TEST STRIPS FOR MEASURING CHLORIDE ION CONCENTRATION

Abstract

TEST STRIPS FOR MEASURING CHLORIDE ION CONCENTRATION ABSTRACT The present disclosure provides a test strip (100) for measuring chloride ion concentration in a sample. The test strip (100) comprises a substrate (102) with a sample port (104) at one end and a sample elution area (106) adjacent to the sample port. The sample elution area (106) comprises a complexing agent, where a concentration of the complexing agent is pre-determined and is variable along the direction of sample elution in the sample elution area (106). The chloride ion concentration in the sample is measured from a detectable visual change in the sample elution area (106) resulting from a reaction of the complexing agent with chloride ions in the sample. A method for measuring chloride ion concentration in a sample using the test strip is provided. A method of preparing the test strip (100) is also disclosed. The test strip (100) finds applications in screening, monitoring and/or diagnosing cystic fibrosis from a salivary sample. [FIG. 1]

Specification

Description:BACKGROUND

FIELD OF THE DISCLOSURE
[0001] Various embodiments of the disclosure relate generally to test strips for measuring chloride ion concentration. More specifically, various embodiments of the disclosure relate to a method of preparing the test strips for measuring chloride ion concentration, in particular chloride ion concentration in biological samples for screening, monitoring and/or diagnosing cystic fibrosis.

DESCRIPTION OF THE RELATED ART

[0002] Cystic fibrosis (CF) is a genetic disorder characterized by the production of abnormally thick and sticky mucus, which obstructs the airways in the lungs and the ducts in the pancreas, leading to severe complications in the human body. If not adequately treated, CF can result in significant lung damage and impaired nutrient absorption, both of which can be life-threatening. Although reliable data on the incidence of CF in India is lacking, based on global incidence rates, it is estimated that approximately 4,000 newborns with CF may be born annually in India, with the vast majority likely remaining undiagnosed.
[0003] Early detection and treatment of CF at birth can significantly reduce long-term effects on the body. Newborn screening for CF involves measuring Immunoreactive Trypsinogen (IRT) levels, which can help identify CF. However, such screening is not universally implemented in India. The reliability of CF newborn screening is highest within the first two months of life, as IRT levels naturally decline with age.
[0004] Genetic testing and sweat tests are two primary methods for diagnosing CF. Though highly effective, genetic tests are expensive and often inaccessible due to limited awareness. While less costly than genetic tests, the sweat test has limited availability in only a few centers across India as this test requires specialized training and is challenging to perform.
[0005] CF transmembrane conductance regulator (CFTR) modulator therapy uses a combination of tezacaftor/ivacaftor (TI), lumacaftor/ivacaftor (LI), elexacaftor, tezacaftor and ivacaftor (ETI) to control and/or correct malfunctioning protein made by the CFTR gene. Sweat test which is the gold standard is also used to monitor the response to CF modulator therapy. The sweat test involves iontophoresis of pilocarpine to stimulate sweat glands to produce sweat for analysis.
[0006] There are several methods for quantifying chloride ion concentration in bodily fluids. Titration, while straightforward, is prone to human error due to the subjective determination of endpoints and has limited sensitivity for detecting low chloride levels. Inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (OES) offer high accuracy but require significant financial investment and specialized operator expertise. Sweat conductivity measurements are another way to determine chloride concentration but the measurements are affected by the presence of other ions, such as bicarbonate and lactate, which can compromise reliability. Further, conductivity measurements may require large sample volumes.
[0007] Microfluidic paper-based analytical devices (µPADs) offer a promising alternative for CF diagnosis due to their ease of use, low cost, rapid results, potential for mass screening, and minimal sample volume requirements. µPADs have the potential to significantly improve patient care by enabling timely and accessible testing.
[0008] Limitations and disadvantages of conventional and traditional approaches will become apparent to one of skill in the art, through comparison of described systems with some aspects of the present disclosure, as set forth in the remainder of the present application and with reference to the drawings.
SUMMARY

[0009] According to embodiments of the present disclosure, a test strip for measuring chloride ion concentration in a sample is provided. The test strip comprises a substrate with a sample port at one end and a sample elution area adjacent to the sample port. The sample elution area comprises a complexing agent, where a concentration of the complexing agent is pre-determined and is variable along a direction of sample elution in the sample elution area. The chloride ion concentration in the sample is measured from a detectable visual change in the sample elution area resulting from a reaction of the complexing agent with chloride ions in the sample.
[0010] In some embodiments, a test strip for measuring chloride ion concentration in a salivary sample is provided. The test strip comprises a substrate with a sample port at one end and a sample elution area adjacent to the sample port. The sample elution area comprises a complexing agent, where a concentration of the complexing agent is pre-determined and is variable along a direction of sample elution in the sample elution area. The chloride ion concentration in the sample is measured from a detectable visual change in the sample elution area resulting from a reaction of the complexing agent with chloride ions in the sample. A minimum concentration measurable by the test strip corresponds to a threshold concentration of 10 mM and a maximum concentration measurable by the test strip is set at 40mM.
[0011] In another embodiment, a method for measuring chloride ion concentration in a sample using the test strip is provided.
[0012] In yet another embodiment, a method of preparing a test strip for measuring chloride ion concentration in a sample is provided. The method comprises a step (i) of providing a substrate. The method further comprises a step (ii) of applying a first solution comprising one of potassium chromate, or potassium dichromate to form a first coating over a sample elution area of the substrate. A pre-determined concentration of the potassium chromate, or the potassium dichromate in the first coating is variable along a direction of sample elution to control a sensitivity of the test strip, or a measurable concentration range of chloride ion by the test strip, or both. The method further comprises a step (iii) of applying a second solution comprising silver nitrate to form a second coating over the first coating. A pre-determined concentration of the silver nitrate is variable along the direction of sample elution and is proportional to the concentration of the potassium chromate, or the potassium dichromate to form silver chromate, or silver dichromate in-situ in the substrate to form the test strip.
BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1 is a schematic representation of a test strip prepared according to embodiments of the present disclosure;
[0014] FIG. 2 depicts schematic diagrams of various test strips, in accordance with embodiments of the present disclosure;
[0015] FIG. 3 is a flow chart illustrating a method of preparing a test strip, in accordance with an exemplary embodiment of the disclosure;
[0016] FIG. 4 is a calibration curve of distance traveled as a function of concentration, according to embodiments of the present disclosure;
[0017] FIG. 5 is a scatter plot of correlation between laboratory salivary chloride values and strip salivary chloride values, according to embodiments of the present disclosure; and
[0018] FIGs. 6a and 6b depict Bland-Altman plots of laboratory salivary chloride values and strip salivary chloride values, according to embodiments of the present disclosure.
[0019] Further areas of applicability of the present disclosure will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description of exemplary embodiments is intended for illustration purposes only and is, therefore, not intended to necessarily limit the scope of the present disclosure.

DETAILED DESCRIPTION OF EMBODIMENTS

[0020] The following description illustrates some exemplary embodiments of the disclosed disclosure in detail. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure that are encompassed by its scope. Accordingly, the description of a certain exemplary embodiment should not be deemed to limit the scope of the present disclosure.
[0021] The term "comprising" as used herein is synonymous with "including," or "containing," and is inclusive or open-ended and does not exclude additional, unrecited elements, or process steps.
[0022] All numbers expressing quantities of ingredients, property measurements, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained.
[0023] These and other features and advantages of the present disclosure may be appreciated from a review of the following detailed description of the present disclosure, along with the accompanying figures in which like reference numerals refer to like parts throughout.
[0024] Microfluidic paper-based analytical devices (µPADs) or test strips have gained much attention for point-of-need monitoring due to their ease of use, low cost, and portability. The most common detection technique used in test strips is colorimetry which distinguishes the changes in color hue and/or intensity with a concentration of an analyte. A distance-based µPAD typically consists of an inlet for adding a sample comprising the analyte and a flow channel containing a specific colorimetric reagent. When a sample is added to the inlet, the sample flows along the flow channel and the analyte present in the sample reacts with the reagent until all of the analyte is consumed. The result is a colored band along the flow channel whose length is proportional to the amount of analyte present in the sample. Various paper-based devices have been reported in the literature, each employing different strategies for analyte deposition.
[0025] M Taghizadeh-Behbahani et al., "A paper-based length of stain analytical device for naked eye (readout free) detection of cystic fibrosis", Analytica Chimica Acta, 1080, pp 38-145, 2019, disclosed a paper-based analytical device where a paper was patterned with a hydrophobic barrier to define a fluid path. The paper was then immersed in silver nitrate, washed, and dried. In a subsequent step, the paper was immersed in a second solution of potassium chromate, washed, and dried. Taghizadeh-Behbahani et al. process is laborious and time-consuming and hence may have difficulty scaling up for mass manufacturing. The process involved significant silver nitrate wastage and the paper-based analytical device may suffer from non-uniform silver loading.
[0026] Chauhan A et al., "In situ synthesis of reagents in paper-based analytical devices using paper stacking", Analytical Methods, 14, pp. 4021-4024, 2022, introduced a paper stack-based method for the in situ synthesis of silver chromate, using a nitrocellulose membrane impregnated with silver nitrate, overlaid with a distributor membrane, and exposed to potassium chromate solution. While this method produced a spatially uniform silver chromate product, it had limitations, including a narrow operational range due to its dependence on optimized reactant concentrations. Chauhan A et al. process is cumbersome and may face challenges in scaling up.
[0027] Phoonsawat et al., "A distance-based paper sensor for the determination of chloride ions using silver nanoparticles," Analyst, 143, pp. 3867-3873, 2018, developed a distance-based paper sensor for chloride ion detection including silver nanoparticles. Silver nanoparticles were manually spotted along a wax-defined flow channel on the paper substrate and dried. Phoonsawat et al. method has limitations such as batch-to-batch variability, scalability challenges, and safety concerns associated with the use of hydrogen peroxide as an additive.
[0028] Rahbar M et al. "Instrument-free argentometric determination of chloride via trapezoidal distance-based microfluidic paper devices," Anal Chim Acta, 1063, pp. 1-8, 2019, employed a wax printing and cutting plotter system to fabricate μPADs with distance-based patterns, including a straight channel and circular sample zone, printed onto paper. Colorimetric reagents were precisely deposited into the channels using technical pens via a plotter. While this method improved reagent placement accuracy and reproducibility, it required an additional wax barrier creation step, thus increasing production costs and complexity. Additionally, the reliance on technical pens for reagent delivery could lead to clogging issues, potentially affecting reagent distribution uniformity, and the approach did not allow for tuning the detection range.
[0029] The term "test strip", as used herein refers to a point-of-use, paper-based microfluidic analytical device (µPAD) to measure a concentration of chloride ions in a sample. The "test strip" of the present disclosure relates to a distance-based µPAD. The "test strip" of the present disclosure, in certain embodiments, may be used to confirm the presence of chloride ions in a sample.
[0030] In one embodiment of the present disclosure, a test strip for measuring chloride ion concentration in a sample is provided. The test strip comprises a substrate with a sample port at one end, and a sample elution area adjacent to the sample port. The sample elution area comprises a complexing agent, wherein a concentration of the complexing agent is pre-determined and is variable along a direction of sample elution in the sample elution area, and wherein the chloride ion concentration in the sample is measured from a detectable visual change in the sample elution area resulting from a reaction of the complexing agent with chloride ion in the sample.
[0031] FIG. 1 is a schematic diagram of a test strip 100, in accordance with embodiments of the present disclosure. The test strip 100 comprises a substrate 102 having a sample port 104, a sample elution area 106, and an optional wicking pad 108.
[0032] The substrate 102 is hydrophilic and porous. The pores (not shown) inherently present in the substrate 102 allow water to move through them via capillary or wicking action. The hydrophilic nature of the substrate 102 further facilitates the wicking action enabling flow of water through the substrate 102. It is known, that pores of smaller size exhibit larger capillary pressure than the pores having larger size. A flow of a liquid through a substrate may depend on parameters of the substrate 102 such as porosity, pore size distribution, and surface characteristics like contact angle.
[0033] In one embodiment, the substrate 102 is single-layered having uniform thickness throughout. The pores of the substrate 102 have substantially uniform pore size. As used herein, the term substantially means about 50%, or more than 50% of the pores having uniform pore size. In one embodiment, the pore size of the substrate 102 is in a range of 5 microns to 10 microns. Examples of substrate 102 comprise cellulose-based paper, nitrocellulose-based paper, and glass fibre-based substrate. In one embodiment, the substrate 102 is nitrocellulose-based paper.
[0034] The sample port 104 is provided at one end of the substrate 102. A sample containing chloride ions is provided in the sample port 104. The sample, in one embodiment, is a biological (bodily) fluid. Examples of biological fluid include sweat, saliva, or combinations thereof. The sample, in another embodiment, is water containing chloride ions. Further, a viscosity of the sample may be adjusted by adding water to enhance flow through the sample elution area 106.
[0035] A portion of the substrate 102 adjacent to the sample port 104 is assigned as sample elution area 106 over which a complexing agent is provided. The sample flows across the sample elution area 106 along a direction of sample elution, where the direction of sample elution is from the sample port 104 towards the wicking pad 108. The term "sample elution area", as used herein refers to an area on the substrate comprising the complexing agent and is defined by its length (along the direction of sample elution) and breadth.
[0036] Examples of complexing agents include silver chromate or silver dichromate. The complexing agent present in the sample elution area 106 reacts with the chloride ions present in the sample indicated by a detectable visual change in the sample elution area 106. In the chemical equation shown below, the complexing agent represented by silver chromate (Ag2CrO4), reacts with chloride ion, as represented by sodium chloride (NaCl) to form silver chloride. Here, 2 moles of chloride ions react with 1 mole of silver chromate to form 2 moles of silver chloride.
Ag2CrO4 + 2 NaCl ⇄ 2 AgCl + Na2CrO4
[0037] Silver chromate imparts a reddish-brown colour on the sample elution area 106. The chloride ions present in the sample reacts with the silver chromate to form white silver chloride along the direction of sample elution on the sample elution area 106. The detectable visual change from reddish-brown to white in the sample elution area 106 is proportional to the concentration of the chloride ions in the sample. In one embodiment, the concentration of the chloride ion is measured from the length of the sample elution area 106 while in some other embodiments, the concentration corresponds to an area defined by a product of the length and the breadth of the sample elution area 106. The length corresponds to a distance along a side of the sample elution area 106 in a direction of flow while breadth corresponds to a distance of a side perpendicular to the direction of flow.
[0038] On one or both sides of the sample elution area 106, markings 112 may be provided to indicate a measurable concentration range of chloride ion by the test strip 100. The markings 112 are arrived at by calibrating the test strip 100 against standard solutions of chloride ions having varying concentrations. In one embodiment, the markings 112 are provided on a backing sheet (not shown) attached to the substrate 102. In some embodiments, the sample elution area 106 is isolated from the substrate 102 using hydrophobic coatings (not shown) to provide the markings 112 on the substrate 102. As shown in FIG. 1, a portion of the reacted complexing agent is marked as 114 on the sample elution area 106 and corresponds to a concentration reading 116 on the markings 112, while 118 corresponds to a portion of unreacted complexing agent on the sample elution area 106.
[0039] In one embodiment, the concentration of the chloride ion is read directly from the markings 112. As, the flow of the sample through the sample elution area 106 may exhibit non-uniformity, leading to a non-linear fluid front, in certain embodiments, an area corresponding to the portion of the reacted complexing agent (such as 114) may be determined to correlate it to the concentration of chloride ion. Consequently, determining the area corresponding to the portion of the reacted complexing agent 114 can be more accurate when considering that the fluid front may not align perfectly linearly with the markings 112. In one embodiment, the area (portion of the reacted complexing agent) may be calculated from an image of the test strip 100 comprising the portion of the reacted complexing agent 114. For example, an imaging device may capture the image of the area (portion of the reacted complexing agent) which may be processed using a computing device to arrive at the concentration of the chloride ions. Examples of imaging devices include a camera, a scanner, or similar such devices. Examples of computing devices include a smart phone, a tablet computer, a laptop computer, a desktop computer, or similar such devices.
[0040] A concentration of the complexing agent is pre-determined and is variable along the direction of sample elution in the sample elution area 106. The concentration of the complexing agent is pre-determined based on a desired sensitivity of the test, or a measurable concentration range of the test, or both. The term "sensitivity", as used herein refers to the ability of a device to detect small changes in concentration of the analyte. In other words, a device or a test strip with high sensitivity can detect small changes in concentration of chloride ions. The term "measurable concentration range", as used herein, corresponds to a concentration range spanning from a lowest concentration of the chloride ion measurable by the test strip to a highest concentration of the chloride ions measurable by the test strip.
[0041] Prior-art paper-based analytical devices have a uniform concentration of complexing agents, and hence the sensitivity of such devices may be enhanced only by increasing a length of an elution area. According to embodiments of the present disclosure, concentration of the complexing agent is variable in the sample elution area 106. By varying concentration of the complexing agent, the sensitivity of the test can be enhanced without varying the length of the sample elution area 106. It is an advantage of the present disclosure by varying the concentration of the complexing agent the test strip can be modified to perform at high sensitivity and desired measurable concentration range of the chloride ion. This eliminates the need for longer test strips thereby reducing costs and avoiding handling issues associated with bulky strips. Another advantage of the present disclosure is shorter sampling time (elution time) when compared to strips having a longer elution area.
[0042] In another embodiment, the measurable concentration range of the test strip 100 is varied by having a variable concentration of the complexing agent along the direction of sample elution in the sample elution area 106. In one embodiment, the test strip has a measurable chloride ion concentration range between 0.01 milliMolar (mM) and 4000 mM. Bureau of Indian Standards (BIS) (IS 10500: 2012) prescribes a chloride ion concentration in drinking water of 250 mg/L (approximately 7 mM) as the acceptable limit which may go up to a permissible limit of 1000 mg/L (approximately 28mM). A chloride concentration higher than 250 mg/L imparts a salty taste to water. The applications of test strip 100 may include measuring chloride ion concentration of samples such as municipal water supply, swimming pools, effluent stream from industries, and similar chloride ion containing samples. In another embodiment, the test strip has a chloride ion concentration range between 10 milliMolar (mM) and 40 mM, when the sample is a biological fluid such as saliva. The test strip 100 finds application in measuring chloride ion concentration in biological samples for screening, monitoring and/or diagnosing cystic fibrosis, and is discussed in detail in Examples section.
[0043] The test strip 100 may further include a wicking pad 108 to absorb excess sample to prevent backflow or re-absorption of the sample in the sample elution area 106, which may adversely affect the measurement of the chloride ions. The wicking pad 108 comprises a material having a higher absorption capacity than the material constituting the substrate 102. In one embodiment, the wicking pad 108 has an absorption capacity of 40 microliters (µL). In some embodiments, the wicking pad 108 has an absorption capacity in a range of 30 µL to 50 µL. In one embodiment, the wicking pad 108 comprises a cellulose fiber sample pad (CFSP). The higher absorption capacity of the wicking pad 108 maintains a capillary suction between the substrate 102 and the wicking pad 108 thus enhancing flow across the sample elution area 106 to completely elute the sample. The enhanced flow across the sample elution area 106 may reduce the sampling time. The term "sampling time", as used herein refers to a time taken for a sample to flow from the sample port 104 to the end of markings 112 proximate to the wicking pad 108. The sampling time may depend on factors such as the material constituting the substrate, a viscosity of the sample, and sample elution area of the substrate.
[0044] FIG. 2 depicts schematic diagrams of various test strips 200, 300, and 400, in accordance with embodiments of the present disclosure. For the sake of brevity, sample ports and wicking pads are not shown in FIG.2. In FIG. 2, the test strip 200 corresponds to a test strip where the complexing agent is provided as discrete bands perpendicular to the direction of sample elution. In test strip 200, bands 202 correspond to regions having the complexing agent.
[0045] In another embodiment, the test strip 300 is provided where a minimum measurable chloride concentration is set at 10 milliMolar (mM). A first marking 302 corresponds to a chloride concentration of 10 mM and marking 304 corresponds to the highest measurable chloride concentration limit of 40 mM. The test strip 300 may be used as a screening, monitoring, and/or diagnosing strip for cystic fibrosis.
[0046] In yet another embodiment, the test strip 400 of varying sensitivity along a length of the test strip 400 is provided. The test strip 400 includes regions 402 and 404 corresponding to lower sensitivity regions where the chloride concentration corresponds to values of 0 to 20 mM and 30 to 50 mM, respectively. In test strip 400, important ranges for detection are between 20 to 30 mM (corresponding to region 406 on the test strip 400), and 50 to 60 mM (corresponding to region 408 on the test strip 400) and have higher sensitivity. The higher sensitivity of regions 406 and 408 is achieved by depositing higher concentrations of complexing agents in lower sensitivity regions 402 and 404, and by increasing lengths of the regions 406 and 408.
[0047] In yet another embodiment, a method of preparing a test strip (for example, test strip 100) for measuring chloride ion concentration in a sample is provided. The method comprises providing a substrate. The method further comprises applying a first solution comprising one of potassium chromate or potassium dichromate to form a first coating over a sample elution area of the substrate. A pre-determined concentration of the first coating varies along a direction of sample elution to control a sensitivity of the test strip, or a measurable concentration range of chloride ion by the test strip, or both. The method further comprises applying a second solution comprising silver nitrate to form a second coating over the first coating. A pre-determined concentration of the second coating varies along the direction of sample elution and is proportional to the first concentration to form one of silver chromate or silver dichromate in-situ in the substrate by reaction between silver nitrate and one of potassium chromate or potassium dichromate to form the test strip.
[0048] FIG. 3 is a flow chart 500 that illustrates a method of preparing a test strip (for example, test strip 100 of FIG. 1) for measuring chloride ion concentration through exemplary steps 502 through 506, according to embodiments of the present disclosure. At step 502, a substrate (for example, substrate 102 of FIG. 1) is provided.
[0049] At step 504, a first solution is applied on the substrate to form a first coating over a sample elution area. The first solution comprises potassium chromate or potassium dichromate. The terms "potassium chromate" and "potassium dichromate", either alone or taken together, are referred to as "potassium salts". A concentration of the potassium chromate, or potassium dichromate in the first coating is variable along a direction of sample elution, and is pre-determined to control a sensitivity of the test strip, or a measurable concentration range of chloride ion by the test strip, or both.
[0050] The first coating is a continuous coating having varying concentration of the potassium salts, in one embodiment. In another embodiment, the first coating is a discrete coating. In some embodiments, an initial concentration of the potassium salt in the first coating is at a threshold concentration and the concentration is maintained a constant at a remaining portion of the first coating (for example, test strip 300). The threshold concentration may correspond to a minimum concentration of the chloride ion anticipated in a sample. In other words, the threshold concentration corresponds to a minimum concentration measurable by the test strip. For example, it is known that normal human beings have a chloride ion concentration of 10 millimolars (mM) in saliva, by keeping the threshold concentration of 10 mM on the test strip avoids misreading and confusion regarding the readings. Patients having cystic fibrosis are known to have heightened levels of chloride ions in biological fluids of more than 10mM, typically in a range of 10 mM to 40 mM. Further, patients undergoing treatment for cystic fibrosis (for example, CFTR therapy) will have lower chloride ions in biological fluid (17 mM to 24 mM). In one embodiment, sensitivity of chloride test strip may be modified to capture patients on treatment and to monitor the effectiveness of CFTR therapy, for example by increasing the sensitivity of the test between 17 mM and 24 mM. In embodiments, where the first coating is discrete, the first coating is provided as discrete bands perpendicular to the direction of sample elution (for example, test strip 200).
[0051] A concentration of the first solution to be dispensed on a portion of the sample elution area is calculated from a volume of a sample that may be dispensed on the test strip, concentration of the potassium salts in the first solution, the measurable concentration range of the test strip desired, the sensitivity of the test, or combinations thereof. For example, for a sample volume of 20 microliter chloride ion-containing solution, if the measurable concentration range of the strip desired is 0 to 30 millimolar (here 30mM translates to highest chloride ion concentration in the sample), one can determine the concentration of silver required on the sample elution area to completely react with the chloride ions present in the sample given the stoichiometric relationship of 2 moles of chloride ions to 1 mole of silver. Once the concentration of silver required for deposition onto the test strip is known, the concentration of potassium salts in the first solution may be calculated.
[0052] In one embodiment, applying the first solution is performed using a plotter controlled by a computer numerical controller (CNC). It is a particular advantage of the present disclosure, using CNC precise dose of the first solution may be dispensed at specific areas on the sample elution area. The variable concentrations of the coating can be achieved using the CNC plotter by changing the concentration of the first solution, by fine-tuning the specific deposition area on the sample elution area, by changing a speed of the plotter, or combinations thereof. For a given concentration of the first solution, a loading (or concentration) of the potassium salts deposited on the sample elution area is varied by changing a speed of the plotter, for example at a slower speed concentration of the potassium salt deposited is more than that at a faster speed of the plotter.
[0053] The plotter in one embodiment is a brush, or a pen containing the first solution.
[0054] At step 506, a second solution comprising silver nitrate is applied to form a second coating over the first coating. A pre-determined concentration of silver nitrate in the second coating is variable along the direction of sample elution and is proportional to the first concentration to form one of silver chromate or silver dichromate in-situ in the substrate by reaction between silver nitrate and one of potassium chromate or potassium dichromate to form the test strip.
[0055] The second coating is applied over the first coating. The silver nitrate present in the second coating reacts with the potassium chromate, or the potassium dichromate of the first coating to form silver chromate, or silver dichromate respectively, on the substrate. The "silver chromate" and/or "silver dichromate" are also otherwise termed as the complexing agent. The second coating is a continuous coating having varying concentration of the silver nitrate, in one embodiment. In another embodiment, the second coating is a discrete coating.
[0056] A concentration of the silver nitrate to be dispensed on a specific area of the first coating is derived from the concentration of potassium salts at the specific area of the first coating for the complete reaction between the silver nitrate and the potassium salts to form the complexing agent. The concentration of the silver nitrate in the second coating is proportional to the concentration of potassium salts in the first solution and is based on the volume of the sample that may be dispensed on the test strip, the concentration of the potassium salts in the first solution, concentration of silver in the second solution, the measurable concentration range of the test strip desired, the sensitivity of the test, or combinations thereof.
[0057] Once the concentration of silver required for deposition onto the test strip is calculated, at step 506, the concentration of silver nitrate in the second solution may be calculated. Further, the silver loading per square centimetre (cm2) on the test strip may be determined from Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), and the method of deposition may be further fine-tuned to obtain the calculated loading of silver on the test strip.
[0058] In one embodiment, applying the second solution at step 506 is performed using a plotter controlled by a computer numerical controller (CNC). The variable concentrations of the second coating can be achieved using the CNC plotter by changing the concentration of the second solution, fine-tuning the specific deposition area over the first coating, by changing a speed of the plotter, or combinations thereof.
[0059] In one embodiment, the method comprises drying the substrate after step 504, or after step 506, or both. In contrast to immersing the whole substrate, calculated amounts of the first solution and the second solution are dispensed on the substrate using the inventive method hence the time required for drying the substrate is shorter.
[0060] In some embodiments, step 506 may be performed before step 504. The method further comprises optionally attaching a wicking pad to an opposite end of the substrate and adjacent to the sample elution area to absorb excess sample after sample elution.
[0061] The process variability in forming the test strip is minimized by the present disclosure by precise dispensing of calculated concentrations of the first and second solutions, rather than using a substrate immersion method. The inventive approach significantly reduces reagent wastage when compared to immersion methods. Moreover, it provides extensive flexibility and versatility to fine-tune the measurable concentration range and sensitivity without the need to increase a length of the strip, unlike other distance-based PADs.

EXAMPLES
EXAMPLE 1
Preparation of a test strip for chloride detection
[0062] A nitrocellulose paper (FF High Performance nitrocellulose membranes from Cytiva) was used as the substrate. To ensure uniform deposition of silver chromate and consistent loading over the substrate, a 2D CNC plotter was employed for the controlled deposition of precursor salt solutions. A Faber-Castell brush pen (size B) was attached to the plotter and served as the dispensing tool. Before use, the pens underwent thorough cleaning. The end caps were removed, and the sponge ink reservoirs and brush nibs were rinsed with tap water to remove any residual black ink. Subsequently, the nibs and reservoirs were dried in a hot air oven for 1 hour. After drying, separate pens were filled with 40mM silver nitrate and 20mM potassium chromate solutions.
[0063] The prepared pens were mounted onto the 2D CNC plotter, and the desired deposition pattern was designed and converted to G-code using Inkscape software. The generated G-code was executed through UGSplatform software to control the plotter's movements. The deposition process involved depositing a layer of potassium chromate onto nitrocellulose paper, followed by a layer of silver nitrate covering an area of 50mm x 50mm. Following deposition, the nitrocellulose paper was supported with a backing paper. Additionally, a 20 mm wicking pad was attached to the end. The wicking pad facilitated the absorption of larger sample volumes. The prepared paper was cut into 80mm x 5mm strips using an MDI SS model M-100 strip cutter to form the test strips.
[0064] The strips were calibrated to measure salivary chloride ions at a measurable range of 0mM (or millimoles per liter (mmol/L)) to 30mM using standard chloride solutions of varying concentrations. A U-100 syringe was used to dispense samples (standard chloride solutions) on the test strip. A calibration curve 600 was generated by plotting distance traveled (cm) as a function of concentration (mM), as shown in FIG. 4. The "distance traveled" in centimeters (cm) is the length of the strip marked by change of colour from reddish-brown to white on the test strip on elution of the standard chloride solutions of varying concentrations. The calibration curve 600 revealed a linear dependence thus confirming the proportionality between the concentration and the distance traveled on the strip.
Testing of test strips for CF screening, monitoring and/or diagnosis
[0065] A study involving 129 children, 61 with CF and 68 without CF was conducted. The salivary samples of children (6 months to 18 years) diagnosed with CF coming to the weekly CF clinics (CF group) and of those without CF (Non-CF group), which included children without CF coming to the clinic and the children of hospital staff who volunteered to participate in the study, were collected after obtaining consent. A minimum of 0.5ml of saliva directly from the mouth using a 2ml syringe was extracted. All other children expectorated into a chloride-free container ensuring a gap of at least 30 minutes after food and 15 minutes after water.
[0066] The testing was further extended to groups of children with CF who were not on any modulator treatment, CF children on Elexacaftor/Tezacaftor/Ivacaftor (ETI) treatment (CF children on ETI), CF children not on any modulator treatment, or children on treatment with two drug combinations, TI/LI) (CF children not on ETI), and the children without CF. The salivary samples were analyzed using the chloride analyzer at the laboratory (denoted as laboratory salivary chloride) and also using the test strips prepared using Example 1 (denoted as strip salivary chloride). Table 1 provides measurement values from laboratory salivary chloride and strip salivary chloride for each of the groups.



Group Description
laboratory salivary chloride
strip salivary chloride

N (mmol/L)
Mean±SD
N (mmol/L)
Mean±SD
children diagnosed with CF 61 24.80±10.95 46 20.20±5.65
CF children not on any modulators 12 37.08±14.57 9 24.22±6.20
CF children not on ETI 25 30.48±13.74 18 23.06±5.94
CF children on ETI 32 20.78±6.07 28 18.68±4.88
children without CF 68 18.0±4.7 25 19.60±4.86

Table 1
[0067] The tests established that a salivary chloride threshold of 20.5 mmol/L using the Corning Chloride Analyser 925, effectively differentiated children with and without CF. The test strips differentiated between children with CF who were not on any modulator treatment and the children who did not have CF. It was also established that chloride levels in the saliva can be effectively used as a screening and/or as a diagnostic tool and also to help monitor the response to CF treatment, especially when on ETI. The detection of salivary chloride levels between the two methods, by way of laboratory or by way of the strips, was found to be very similar and therefore, establishes the strips as an acceptable method to screen, diagnose, and/or monitor treatment response.
[0068] FIG. 5 is a scatter plot 700 of correlation between laboratory salivary chloride values and strip salivary chloride values. A strong positive correlation (Pearson correlation, r=0.66, p<0.01) was observed between laboratory salivary chloride and strip salivary chloride values, as shown in FIG.5. Both the values, namely laboratory salivary chloride and strip salivary chloride values, were largely in agreement. However, certain variations in strip salivary chloride values compared with the laboratory salivary chloride values were seen which may be due to higher viscosity in CF salivary samples compared to the non-CF salivary samples and due to initial test strips having a maximum concentration limit of 30 mmol/L, due to which several CF patients showing higher values of chloride concentration were missed out.
[0069] The Bland-Altman plots 800 and 900 were used to assess the agreement between laboratory salivary chloride and strip salivary chloride values, as shown in FIGs 6a and 6b. In FIG. 6a, the Non-CF cohort (n=23), the mean bias was 0.21 with a standard deviation (SD) of 2.9 and limits of agreement between 6.09 and -5.66, which was well within the acceptable levels of agreement. In FIG. 6b, the CF cohort (n= 47), the mean bias was -1.17 with SD 4.52 and limits of agreement 7.7 and -10.04. The Bland-Altman plots 800 and 900 show that the chloride detection strips have an acceptable level of agreement with the laboratory method for both Non-CF and CF groups, thus confirming that the inventive strips can be effectively used for salivary chloride measurement in CF screening.
[0070] It is to be understood that the above description is intended to be illustrative, and not restrictive. Furthermore, many other embodiments will be apparent to those of skill in the art upon reading and understanding the above description. Although the present disclosure has been described with reference to specific exemplary embodiments, it will be recognized that the disclosure is not limited to the embodiments described, but can be practiced with modification and alteration within the scope of the appended claims.
, Claims:CLAIMS

I/We Claim:

1. A test strip (100) for measuring chloride ion concentration in a sample comprising:
a substrate (102) with a sample port (104) at one end; and
a sample elution area (106) adjacent to the sample port (104), wherein the sample elution area (106) comprises a complexing agent, wherein a concentration of the complexing agent is pre-determined and is variable along a direction of sample elution in the sample elution area (106), and wherein the chloride ion concentration in the sample is measured from a detectable visual change in the sample elution area (106) resulting from a reaction of the complexing agent with chloride ion in the sample.
2. The test strip (100) as claimed in claim 1, wherein the test strip (100) optionally comprises a wicking pad (108) attached to an opposite end of the substrate (102) and adjacent to the sample elution area (106) to absorb excess sample after sample elution.
3. The test strip (100) as claimed in claim 1, wherein the substrate (102) is single-layered and has substantially uniform pore size.
4. The test strip (100) as claimed in claim 1, wherein the substrate (102) comprises cellulose-based paper, or nitrocellulose-based paper, or glass fibre-based substrate.
5. The test strip (100) as claimed in claim 1, wherein the complexing agent comprises silver chromate, or silver dichromate, or combinations thereof.
6. The test strip (100) as claimed in claim 1, wherein the sample comprises a biological fluid comprising sweat, saliva, or combinations thereof.
7. The test strip (100) as claimed in claim 6, wherein the sample comprises saliva.
8. The test strip (100) as claimed in claim 1, wherein the concentration of the complexing agent is pre-determined to control a sensitivity of the test strip, or a measurable concentration range of chloride ion by the test strip, or both.
9. The test strip (100) as claimed in claim 1, wherein the sample elution area (106) comprises complexing agent provided as discrete bands perpendicular to the direction of sample elution.
10. The test strip (100) as claimed in claim 1, wherein the test strip (100) has a measurable chloride ion concentration in a range of 0.01 milliMolar (mM) to 4000 mM.
11. The test strip (100) as claimed in claim 6, wherein the test strip (100) has a measurable chloride ion concentration in a range of 10 milliMolar (mM) to 40 mM when the sample comprises saliva.
12.A test strip (100) for measuring chloride ion concentration in a salivary sample comprising:
a substrate (102) with a sample port (104) at one end; and
a sample elution area (106) adjacent to the sample port (104), wherein the sample elution area (106) comprises a complexing agent, wherein a concentration of the complexing agent is pre-determined and is variable along a direction of sample elution in the sample elution area (106), wherein the chloride ion concentration in the sample is measured from a detectable visual change in the sample elution area (106) resulting from a reaction of the complexing agent with chloride ion in the sample, and wherein a minimum concentration measurable by the test strip (100) corresponds to a threshold concentration of 10 mM and a maximum concentration measurable by the test strip is 40mM.
13. A method for measuring chloride ion concentration in a sample using the test strip (100) as claimed in claim 1.
14. The method as claimed in claim 13, wherein the sample comprises a biological fluid comprising sweat, saliva, or combinations thereof.
15. A method of preparing a test strip for measuring chloride ion concentration in a sample comprising steps of:
(i) providing a substrate (502);
(ii) applying a first solution comprising potassium chromate or potassium dichromate to form a first coating (504) over a sample elution area of the substrate, wherein a pre-determined concentration of the potassium chromate, or the potassium dichromate in the first coating is variable along a direction of sample elution to control a sensitivity of the test strip, or a measurable concentration range of chloride ion by the test strip, or both; and
(iii) applying a second solution comprising silver nitrate to form a second coating over the first coating (506), wherein a pre-determined concentration of the silver nitrate in the second coating is variable along the direction of sample elution and is proportional to the concentration of the potassium chromate, or the potassium dichromate in the first coating to form silver chromate, or silver dichromate in-situ in the substrate to form the test strip.
16. The method as claimed in claim 15, wherein the method comprises drying the substrate after step (ii), or step (iii), or both.
17. The method as claimed in claim 15, wherein the step (ii) and step (iii) are performed in any order.
18. The method as claimed in claim 15, wherein the method comprises optionally attaching a wicking pad to an opposite end of the substrate and adjacent to the sample elution area to absorb excess sample after sample elution.
19. The method as claimed in claim 15, wherein applying the first solution at step (ii), and applying the second solution at step (iii) are performed using a plotter controlled by computer numerical controller (CNC).
20. The method as claimed in claim 15, wherein the silver chromate or silver dichromate reacts with chloride ion in the sample to form silver chloride indicated by a detectable visual change in the sample elution area which is proportional to the chloride ion concentration.

Dated this 07th day of November 2024

Bikash Lohia
IN/PA - 1714
Agent for the Applicant


 
TEST STRIPS FOR MEASURING CHLORIDE ION CONCENTRATION
ABSTRACT

The present disclosure provides a test strip (100) for measuring chloride ion concentration in a sample. The test strip (100) comprises a substrate (102) with a sample port (104) at one end and a sample elution area (106) adjacent to the sample port. The sample elution area (106) comprises a complexing agent, where a concentration of the complexing agent is pre-determined and is variable along the direction of sample elution in the sample elution area (106). The chloride ion concentration in the sample is measured from a detectable visual change in the sample elution area (106) resulting from a reaction of the complexing agent with chloride ions in the sample. A method for measuring chloride ion concentration in a sample using the test strip is provided. A method of preparing the test strip (100) is also disclosed. The test strip (100) finds applications in screening, monitoring and/or diagnosing cystic fibrosis from a salivary sample.
[FIG. 1]

Documents