of the test strip through the device and therefore drives the capillary flow from the start zone through the reaction zone to the wick to maximise the amount of product formed. The wick is typically composed of materials similar to those employed for the dry matrix material of the test strip, e.g. the sample pad of the start zone, whereby in this case the oxidation stability of the material is not relevant. In a preferred embodiment, the wick is composed of an absorbent paper or other cellulose based material.

It has been found that lateral flow devices according to the present invention which comprise a wick as discussed above and in which at least the reaction zone is impregnated with a buffer having a pH between 4 and 6 provide especially sensitive tests. Ideally, the test strip also comprises a reaction zone on which two or more bands are present with an immobilized redox dye.

In one embodiment the test strip further comprises a plastic case or other housing to provide additional strength and rigidity to the device and to facilitate handling of the device without contaminating it. Preferably, the test strip is only partially covered by the housing. The start zone is preferably not covered by the housing to ensure easy application of the sample. The various absorbent zones of the test strip of the lateral flow device are preferably all in the same plane, allowing capillary flow of a liquid sample between the zones. In the process the redox dye should remain immobilized in the reaction zone. All zones may contain further reagents like stabilising agents or buffers which e.g. support the storage of the components or provide reaction conditions (e.g. pH) that are suitable for the colour forming reaction.

In one embodiment, all zones of the test strip of the lateral flow device are made of one continuous dry porous matrix material. This ensures a continuous flow through the whole test strip. To provide certain properties to the certain zones of the matrix material, it might be coated or impregnated with reagents. For example, the dry porous matrix of the whole test strip might be composed of a glass fiber material or a polyester polymer like PET. To ensure that the reagent zone is nevertheless suitable for non-covalent attachment of a redox dye, it might be coated with a composition like an ink comprising cellulose fibers and/or cellulose particles and/or a cellulose derivative like cellulose acetate. The coating material preferably comprises hydroxypropylcellulose, hydroxyethylcellulose, hydroxybutylcellulose, or sodium carboxymethylcellulose or most preferred cellulose acetate. The coating might be applied to the base material by any suitable mechanical or chemical method like pressing, gluing or impregnation. In a preferred embodiment the coating is applied to the matrix material by printing or dispensing a liquid, e.g. an ink onto it that comprises the cellulose material, e.g. cellulose fibers and/or cellulose powders and/or cellulose acetate. The zones of the test strip might also be made of different materials.

Typically the test strip made of one or more porous materials which enable the flow of liquid through the material by capillary action as described above is fixed on a non-porous, non-bibulous support or backing, e.g. a plastic sheet like a polystyrene sheet, which ensures mechanical stability, also called backing card.

The backing card typically has the same size as the porous matrix and the porous matrix is fixed onto the backing card, as e.g. can be seen in Figure 1.

The width of the test strip is typically between 0.3 and 1.5 cm, preferably between 0.5 and 1 cm. The length is typically between 5 and 20 cm. A preferred dimension of a flow test is for example 0.5x9 cm ² . The volume of the analyte passing the dye can be increased by an absorbent pad or wick glued or otherwise fixed between the flow substrate in form of the dry porous matrix and the backing card with a typical dimension of 0.5x4.5 cm ² . Figure 1 is a schematic, illustrative view of a lateral flow test strip according to the present invention. On a self-adhesive plastic support (backing) is located a dry porous matrix. In version a) the start zone and the reaction zone are made of the same dry porous matrix material. In the reaction zone, the redox-dye is immobilized in 5 separate zones. These zones might also comprise a coating or impregnation with a material to support the stable, non-covalent attachment of the redox dye. Due to the fact that the whole test strip is made of one type of porous base matrix the flow of the liquid through the test strip is very homogenous. By impregnating certain areas of the porous base matrix its properties can be locally changed.

In version b) the start zone of the test strip is made of a material different from the dry porous material of the rest of the test strip. In addition, a wick is inserted in the area after the reaction zone. The wick draws liquid sample from the matrix of the test strip through the device and therefore drives the capillary flow from the start zone through the reaction zone to the wick. It allows more sample liquid to flow through the reaction zone and thus maximises the amount of product formed.

The present invention is further directed to a method for detecting the presence of redox agents in a sample characterised in that a lateral flow test device according to the present invention is contacted with the sample and the development of a colour is detected in the reaction zone of the lateral flow device. A sample can be any liquid sample potentially comprising a redox agent. Typically the sample is an aqueous liquid. Application of the sample to the start zone can be done by dipping the start zone into the sample or by applying the sample onto the start zone e.g. with a pipette. Typically, about 50 to 500 pi of the aqueous sample are applied to the start zone. In case the test strip is dipped into the aqueous sample solution it has to be removed after a certain time, typically after the complete test strip is wetted. To facilitate the determination of the right time to remove the test strip, a sensor for water, e.g. a band of anhydrous C0CI2 _, can be positioned at the distant end of the test strip to indicate the presence of the sample solution at the distant end of the test strip and thus the end of the measurement. The flow of the liquid through the test strip starts automatically due to capillary forces.

When the sample reaches the reaction zone, the redox agents present in the sample liquid react with the redox dye and a coloured reaction product is formed which can be detected e.g. visually.

In a preferred embodiment the presence of redox agents is not only detected but the level of redox agents is also measured by measuring the intensity of the colour and/or the width of the colour band and/or the number of colour bands. It was found that next to the color intensity, the width of the colored pad also depends on the concentration of the redox agent. For a better readability based on colour intensity only, it is helpful to use a sheath around the paper strip with an aperture that the user focuses on the color intensity to interpret the results. Alternatively, it is possible to print an even broader dye pad and use a distance-based detection readout. Printing separate dye lines or bands like in a barcode flow test instead of one dye section, facilitates the readout by eye as both the color intensity and the number of lines can be compared for semiquantitative analysis.

In a preferred embodiment, the colour intensity of the one or more colour bands is measured.

The lateral flow device and the method of the present invention provide a reliable, easy to use test for free chlorine and other redox agents. The sensitivity can be increased to go below 0.05 ppm. Typically it is between 0.05 and 1 ppm. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilise the present invention to its fullest extent. The preferred specific embodiments and examples are, therefore, to be construed as merely illustrative, and not limiting to the remainder of the disclosure in any way whatsoever.

The entire disclosures of all applications, patents, and publications cited above and below, as well as the corresponding EP 19194240.8 filed on August 29, 2019, are hereby incorporated by reference.

Examples

The following examples represent practical applications of the invention.

Example 1 (Best mode with cellulose acetate on GF or PET substrate): Tests were prepared using as porous matrix material a PET (polyethylene terephthalate) substrate from Ahlstrom 6613H and the glass fiber GF MN 85/90 BF from Macherey-Nagel. Some substrates were immersed for 5 s in a phosphate buffer pH 5. The substrates were dried overnight on a glass plate. All substrates were cut to 9x5 cm ² pieces and glued on the backing cards supplied from Lohmann (polystyrene with GL-187 acrylic PSA, 9x5 cm ² ). Wicks (GF or cotton linters paper) of 4.5x5 cm ² were glued between backing cards and substrates leaving 1 cm free from the top edge.

Printing was conducted on a Hyrel System30M 3D-Printer using a custom- made syringe-based print head. A Flamilton 500 pi syringe was used with a flexible polypropylene needle (length 1”, diameter 0.25 mm, needle hold at 20°). The substrate was fixed on the printing table by vacuum and a 4 mM syringaldazine ink in acetone with 3 % cellulose acetate was printed at 1 ;

1.5; 2; 2.5 and 3 cm from the bottom edge with the dosing of 2 mI/cm. The substrates were cut into 9x0.5 cm ² stripes and analyzed the next day by dipping them into free chlorine solutions prepared by dissolving dichloroisocyanuric acid sodium salt dihydrate in purified water.

The detection limit was 0.1 ppm free chlorine for the PET substrate impregnated with buffer and a paper wick and 0.05 ppm for the GF substrate without buffer immersion and a GF wick.

Example 2 (Hybrid Flow Test):

Tests were prepared using cotton linters paper Schleicher & Schuell 2992 and Ahlstrom DBS-paper TFN as porous matrix material, and Ahlstrom PET 6613H and Macherey-Nagel glass fiber GF MN 85/90 BF as start zone material.

All tests had a total size of 9x0.5 cm ² . For tests with separate start zones, the PET pad had a size of 1.2x0.5 cm ² and the paper a size of 8x0.5cm ² in order to obtain an overlap of the two of 1-2 mm. Some tests were assembled with an additional 4 cm long wick as this further increased the sensitivity. Some paper substrates were immersed in phosphate buffer pH 5 for 15 s and dried on a glass plate. The samples were glued on the backing cards supplied from Lohmann (polystyrene with GL-187 acrylic PSA, 9x5 cm ² ). 2 mM SA solution in absolute ethanol was freshly prepared. Printing was conducted on a Hyrel System30M 3D-Printer using a custom-made syringe- based print head. A Flamilton 250 pi syringe was used with a flexible polypropylene needle (length 1”, diameter 0.25 mm, needle hold at 20°). The paper was fixed on the printing table by vacuum and the dye solution was printed onto paper in 5 stripes with the dosing of 4x0.5 mI/cm and a distance of 5 mm. The first stripe was printed at 1.4 cm. After printing, the paper was cut into 9x0.5 cm ² stripes and analyzed by dipping them into free chlorine solutions prepared by dissolving dichloroisocyanuric acid sodium salt dihydrate in purified water. The detection limit was 0.2 ppm free chlorine for hybrid tests with buffer impregnation and wick. Example 3 (Comparative Example Barcode Flow Test):

For comparison, a test with a non-oxidation stable paper matrix without stable start zone is prepared. Therefore, cotton linters paper Schleicher & Schuell 2992 was cut to 9x5 cm ² pieces and glued on the backing cards supplied from Lohmann (polystyrene with GL-187 acrylic PSA, 9x5 cm ² ).

Printing was conducted on a Hyrel System30M 3D-Printer using a custom- made syringe-based print head. A Flamilton 500 pi syringe was used with a flexible polypropylene needle (length 1”, diameter 0.25 mm, needle hold at 20°). The substrate was fixed on the printing table by vacuum and a 2 mM syringaldazine ink in ethanol was printed at 1 ; 1.5; 2; 2.5 and 3 cm from the bottom edge with the dosing of 2 mI/cm. The substrates were cut into 9x0.5 cm ² stripes and analyzed the next day by dipping them into free chlorine solutions prepared by dissolving dichloroisocyanuric acid sodium salt dihydrate in purified water. The detection limit was 0.3 ppm free chlorine for non-oxidation stable paper-based barcode flow test.

Example 4 (Comparative Example Merck dip test):

For comparison, commercial colorimetric chlorine dip test strips (1.17925, MQuant, Merck KGaA) were used as received. The detection limit was 0.5 ppm for dip test strips.

Example 5 (Quantitation of purple intensity):

After removal of the different flow tests from the chlorine solution, they were immediately scanned with the scanner CanoScan 9000F Markll (Canon, 600 dpi without any image correction). The intensity of the purple color of the oxidized syringaldazine was determined by ImageJ using the color deconvolution plugin. The RGB stain vector was determined to [0.380.89 0.24] on paper and [0.370.860.35] for PET and GF and the RGB intensity value determined from the deconvoluted “purple” image, on which only this color remained in order to avoid interference from grey background due to water stains on the substrate. The purple intensity for different chlorine concentration of the five tests described in Example 1-4 is plotted in Figures 2 and 3, whereby Figure 2 shows the intensity versus the chlorine concentration over the whole tested range and Figure 3 provides an enlarged view of the area with low chlorine concentrations.

Example 6 (Flow tests with TMB):

Tests were prepared using the PET substrate from Ahlstrom 6613H and the paper 2992 from Schleicher & SchCill for comparison. All substrates were cut to 9x5 cm ² pieces and glued on the backing cards supplied from Lohmann (9x5 cm ² ).

40 mM TMB (Merck KGaA) was dissolved in acetone. 5 % 2-hydroxyethyl cellulose (HEC, 434965, Aldrich, 90000 g/mol) was dissolved in water (300 mg in 6 g solution). Printing was conducted on a Hyrel System30M 3D- Printer using a custom-made syringe-based print head. A Flamilton 500 pi syringe was used with a flexible polypropylene needle (length 1”, diameter 0.25 mm for inks with TMB and 0.6 mm for FIEC ink, needle hold at 20°). The substrate was fixed on the printing table by vacuum and the 40 mM TMB-ink in acetone was printed first at 1 ; 1.5; 2; 2.5 and 3 cm from the bottom edge with the dosing of 2 mI/cm. After drying, the FIEC ink was printed on top of the TMB bands at 1 ; 1.5; 2; 2.5 and 3 cm with the dosing of 2 mI/cm. The substrates were cut into 9x0.5 cm ² stripes and analyzed the same day by dipping them into free chlorine solutions prepared by dissolving dichloroisocyanuric acid sodium salt dihydrate in purified water. The detection limit was 0.1 ppm free chlorine for the PET substrate and 0.2 ppm for the paper substrate.