E–Eye for Point–of–Care sensing of Chromium in Environmental, Food & Beverages and Biological samples

Abstract

ABSTRACT TITLE: E–Eye for Point–of–Care sensing of Chromium in Environmental, Food & Beverages and Biological samples An optical portable, point-of-care device and method for the onsite detection of chromium in the aqueous phase in the environmental, clinical, and food samples are provided. The device comprises a casing for holding cuvettes where the colorimetric reaction takes place.A circuit consisting of LED–LDR with a display unit is interfaced with a microcontroller unit Arduino Uno. The microcontroller is algorithimically programmed to gibe digital readout for an easy user-friendly interface. The system is equipped with a solar pannelto operate in resource limited setting and a smartphone based application for digital storage of data. The device has been tested against more than 2000 field samples and the results accords with gold standards. The invention enables rapid, low-cost, selective and instrument-free detection of chromium in the field, with high specificity, sensitivity, and neglisible interference in the range of 5 µgL-1 to 2.5 mgL-1. It is particularly suited for deployment in remote, resource-limited, or healthcare-relevant environments where conventional analytical instrumentation is inaccessible. Figure 1

Description

Description:FIELD OF INVENTIONThe present invention entails an E–Eye for the Point–of–Care testing (POCT) of chromium (Cr) in biological samples. More specifically, the E–eye pertains to user-friendly colorimetric chromium sensor system as a portable, cost-effective, user-friendly device designed for real-time monitoring of chromium concentration in non-invasive body fluids for healthcare applications. The device utilizes a reagent-based optimized colorimetric reaction with 1,5-diphenylcarbazide (DPC) under an acidic environment. The device's hardware comprises an integrated circuit consisting of a light-emitting diode (LED) and a light-dependent resistor (LDR / photoresistor) interfaced with an Arduino Uno microcontroller. The reagent-based colorimetric reaction integrated with the electronic circuit provides a platform for selective, sensitive, and quick sensing of chromium in biological samples.BACKGROUND ARTChromium is a heavy metal found naturally in soil, rocks, sediments, and plants,primarily in its stable oxidation states of Cr(III) and Cr(VI). Cr(III) between Cr(III) and Cr(VI) is essential for promoting insulin actionin the most red-blooded creatures, including humans,which helps for the healthy metabolism of glucose, proteins, and fats [Shrivas, et al., "Localized surface plasmon resonance of silver nanoparticles for sensitive colorimetric detection of chromium in surface water, industrial waste water and vegetable samples", Journal 8, 2088-2096; 2016, Shrivastava, et al., "Effects of chromium on the immune system", Journal 34, 1-7; 2002]. Optimized dietary intake of chromium leads to improved blood lipid profile and reduces the requirement for insulin [Anderson, "Nutritional role of chromium", Journal 17, 13-29; 1981].Inside the human body, it is bound to red blood cells as chromate ions, and in the serum, these are found in the 𝛽-globulin fraction attached to serum transferrin [Burrows, "Chromium: metabolism and toxicity", Journal 137, 1983]. However, Cr(VI), on the other hand, is a potent carcinogen. The reduction of Cr(VI) to Cr(III) induces oxidative stress, causing cell transformation and ultimately leading to Apoptosis. At the microscopic and cellular level, it leads to DNA damage, gene alteration, and mutation in the nucleus [Shrivas, et al., "Localized surface plasmon resonance of silver nanoparticles for sensitive colorimetric detection of chromium in surface water, industrial waste water and vegetable samples", Journal 8, 2088-2096; 2016, Iyer, et al., "A review of chromium (Cr) epigenetic toxicity and health hazards", Journal 882, 163483; 2023]. Overexposure to hexavalent chromium causes ulcers of the skin and cancer of the respiratory tract [Shekhawat, et al., "Chromium toxicity and its health hazards", Journal 3, 167-172; 2015]. On the other hand,chromium deficiency can increase blood glucose, triglycerides, and cholesterol levels and increase the risks of diabetes and cardiac disorders [Anderson, "Chromium as an essential nutrient for humans", Journal 26, S35-S41; 1997]. The threshold limit of chromium in drinking water recommended by WHO is 0.05 mgL-1[Edition, "Guidelines for drinking-water quality", Journal 38, 104-8; 2011]. In the human body, the chromium content differs depending on what biological sample we are considering. In the whole blood, it lies in the range of 0.7-28 µgL-1, whereas in serum it is about 1 µgL-1. Chromium concentration in the saliva of a healthy person is in the range of 0.3 µgL-1 and in the hair it is in the range of 0.2 to 2 mg / kg. The normal range of chromium excreted in urine is 0.4-50 µgL-1[Burrows, "Chromium: metabolism and toxicity", Journal 137, 1983, Pechancová, et al., "Recent advances in chromium speciation in biological samples", Journal 152, 109-122; 2019]. It could be toxic if chromium content in the body exceeds these threshold limits. Chrome plating, stainless steel, pigment industries, etc. are major anthropogenic sources of chromium pollution. Humans are exposed to chromium from the environment via ingestion, inhalation, and cutaneous contact [Paustenbach, et al., "Human health risk and exposure assessment of chromium (VI) in tap water", Journal 66, 1295-339; 2003].Hence there is a need to explore for Point–of–Care testing (POCT) system towards user–friendly, rapid, selective, sensitive, and reliable sensing of chromium non–invasively in test samples including biological and environmental samples via colorimetric approach.OBJECTS OF INVENTIONThe prime objective of the present invention is to develop a POCT device to facilitate user–friendly, rapid, selective, sensitive, and reliable sensing of chromium in non–invasive body fluids using a colorimetric approach.Another important object of the invention is to provide photoresistor-based digital sensing of chromium in biological and environmental samples.Another important object of the invention is to design and develop an optical sensor that quantifies chromium based on the resistance offered by the light absorbed by the sample, akin to the UV-visible (UV-vis) Spectroscopy principle with digital readout.A further object of the invention is to design and develop a cost-effective system for onsite analysis that could mimic the spectroscopic system for quantification of target analyte in resource-limited settings.Another essential object of the invention is to provide quick, user-friendly, and cost-effective chromium sensing without employing tedious methods such as the synthesis of nanoparticles.Yet another object of the present invention is to precisely optimize the pH and reaction mixtures to provide the least documented limit of detection (LOD) value using the most straightforward colorimetric route. Another object of the present invention is to provide better sensitivity, the least count, and response and recovery time by incorporating hardware and software filters using microcontrollers. SUMMARY OF THE INVENTIONThus according to the basic aspect of the present invcention there is provided an user-friendly colorimetric chromium sensor system comprising a point of care optical sensor including acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor enabled for lock and key colorimetric reaction with chromium for generating concentration of chromium based colour intensity dependent purple-coloured complex as a colorimetric sensor of chromium.Preferably said user-friendly colorimetric chromium sensor system is provided wherein said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) colorimetric sensor is capable of said colorimetric detection of chromium in the pH range of 1.15-1.39, preferably 1.38, stability time of 5 mins to 5 days and temperature range of 25C-85C.More preferably said user-friendly colorimetric chromium sensor system is provided adapted as a Point–of–Care testing (POCT) system comprises photoresistor based chromium concentration / level detector for digital sensing of chromium concentrations based on said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) enabled lock and key colorimetric reaction with chromium generating concentration of chromium based colour intensity dependent purple coloured complex.Preferably said user-friendly colorimetric chromium sensor system is provided wherein said photoresistor device includes said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor including:a cuvette for containing test sample and said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor;said cuvette containing test sample operatively connected to an LED-LDR sensing mechanistics wherein the light-emitting diode (LED) is disposed opposite to the light-dependent photoresistor (LDR) across said cuvette for containing test sample such that LED light passing through sample in cuvette is absorbed and sensed by the photoresistor LDR;calibrator means operatively connected for converting said photoresist measured by LDR into said concentration of chromium in said test sample.More preferably said user-friendly colorimetric chromium sensor system is provided wherein said photoresistive device with its hardware components are electrically integrated to said LED-LDR sensing mechanistics by including said light-emitting diode (LED) (303) and said light-dependent photoresistor (LDR / photoresistor) LDR (305) detector disposed at the opposing faces of sample holder (304) holding cuvette (306) for containing Chromium based samples;said light-emitting diode (LED) (303) connected to a power source (301) via resistors (302);said light-dependent resistor (LDR / photoresistor) LDR (305) detector interfacing with Arduino Uno microcontroller (307) across a printed circuit board (PCB) adapted as central processing unit (CPU) in turn connected to chromium quantifying processor (309) based calibrator via jumper cables (308).According to another preferred aspect of the present invention there is provided said user-friendly colorimetric chromium sensor system wherein said electrically integrated hardware components of photoresistive device is housed by enclosure members with a lid impermeable to external light allowing sample entry based on opening and closure of the lid, said enclosure member equipped with a read-out display preferably LCD display compatible with smartphone applications interfacing said chromium quantifying processor (309) enclosed within to display chromium concentration of the sample empirically related to the light-dependent photoresistance encountered from chromium based samples including biological and environmental samples.Preferably said user-friendly colorimetric chromium sensor system is provided wherein said device is powered by plurality of sources via a solar panel (311) mounted on top of enclosure member connected to a power source (301) preferably a 12 V battery via universal serial bus (USB) cable (312).More preferably said user-friendly colorimetric chromium sensor system is provided wherein upon illumination of LED light (400–700 nm) the same passes through the sample contained in the cuvette and gets absorbed providing colorimetric Cr (VI) concentration-colour intensity responsive corresponding LDR detector signal for algorithmic conversion to digital signal by empirical Cr (VI) quantifying processor (309) for display at the LCD interface in ppb scale (µgL-1), said sensor is sensitive to Cr in the range of 5 ppb to 2.5 ppm providing a sharp peak at 543 nm that is selectively unresponsive to interfering metal ions including Fe(III), Pb(II), and Cu(II), with the limit of detection (LOD) of the sensor being as low as 6 ppb / 6.1 µgL-1, Limit of Quantification (LOQ) 18.48 µgL-1 respectively in ppb scale being the least reported for colorimetric sensing.According to another preferred aspect of the present invention there is provided said user-friendly colorimetric chromium sensor system wherein said generation of intensity based pH stabilized 1, 5-diphenylcarbazide (DPCA) chromogenic sensor is adaptive to Cr (VI) ion sensing upon forming 1,5-diphenylcarbazide (DPCA)-Cr(III) complex thereby favouring selective, sensitive, and quick sensing of chromium in said samples.According to another aspect of the present invention there is provided a method for chromium sensing in samples including environmental samples of ground water including user-friendly colorimetric chromium sensor system comprising Providing a point of care optical sensor including acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor;Providing Chromium samples including pre-treated chromium samples for interaction with said sensor enabling a lock and key colorimetric reaction with chromium and generating concentration of chromium based colour intensity dependent purple-coloured complex as a colorimetric sensor of chromium for related detection.Preferably in said method wherein said optical sensor is prepared based on 0.5% w / v 1,5-diphenylcarabzide (DPC) involving 250 mg of DPC dissolved in 25 mL acetone, then diluted up to the mark of 50 mL with milli-Q water followed by preparing a solution of 0.15 M H2SO4 for necessary pH stabilization in acidic range.More preferably said method is provided wherein said pre-treated chromium samples are pre-treated post collection based on the following:environmental samples that are collected in acid-washed polyethylene or Teflon containers, followed by optional membrane filtration (0.45 µm) for dissolved metal analysis, followed by acidification involving concentrated nitric acid to lower the pH below 2, thereby stabilizing metal ions and preventing microbial activity or precipitation whereby total Cr in samples undergo acid digestion using concentrated nitric acid optionally involving hydrogen peroxide under controlled heating (85–95°C) until a clear solution is obtained followed by filtering the clear samples with deionized water, stored at 4°C in acid-cleaned containers for analysis within a recommended holding time;urine samples collected and filtered with Whatman filter paper grade-1 of pore-size 20-25 µm to remove cellular extracts and any particulate matter leading to turbidity followed to which it is centrifuged at 10000 rpm for 30 minutes and then digested with nitric acid (65%) in a volume ratio of 1:19 to suppress any organic interference at a temperature of 80 °C that is digested and stored at 4 °C diluted 10-fold with mili-Q water before investigation;hair samples of 1 gram of hair samples were finely cut into small pieces and soaked in the solution mixture consisting of diethyl ether, acetone, and DI water in a ratio of 3:1:20 under an ultrasonication bath for 1 hour followed by drying in a glass Petri dish at 105 °C for 24 hours that was thereafter digested with nitric acid (HNO3) and hydrogen peroxide (H2O2) in a ratio of 3:1 (v / v) with said digested hair samples dried at 80 °C near to dryness, to which dried samples was added 10 mL of 0.1 M HNO3 and filtered using Whatman filter paper grade-1 of pore-size 20-25 µm and stored at 4 °C until further analysis.More preferably said method is provided wherein said chromium sensing is carried out based on Providing said samples / pre-treated test samples in a cuvette together with said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor followed by adding Cr(VI) solution to the cuvette for producing purple-colored complex [DPCA-Cr(III) complex];Illuminating said cuvette by LED-LDR sensing mechanistics wherein the light-emitting diode (LED) disposed opposite to the light-dependent photoresistor (LDR) across said cuvette allows passing of LED light through sample in cuvette that is absorbed and sensed by the photoresistor LDR as light-dependent photoresistance encountered from chromium based samples;Obtaining corresponding chromium concentration of the test sample linearly related to and empirically converted from said light-dependent photoresistance as displayed in the display of the calibrator.BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES OF THE INVENTIONFigure 1 depicts the schematic and mixing scheme of the reaction.Figure 2 (a–c) depicts optimizing different parameters of the sensor: (a) Effect of pH, (b) Temperature,and (c) Time, based on UV-vis spectroscopic absorbance.Figure 3 (a–d) depicts the design and development of the device. (a) Sensor Set-up. (b) External Sketch. (c) Schematic of the circuit. and (d) Image of the Device.Figure 4 depicts the selectivity study of the device. Inset UV Spectra of Different Elements.Figure 5 (a-c) depicts the UV characterization of the DPCA-Cr(III) complex: (a) Colorimetric Intensity of the complex, (b) UV Spectra, and (c) UV Calibration.Figure 6(a-d) depicts the Calibration of E-Eye. (a) Calibration Plot using prototype up to a concentration of 500 µgL-1, (b) Calibration Plot using prototype w.r.t UV, (c) Calibration Plot using prototype up to a concentration of 2.5 mgL-1, and (d) Calibration Plot using prototype w.r.t AAS.Figure 7 depicts theSensor validation with spiked DI water.Figure 8 (a-e) depicts theSensor validation with Environmental Samples: (a) Brahmaputra water, (b) Lake water, (c) Tap water, (d) Borewell 1, and (e) Borewell 2.Figure 9 (a-b) depicts theSensor validation with Clinical Samples: (a) Urine, (b) Extracts of Hair.Figure 10 (a-b) depicts theSensor validation with Food Samples: (a) Cowmilk, (b) Extracts of Peanuts.DETAILED DESCRIPTION OF THE INVENTIONAs discussed hereinbefore, the present invention thus discloses a portable, selective, and digital E-Eye for sensing chromium in samples including biological / environmental samples. Colorimetric sensing of the analyte has been established, akin to the spectrophotometric absorption principle. Chromium in the sample is measured by forming a purple-colored complex resulting from a lock and key-reaction between 1,5-diphenylcarbazide and chromium. The present invention provides for a system comprising of an optical source (Light-Emitting diode (LED)), a photoresistor (Light Dependent Resistor (LDR)), and a programmable microcontroller with a display unit Liquid Crystal Display (LCD) to give digital output. The system comprises a three-dimensional (3D) printed enclosure for the electronic printed circuit board (PCB), with a confined slot for LED geometrically opposite to LDR and a quartz cuvette of 10 mm × 10 mm between the LED and LDR.Figure 1 shows the schematic of the reaction. The reaction mixture consists of H2SO4 and DPC solution poured into a cuvette. Then, a chromium (VI) solution is added to this reaction mixture, which leads to a colorimetric reaction between chromium (VI) and the reaction mixture to form a purple complex. The produced color complex is the basis of the colorimetric detection of chromium.Figure 2 refers to the optimization parameters of the colorimetric reaction. Various reaction parameters (such as pH, stability time, temperature, etc.) were tuned to establish the optimum reaction condition. ThepH of the reaction was optimized by varying the concentration of H2SO4, keeping the other parameters of the reaction constant. The effect of temperature on the complex was studied by changing the temperature from room temperature to elevated temperature keeping other parameters constant. Similarly, the time stability of the complex was also investigated up to 5 days.Figure 2a refers to the optimization of pH. Figure 2a reveals that the DPCA-Cr(III) complex absorbs maximum intensity at pH 1.38, which is achieved at the concentration of 0.15M H2SO4.That is why pH 1.38 is accepted as an optimized pH, and the sensor has been developed at this pH.Figure 2b reveals that temperature optimization demonstrates minor changes in absorbance value within the experimental temperature range (25 to 85 ). Hence, the room temperature (25 ) was finalized as the sensor's operating temperature. Time optimization is essential for developing a stable, reproducible, and reliable digital sensor. For this purpose, UV-absorbance data of the complex at different time intervals was collected. Figure 4c shows that the stability of the DPCA-Cr(III) complex is viable for up to six hours. So, all the measurements are mandated to be completed within six hours only.Figure 3a–d refers to the design and development of the sensor herein referred to as E–Eye for quantification of chromium in biological samples. The photoresistive device prototype is based on an LED-LDR sensing mechanism. The electronic hardware of the E-Eye includes LED (303), LDR (305), LCD (309), resistors (302), and jumper cables (308). These elements are interfaced with the Arduino Uno microcontroller (307) across a printed circuit board (PCB) to form the device's central processing unit (CPU).Figure 3d reveals the 3D printed enclosure that has a well-defined slot for holding the CPU and other components such as LED, LDR, and the cuvette (306). Figure. 3a shows the LED was held opposite to LDR across a cuvette holder (304) in which the cuvette-containing sample was placed. A cuvette (306) of standard dimension with a path length of 10 mm was used.Figure. 3b shows the external sketch of the circuit, and a schematic diagram of the circuit is shown in Figure 3c. Figure 3d shows the finalform of the digital sensor (310) for measuring chromium concentration. The device can be powered with multiple sources via a solar panel (311) mounted on top of it, a 12 V battery (301), and a universal serial bus (USB) cable (312). When the LED is illuminated, light passes through the sample present in the cuvette and gets absorbed. LDR measures the sample's resistance and records data to prepare a calibration curve. This Calibration is used in an algorithm to convert the photoresistance to the concentration of the sample, and the concentration value is displayed on the LCD of the E-Eye in the ppb scale (µgL-1).The experiments were performed based on the scheme shown in Fig. 3b. The reaction mixture of H2SO4 and DPC was poured into a cuvette. Then, a chromium(VI) solution was added to this mixture, which produced a purple complex. This complex was analyzed by UV vis spectrophotometer (Shimadzu UV 2600) at a wavelength (𝜆max) of 543 nm. UV absorbance was recorded in the standard samples of known concentrations ranging from 10 µgL-1 to 2000 µgL-1. All the readings were taken in triplicate, and a calibration plot was prepared at maximum absorbance. Similar experiments under the same conditions were performed using the indigenously developed E-Eye-based digital chromium sensor. Based on the working principle of the E-Eye, the sensor prototype (Figure 3b) measured the photo resistance of the sample corresponding to its color intensity. The resistance value was then correlated with the samples’ concentrations, and a calibration plot for the E-Eye-based digital sensor was developed, which was used to get the concentration of the unknown samples.Figure 4 depicts the selectivity study of the present invention. To explore the selectivity of the present invention, the reaction was carried out at a higher concentration of possible interfering agents present in the environmental and biological samples. The solutions of the interfering elements such as As(III), Co(II), Cu(II), Fe(II), Fe(III), Ni(II), Pb(II), Zn(II), Na(+), and F(-) were prepared at a concentration of ten times higher than the corresponding threshold value recommended by WHO. Similarly, for organic interferants such as urea and glucose, its solutions were prepared in a concentration higher than the threshold value recommended by the American Association of Clinical Endocrinologists (AACE).Selectivity was also tested against urea and glucose as an interfering element in urine. The study reveals that (Figure 4) none of these agents show any interferenceat a wavelength of 543 nm, and a distinct peak at the given wavelength was observed in the case of chromium only. UV spectra of this study arealso shown in the inset of Figure 4. Since the absorbance of interfering elements was< 5%, the present invention could be a highly selective device for sensing the application of chromium in environmental and biological samples.Figure 5 depicts the UV visible spectrophotometer study for the said DPCA-Cr(III) complex. At the outset, aqueous Cr(VI) solution were prepared in different concentrations and added to the reaction mixture. The appearance of purple color in the reaction confirms the accomplishment of the reaction. The color intensity of the complex increased as we increased the chromium concentration, as shown in Figure 5a.UV-vis spectroscopy wasperformed to study the characteristics of the complex, and a predominant peak at the wavelength (𝜆max) of 543 nm (Figure 5b) confirms the formation of DPCA complex with chromium. A hyperchromic shift was observed as we increased chromium concentration in the aqueous solution. As we increased the concentration of chromium ions from 0 to 1.5 mgL-1, a smooth curve in the UV-vis spectra was recorded. A noise in the spectra was observed (Figure 5b) on further increasing the concentration beyond 1.5 mgL-1, which was more pronounced at higher concentrations. It may be due to reaching the saturation point of the UV absorbance of the colorimetric solution. Hence, the upper absorbance limit was marked as 2.5 mgL-1.A standard calibration plot was prepared (Figure 5c) employing these UV-vis absorbance data for future use. The calibration plot shows an outstanding linear response with a regression coefficient (R2) of 0.999. UV-vis spectroscopy has been used as a gold standard method for estimating unknown concentration of real samples. Concentrations of the actual field samples were estimated with the help of this plot and used to validate the performance of the E-Eye digital chromium sensor.Figure 6 shows the Calibration of the E-Eye prototype and validation with UV and AAS data. The indigenous E-Eye prototype was calibrated with different chromium concentrations ranging from 10 µgL-1 to 500 µgL-1,as shown in Figure 6a. The calibration plot shows an excellent linear response with a regression coefficient (R2) value of 0.9975. The calibration equation is also displayed on the graph (Figure 6a), which is incorporated in the algorithm of the digital chromium sensor. To judge the wide applicability of this Calibration, a maximum limit was set at 500 µgL-1 (Figure 6a), which is ten times higher than the WHO recommended value. The plot calculates the Limit of Detection (LOD) and Limit of Quantification (LOQ). The LOD and LOQ of the sensor were calculated using the equation given below:LOD=3.3×(standard deviation (σ)) / (slope (m)) (1)LOQ=10×(standard deviation (σ)) / (slope (m)) (2)where ‘σ’is the slandered deviation of the blank sample. The LOD and LOQ of the digital sensor using the above equation were reported to be 6.1 µgL-1 and 18.48 µgL-1, respectively. These values are the least reported values in literature following the DPC route. This calibration plot was further validated with UV-vis spectroscopy as well as AAS data. For that, the calibration data of the prototype was compared with that of UV-vis and AAS calibration data in graphical form, as shown in Figure 6b and Figure 6d. An excellent linear response was observed between them with an R2 coefficient of 0.9955 and 0.9971 for UV-vis and AAS, respectively, thus confirming the authenticity of the prototype of the E-Eye chromium sensor. Figure 7 illustrates and establishes usefulness and practical applications of the indegeneously fabricated device. The performance of the developed E-Eye POCT was tested with various real samples, namely environmental, clinical samples, and food samples. Two types of environmental samples were collected- surface and underground water. Among the clinical samples, urine and hair extracts were investigated for the non-invasive detection of chromium. The test results were also compared with the gold standard (both UV-vis spectroscopic and AAS) results to scrutinize the performance of the chromium sensor. Before going to real samples, test runs were conducted with the synthesized samples spiked in DI water. For this purpose, the various samples of DI water were spiked with unknown chromium concentrations. The concentrations of synthesized samples were measured with the indigenously developed prototype, UV-vis spectrophotometer, and AAS. All the results were compared and shown in Figure7. The study reveals that the prototype results coincide with UV and AAS results, and the POCT could successfully report concentration well below the threshold value of 50 µgL-1 recommended by WHO. The red horizontal line given in the plot indicates the WHO recommended value, and the black dotted line represents the LOD value. However, the prototype’s reading slightly deviates from the UV-vis results. The deviations were calculated in terms of % error. The absolute error of the individual synthesized samples was estimated using Eq. (3). Then, an average value was evaluated based on all the synthesized samples. The table indicates only a 6.67% error for synthesized samples in DI water for UV and 1.24% for AAS. This performance of the prototype encourages us to check its efficacy on the environmental and clinical samples, as discussed in the following experimental studies.% Error=|(〖Equipment〗_reading -〖 prototype〗_reading) / 〖Equipment〗_reading ×100| (3)N.B Equipment used here is UV and AASEXAMPLESEXPERIMENTAL STUDIES Example 1Validation of Indigenously Developed Chromium Sensor on Surface WaterSurface water samples were collected from the Brahmaputra river (Guwahati, Assam) and lake inside IIT Guwahati. Domestic municipal water supply (tap water) was also investigated. There are a total of 35 samples- 12 from the Brahmaputra river, 11 from the lake inside IIT Guwahati, and 12 from tap water. These samples were pretreated. The surface water from this region suffers contamination from heavy metals and total dissolved solids. Chromium was absent in the raw samples, as detected by AAS and UV-vis spectroscopic analysis. Due to this, several spiked samples were prepared for investigation by adding 2 mL of chromium solution with different concentrations prepared from a stock solution. Then, the chromium concentration of the spiked samples was measured using the indigenously developed sensor prototype and UV-vis spectroscopy. Figure 8a–c illustrates all the results (prototype, UV-vis spectroscopy, and AAS) compared and presented as a bar diagram. All the readings in the figures are triplicate with a small standard deviation, as shown in the figures with the vertical bars of corresponding samples (Figure 8a–c). The statistics ensure that the sensor prototype gives good accuracy in measurement as its reading (green bars) accords with UV-vis spectroscopy measurement (purple bars) and AAS measurements (gray bars). The absolute error of all the samples was also calculated using Eq. 3. The prototype gives an average absolute error of 6.23%, 10.31%, and 9.64% for the water of Brahmaputra River, a lake inside IIT Guwahati, and tap water, respectively using UV-vis measurements and the corresponding error using AAS are reported to be 0.66%, 0.77% and 1.51%. Black dotted horizontal lines on the plots in Figure 8a–c indicate the LOD value of our indigenously developed sensor prototype. The chromium sensor can detect chromium concentration below the threshold value recommended by WHO, as indicated by a horizontal red line in the figures.Example 2Validation of Indigenously Developed Chromium Sensor on Underground WaterA large population depends on the underground water for their daily needs and agricultural works. So, attention was paid to checking its chromium content. Underground water was collected from Kamrup Metropolitan and Kamrup Rural, which lie on the opposite banks of the Brahmaputra River. Eleven samples from each source, namely Kamrup Metropolitan and Kamrup Rural, have been collected and labeled as bore-well 1 and 2, respectively. The samples were pretreated. After the pretreatment, the chromium concentration of raw samples was measured using UV-vis spectroscopy and AAS. The analysis showed that the samples contained a very small amount of chromium (2.6 µgL-1), well below the WHO limit. Then, the samples were spiked with chromium from the previously prepared stock solution to examine the sensor performance further. For this, the concentration of the samples was measured with the indigenously developed sensor prototype, and the recorded values were verified with UV-vis spectroscopy and AAS. All the measurements were in triplicate. The corresponding error bars have also been included in the figure (Figure 8d–e). The study results are pictorially represented as a bar graph in Figure 8d and Figure 8e. The study shows that the prototype readings agree with the UV-vis spectroscopy and AAS with a slight deviation. The absolute error was evaluated using Eq. (3), and the average value of 5.68% and 3.38% was found for bore-well 1 and bore-well 2, respectively, with respect to UV. The absolute error concerning AAS is 1.3% and 0.9% for bore-well 1 and 2, respectively. The sensor can detect chromium up to 14 µgL-1, well below the threshold limit recommended by WHO, as indicated in the figure.Example 3Validation of Indigenously Developed Chromium Sensor on Clinical SamplesThe E-Eye prototype's success with the environmental samples also prompted us to extend our study to clinical samples. Chromium plays a vital role in insulin's activity in the human body. However, the excess chromium harms human health, as deliberated in the background art. The excess chromium may be excreted via body fluids or accumulated in the nails, hair, bones, etc.Therefore, samples of urine and hair extracts were chosen to study for the non-invasive detection of chromium in biological samples. A total of 23 samples of urine and hair were collected from healthy volunteers. Of these, 13 samples are for urine, and 10 are for hair. Pretreatment of urine and hair was done as per the procedure mentioned. The samples of treated colourless urine and hair extract were found to be free from chromium with the help of AAS and UV-vis spectroscopy analysis. These samples were then synthesized following the spiking technique. The chromium concentration of these synthesized samples was measured with the E-Eye prototype, UV-vis spectroscopy, and AAS. The results are compared in Figure 9. The results show good accord with UV-vis spectroscopy and AAS data. The green vertical bar represents the prototype's reading, whereas the UV- vis and AAS readings are represented by purple and gray, respectively. The absolute average % error was also calculated and was reported to be 5.22% and 2.75% in urine and hair, respectively,w.r.t UV-vis, and the corresponding value using AAS was reported to be 0.6% and 1.07%. The developed prototype could sense chromium below the threshold limit of 45 µgL-1 in urine, as recommended by the American Conference of Governmental Industrial Hygienists (ACGIH).Example 4Validation of Indigenously Developed Chromium Sensor on Food SamplesThe success of environmental and food samples prompted us to investigate food samples as well. Figure 10 refers to the investigation offood samples. The cowmilk (Figure 10a) and peanut (Figure 10b) extracts were chosen for this purpose. A total of 20 samples, 10 of each cowmilk and peanut, were investigated with the chromium sensor. The samples were prepared using the spiking technique, and the results were validated to gold standards. The results show good accord with UV-vis spectroscopy data. The green vertical bar represents the prototype's reading, whereas the UV- vis readings are represented by purple.The absolute average % error was also calculated and reported to be 8.27% and 7.85% in cowmilk and peanuts, respectively.EXPERIEMENTAL METHODS AND PROTOCOL Preparation of the Reagents Pretreatment of Environmental SamplesThe method comprises the collection of samples in acid-washed polyethylene or Teflon containers, followed by optional membrane filtration (0.45 µm) for dissolved metal analysis. Samples are immediately acidified in the field using concentrated nitric acid to lower the pH below 2, thereby stabilizing metal ions and preventing microbial activity or precipitation. For total Cr analysis, the sample undergoes acid digestion using concentrated nitric acid, optionally with hydrogen peroxide, under controlled heating (85–95°C) until a clear solution is obtained. The digested or filtered samples are then diluted with deionized water, stored at 4°C in acid-cleaned containers, and analyzed within a recommended holding time. Pretreatment of Urine SamplesThe urine sample of a healthy person was collected and filtered with Whatman filter paper grade-1 of pore-size 20-25 µm. This filtration is essential to remove cellular extracts and any particulate matter that could lead to turbidity of urine. The filtered urine was centrifuged at 10000 rpm for 30 minutes and then digested with nitric acid (65%) in a volume ratio of 1:19 to suppress any organic interference at a temperature of 80 . The digested urine was then stored at 4 , diluted 10-fold with mili-Q water before investigation. Pretreatment of Hair SamplesTypically, 1 gram of hair samples were finely cut into small pieces and soaked in the solution mixture consisting of diethyl ether, acetone, and DI water in a ratio of 3:1:20 under an ultrasonication bath for 1 hour. Following this, the hair samples were kept in a glass Petri dish for drying at 105 for 24 hours. The dried hair samples were then digested with nitric acid (HNO3) and hydrogen peroxide (H2O2) in a ratio of 3:1 (v / v). The digested hair samples were dried at 80 near to dryness. To this digested sample, 10 mL of 0.1 M HNO3 was added, and the extract was filtered using Whatman filter paper grade-1 of pore-size 20-25 µm and stored at 4 ℃ until further analysis. Preparation of the ReagentsA solution of 0.5% w / v 1,5-diphenylcarabzide (DPC) was prepared. For this, 250 mg of DPC was measured and dissolved in 25 mL acetone, then diluted up to the mark of 50 mLwithmilli-Q water. A solution of 0.15 M H2SO4 was prepared. A stock solution of 200 mgL-1 (200 ppm) Cr(VI) was prepared by dissolving K2Cr2O7. Similarly, a stock solution of 200 mgL-1 of other heavy metal ions was prepared for selectivity study. All the stock solutions were used by serial dilution. All the experiments were carried out at room temperature (25 ). Preparation of the SensorA handheld POCT device, i.e., the E-Eye, was designed based on the colorimetric reaction between DPCA and Cr(III). The photoresistive device prototype was based on an LED-LDR sensing mechanism. The necessary electronic components for this prototype are LED, LDR, LCD, resistors, etc. White LED was used throughout the experiments, covering the entire visible spectrum from 400–700 nm. The chromium complex absorbs sharply at 543 nm (Figure 5b). A substantial portion of the white LED emission overlaps with the green region (500–570 nm).The components were interfaced with the Arduino Uno microprocessor across a voltage divider circuit. The LED was fixed opposite to LDR across a cuvette holder (Fig.1a), in which a cuvettecontaining the sample was placed to hold the sample. Figure3b shows the external sketch of the circuit, and a schematic diagram of the circuit is shown in Figure 3c. Figure 3d shows the actual image of the digital sensor for measuring chromium concentration. Light is passed through the sample present in the cuvette when light is illuminated, and it gets absorbed. LDR measured the resistance of the light falling on it and was recorded to prepare a calibration curve, as discussed in section 2.5. This Calibration was used in an algorithm to convert the photoresistance to the concentration of the sample, and the concentration value was displayed on the LCD of the E-Eyesensor prototype in the ppb scale (µgL-1). Procedure for Testing of ChromiumThe experiments were performed based on the scheme shown in Figure 3b. The reaction mixture consisting of H2SO4 and DPC was taken into a cuvette. Then, Cr(VI) solution was added to this mixture, which produced a purple-colored complex (DPCA-Cr(III) complex). This DPCA-Cr(III) complex was confirmed by a UV-vis spectrophotometer (Shimadzu UV 2600) at a wavelength (𝜆max) of 543 nm. UV absorbance was recorded in the samples of different known concentrations ranging from 10 µgL-1 to 2000 µgL-1. All the readings were taken in triplicate, and a calibration plot was prepared at maximum absorbance. Similar experiments under the same conditions were performed using the indigenously developed E-Eye digital chromium sensor. Based on the working principle of the E-Eye, as described in paragraph above, the sensor prototype (Fig.1b) measured the photo resistance of the sample corresponding to its color intensity. The resistance value was then correlated with the samples' concentrations. A calibration plot for the E-Eyesensor prototype was developed and used to get the concentration of the unknown samples. The device readings were compared with the results obtained from UV-Vis Spectrophotometer (Shimadzu UV-2600) Atomic absorption spectroscopy (AAS, Shimadzu AA-6880) for further confirmation.. UV-vis spectroscopy and Atomic Absorption Spectroscopy (AAS) are commonly used high-end standard techniques in this field. Concentrations of the actual field samples were estimated with the help of Figur 6 and used to validate the performance of the E-Eye digital chromium sensor. MaterialsPotassium dichromate (K2Cr2O7) was purchased from Himedia, India. Acetone (CH2COCH2) and 1,5-diphenylcarbazide (C6H5NHNHCONHNHC6H5) were purchased from Sigma Aldrich, India. Sulphuric acid (H2SO4)(98%), nitric acid (HNO3), hydrogen peroxide (H2O2) and diethyl ether (C2H5OC2H5) were procured from Finar, India. All reagents were of analytical grade and used without any further purification. Milli-Q grade water (18.2 MΩ) was used throughout the experiment unless stated otherwise. Arduino UNO development board, breadboard, liquid crystal display (LCD), potentiometer, jumper wires, resistors, multimeter, light emitting diode (LED), and light-dependent resistors (LDRs) were purchased from Rhydo Labz, India. Quartz cuvettes for spectroscopy study were procured from Axiva, India.It is thus possible for the present invention to provide for Point–of–Care testing (POCT) of chromium (Cr) in biological samples that pertains to user-friendly colorimetric chromium sensor system as a portable, cost-effective, user-friendly device configured for real-time monitoring of chromium concentration non-invasively in body fluids for healthcare applications and also in environmental samples by utilizing reagent-based pH stabilized and selective optical sensor.

Claims

, Claims:We Claim:

1. An user-friendly colorimetric chromium sensor system comprising a point of care optical sensor including acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor enabled for lock and key colorimetric reaction with chromium for generating concentration of chromium based colour intensity dependent purple-coloured complex as a colorimetric sensor of chromium.

2. The user-friendly colorimetric chromium sensor system as claimed in claim 1 wherein said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) colorimetric sensor is capable of said colorimetric detection of chromium in the pH range of 1.15-1.39, preferably 1.38, stability time of 5 mins to 5 days and temperature range of 25C-85C.

3. The user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 or 2 adapted as a Point–of–Care testing (POCT) system comprises photoresistor based chromium concentration / level detector for digital sensing of chromium concentrations based on said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) enabled lock and key colorimetric reaction with chromium generating concentration of chromium based colour intensity dependent purple coloured complex.

4. The user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 to 3 wherein said photoresistor device includes said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor including:a cuvette for containing test sample and said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor;said cuvette containing test sample operatively connected to an LED-LDR sensing mechanistics wherein the light-emitting diode (LED) is disposed opposite to the light-dependent photoresistor (LDR) across said cuvette for containing test sample such that LED light passing through sample in cuvette is absorbed and sensed by the photoresistor LDR;calibrator means operatively connected for converting said photoresist measured by LDR into said concentration of chromium in said test sample.

5. The user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 to 4 wherein said photoresistive device with its hardware components are electrically integrated to said LED-LDR sensing mechanistics by including said light-emitting diode (LED) (303) and said light-dependent photoresistor (LDR / photoresistor) LDR (305) detector disposed at the opposing faces of sample holder (304) holding cuvette (306) for containing Chromium based samples;said light-emitting diode (LED) (303) connected to a power source (301) via resistors (302);said light-dependent resistor (LDR / photoresistor) LDR (305) detector interfacing with Arduino Uno microcontroller (307) across a printed circuit board (PCB) adapted as central processing unit (CPU) in turn connected to chromium quantifying processor (309) based calibrator via jumper cables (308).

6. The user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 to 5 wherein said electrically integrated hardware components of photoresistive device is housed by enclosure members with a lid impermeable to external light allowing sample entry based on opening and closure of the lid, said enclosure member equipped with a read-out display preferably LCD display compatible with smartphone applications interfacing said chromium quantifying processor (309) enclosed within to display chromium concentration of the sample empirically related to the light-dependent photoresistance encountered from chromium based samples including biological and environmental samples.

7. The user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 to 6 wherein said device is powered by plurality of sources via a solar panel (311) mounted on top of enclosure member connected to a power source (301) preferably a 12 V battery via universal serial bus (USB) cable (312).

8. The user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 to 7 wherein upon illumination of LED light (400–700 nm) the same passes through the sample contained in the cuvette and gets absorbed providing colorimetric Cr (VI) concentration-colour intensity responsive corresponding LDR detector signal for algorithmic conversion to digital signal by empirical Cr (VI) quantifying processor (309) for display at the LCD interface in ppb scale (µgL-1), said sensor is sensitive to Cr in the range of 5 ppb to 2.5 ppm providing a sharp peak at 543 nm that is selectively unresponsive to interfering metal ions including Fe(III), Pb(II), and Cu(II), with the limit of detection (LOD) of the sensor being as low as 6 ppb / 6.1 µgL-1, Limit of Quantification (LOQ) 18.48 µgL-1 respectively in ppb scale being the least reported for colorimetric sensing.

9. The user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 to 8 wherein said generation of intensity based pH stabilized 1, 5-diphenylcarbazide (DPCA) chromogenic sensor is adaptive to Cr (VI) ion sensing upon forming 1,5-diphenylcarbazide (DPCA)-Cr(III) complex thereby favouring selective, sensitive, and quick sensing of chromium in said samples 10. A method for chromium sensing in samples including environmental samples of ground water including user-friendly colorimetric chromium sensor system as claimed in anyone of claims 1 to 9 comprising Providing a point of care optical sensor including acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor;Providing Chromium samples including pre-treated chromium samples for interaction with said sensor enabling a lock and key colorimetric reaction with chromium and generating concentration of chromium based colour intensity dependent purple-coloured complex as a colorimetric sensor of chromium for related detection.

11. The method as claimed in claim 10 wherein said optical sensor is prepared based on 0.5% w / v 1,5-diphenylcarabzide (DPC) involving 250 mg of DPC dissolved in 25 mL acetone, then diluted up to the mark of 50 mL with milli-Q water followed by preparing a solution of 0.15 M H2SO4 for necessary pH stabilization in acidic range.

12. The method as claimed in anyone of claims 10 or 11 wherein said pre-treated chromium samples are pre-treated post collection based on the following:environmental samples that are collected in acid-washed polyethylene or Teflon containers, followed by optional membrane filtration (0.45 µm) for dissolved metal analysis, followed by acidification involving concentrated nitric acid to lower the pH below 2, thereby stabilizing metal ions and preventing microbial activity or precipitation whereby total Cr in samples undergo acid digestion using concentrated nitric acid optionally involving hydrogen peroxide under controlled heating (85–95°C) until a clear solution is obtained followed by filtering the clear samples with deionized water, stored at 4°C in acid-cleaned containers for analysis within a recommended holding time;urine samples collected and filtered with Whatman filter paper grade-1 of pore-size 20-25 µm to remove cellular extracts and any particulate matter leading to turbidity followed to which it is centrifuged at 10000 rpm for 30 minutes and then digested with nitric acid (65%) in a volume ratio of 1:19 to suppress any organic interference at a temperature of 80 ℃ that is digested and stored at 4 ℃ diluted 10-fold with mili-Q water before investigation;hair samples of 1 gram of hair samples were finely cut into small pieces and soaked in the solution mixture consisting of diethyl ether, acetone, and DI water in a ratio of 3:1:20 under an ultrasonication bath for 1 hour followed by drying in a glass Petri dish at 105 °C for 24 hours that was thereafter digested with nitric acid (HNO3) and hydrogen peroxide (H2O2) in a ratio of 3:1 (v / v) with said digested hair samples dried at 80 °C near to dryness, to which dried samples was added 10 mL of 0.1 M HNO3 and filtered using Whatman filter paper grade-1 of pore-size 20-25 µm and stored at 4 °C until further analysis.

13. The method as claimed in anyone of claims 10 to 12 wherein said chromium sensing is carried out based on Providing said samples / pre-treated test samples in a cuvette together with said acidic pH stabilized 1, 5-diphenylcarbazide (DPCA) optical sensor followed by adding Cr(VI) solution to the cuvette for producing purple-colored complex [DPCA-Cr(III) complex];Illuminating said cuvette by LED-LDR sensing mechanistics wherein the light-emitting diode (LED) disposed opposite to the light-dependent photoresistor (LDR) across said cuvette allows passing of LED light through sample in cuvette that is absorbed and sensed by the photoresistor LDR as light-dependent photoresistance encountered from chromium based samples;Obtaining corresponding chromium concentration of the test sample linearly related to and empirically converted from said light-dependent photoresistance as displayed in the display of the calibrator.Dated this the 30th day of June, 2025 Anjan Sen Applicants Agent & Advocate IN / PA-199