DESC:FIELD OF THE INVENTION
The present invention relates to a lightweight, pocket-sized, low-power electronic meter for analyte quantification on colourimetric devices. More specifically, the present invention discloses the design, development and fabrication of an accurate, lightweight, pocket-sized, low-power electronic meter, for analyte quantification on colourimetric Paper Analytical Devices (PADs), display and wireless transfer of data to nearby smart devices.

BACKGROUND OF THE INVENTION
The field of medical diagnosis has undergone many visible developments in the past years with an increase in the number of rapid tests for medical diagnosis as a response to the frequent outbreak of several infections. Rapid diagnostic tests offer great potential for fast and accurate identification of infectious organisms and for the evaluation of antimicrobial susceptibility in patients. These tests can be conducted close to the site of patient care, often referred to as the point-of-care (POC), which may be a doctor’s office/clinic setting and are designed to provide results using patient samples that are easy to collect and can enable immediate treatment in case of a positive test. This has led to the intense development of millions of Point-of-Care Testing (PoCT) systems.

The term ‘Point-of-Care Testing (POCT)’, also known as ‘near-patient testing’ or ‘bedside testing’, is a type of rapid diagnostic performed with the patient, outside of a laboratory setting. In general, the term can encompass any medical test that is given ad hoc and provides quick results. POC diagnostic devices are used by healthcare professionals, patients, and their families because of their user-friendly features. POCT is often accomplished through the use of transportable, portable, and handheld instruments called Point-of-Care (POC) diagnostic devices which are used in Point-of-Care testing to obtain diagnostic results while with the patient or close to the patient.

POC technology encompasses a wide range of analytical devices and systems, including handheld diagnostic devices, portable ultrasound machines, and mobile health apps. In developing countries, such as India, POC tests and devices play a pivotal role in the healthcare system. As a result, nowadays, POC diagnostic including paper analytical devices devices (PADs) are used widely and available for a large variety of analytes, their utilities ranging from intensive care units to outpatient clinics to personal care.

Majority of the current diagnostic assays require sophisticated laboratory infrastructure and expensive reagents which may not be suitable for resource-constrained settings with limited financial resources, basic health infrastructure, and few trained technicians. In such settings as well as to support rapid diagnosis of critical diseases requiring immediate assistance, cellulose and flexible transparency paper-based analytical devices (PADs) have demonstrated enormous potential for developing robust, inexpensive and portable devices for disease diagnostics. These devices offer promising solutions to disease management in resource-constrained settings where the vast majority of the population cannot afford expensive and highly sophisticated treatment options.

Over the last 15 years, Paper-based analytical devices (PADs) have emerged as a promising tool for Point-of-Care testing. In PADs, reagents and samples can be spotted easily on paper either manually or using high-speed printing techniques, i.e., paper is used as a substrate for analyzing the samples. These are portable devices that can detect selected substances in a sample and quickly display results, thus making them a powerful platform for point-of-need testing. Moreover, PADs are inexpensive and can be fabricated in different shapes and miniaturized sizes, ensuring better portability. PADs have an appreciable form of detection, consisting of colorimetric, fluorometric, electrochemical, voltammetry, amperometry, chemiluminescence and fluorescence. However, results obtained on PADs are semi-quantitative and are usually prone to error as specific interaction between the target and reagents produces a colour change that is assessed by the naked eye.

To address this issue, nowadays PADs are being incorporated into digital detection systems with enhanced accuracy and at the same time being cost-effective. In one such system, the quantification of analytes on PADs using smartphones attached to analytical cartridges has gained popularity. The image captured by the smartphone is input to a dedicated software which converts the image to different colour models, followed by the display of the target analyte concentration. However, the accuracy of these optical techniques is influenced by gamma correction, focal length, illumination, and the camera resolution employed along with different environmental conditions, including the intensity and colour of the ambient light. Nevertheless, creating a trained model for these techniques demands vast experimental data.
An alternative method to address these limitations is to use desktop scanners for image acquisition. However, their use is limited in a typical POC setting due to poor portability. Lately, portable scanners have been employed to quantify colour developed on PADs, yet the output data can only be processed into a human-readable format with the assistance of laptop or desktop computers. Dedicated electronic colour readers have been developed to address the limitations associated with the aforementioned detection techniques. These colourimeters deploy either transmission-based or reflectance-based optical methods for measurement.

Analyte quantification in colourimetric POC devices involves the use of image processing software such as Image and MATLAB and open-source programs like ColorScan. However, the process is tedious and involves multiple steps, including image acquisition and transfer to a computer, selection of the region of interest and extensive image processing. To enhance the accuracy, precision and sensitivity of colorimetric quantification in POC devices, deep learning and machine learning methods are nowadays being utilized.

In a transmission-based optical system, the quantification of colour developed on the PAD is carried out by measuring the intensity of light transmitted through the paper using a handheld device. However, the optical setup can suffer from significant light energy loss, leading to a substantial reduction in the signal. Alternatively, reflectance-based detection under controlled lighting conditions is more suitable for colourimetric analysis as it minimizes signal loss.
The colourimetric data obtained can also be represented as colour models or as units of absorbance based on the colour obtained. Expressing target analyte concentration in absorbance units is a more feasible approach as it helps correct variations in background lighting conditions. Also, the use of absorbance-based methods in PADs has a distinct advantage, as the results can be directly compared to those obtained from laboratory spectrophotometer-based assays.

In recent work by V.S. Siu et.al. 2022, portable scanners were employed to quantify colour developed on PADs. However, the output data could only be processed into a human-readable format with the assistance of laptop or desktop computers.

Dedicated electronic colour readers were developed by A.K. Ellerbee et. al., 2009, to address the limitations associated with colourimetric detection techniques. These colourimeters deploy either transmission-based or reflectance-based optical methods for measurement.

Although there have been advancements in recent years, with respect to indigenously developed and commercial analytical devices used for colourimetric quantification, an unmet gap still exists for the smooth integration of the paper substrate with portable colourimeters to facilitate easy, accurate and quick analysis of samples.

OBJECT OF THE INVENTION
In order to obviate the drawbacks of the existing state of the art, the present invention discloses the design and fabrication of an indigenous, lightweight, hand held pocket-sized, low-power electronic meter for analyte quantification on colourimetric devices.

The main object of the present invention is to provide an indigenous, lightweight, pocket-sized, low-power electronic colourimeter for the quantification of colourimetric assays on Paper Analytical Devices (PADs).

Another object of the invention is to provide an indigenous, handheld, portable electronic colourimeter whose accuracy is unaffected by ambient light conditions.

Another object of the invention is to provide an indigenous, handheld, portable electronic colourimeter for point-of-care colourimetric quantification from real life samples.

Yet another object of the invention is to provide an indigenous, handheld, portable electronic colourimeter that can quantify as well as display the concentration of the analyte on an organic light-emitting-diode (OLED) display.

Yet another object of the invention is to provide a handheld, portable electronic colourimeter wherein the data acquisition, processing, and transmission of the data to a smartphone is achieved expeditiously.

Yet another object of the invention is to provide a handheld, portable electronic colourimeter which can be configured to quantify different analytes on PADs.
SUMMARY OF THE INVENTION:
The present invention discloses the design, development and fabrication of an accurate, hand held lightweight, pocket-sized, low-power electronic meter for analyte quantification on colorimetric devices Paper Analytical Devices (PADs). The Colourimeter displays the results of the quantitative assessment on an organic light-emitting-diode (OLED) display and enables wireless transfer of data to nearby smart devices. The accuracy of the colorimeter is unaffected by ambient light conditions. The device works on the principle of light reflection from the sample wherein the colorimetric data is expressed in terms of absorbance values that are directly related to the concentration of the analyte and facilitates data acquisition, processing, and transmission of the data to a smartphone using Bluetooth in less than 7 seconds. The colorimeter assesses the analyte concentration from a sample placed on Leak-proof Paper analytical device (PAD) comprising Whatman chromatographic paper which is developed by wax printing and created using Clewin software. The colorimeter comprises of a 3D printed platform for optical arrangement, a highly integrated six-channel spectral sensor chip, LEDs to illuminate the sample and OLED display for displaying the analyte concentration. A low-power microcontroller accesses the controls and spectral data of the device and communicates with Bluetooth that helps transmit data to a suitable smartphone.

The accuracy of the device was validated by testing different concentrations of alkaline phosphatase (ALP) and total protein on PADs explicitly developed for this study. The colourimeter exhibited linearity with R2 values of 0.98 and 0.97 for ALP and total protein, respectively. Moreover, by reconfiguring the calibration, the developed device can be used to quantify different analytes on PADs. The handheld colourimeter of the invention is a very promising tool for point-of-care colourimetric quantification as the absorbance values generated correspond to the analyte concentration and can be easily compared to results generated from a clinical laboratory as observed from real sample analysis. The electronic design of the colorimeter employs commercially available components and has added advantages of ease of use, portability, and elimination of interference from ambient light conditions, together with the possibility of measuring different analytes on PADs.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1: depicts the The working principle as well as cross-sectional view of the developed handheld colourimeter

Figure 2: depicts the schematics of the electronic module of the handheld colourimeter.

Figure 3: depicts the comparison of the reflected light intensity values measured from Pantone colour cards with CMYK values of (0,0,78,0), (0,0,66,0), and (0,0,44,0) for ten consecutive days using the developed colourimetric device.

Figure 4(A): depicts the image of the paper strip obtained with the testing of ALP.

Figure 4(B): depicts the colour band generated for the visual interpretation of ALP.

Figure 5(A): depicts the UV-DRS reflection spectra of PNPP and PNPP-ALP complex with ALP concentrations ranging from 60 U/L to 700 U/L on paper substrate.

Figure 5(B): depicts the UV-DRS reflection spectra of TBPB and TBPB-total protein complex with total protein concentrations ranging from 1 g/dL to 12 g/dL on paper substrate.
Figure 6(A): depicts the photograph of the PAD used for the colourimetric assay of total protein (0 to 12g/dL) with laboratory protein sample.

Figure 6(B): depicts the colour band created using the PAD.

Figure 7(A): depicts Plots of reflected light intensity captured by the violet channel of the colourimetric device vs. different ALP concentrations.

Figure 7(B): depicts Plots of reflected light intensity captured by the orange channel of the colourimetric device vs total protein concentrations.

Figure 7(c): depicts the Calibration plot of absorbance vs. logarithm of ALP concentration ranging from 40 U/L to 600 U/L (n=4).

Figure 7(D): depicts a plot of absorbance vs. log concentrations of serum spiked protein on PAD.

Figure 8: depicts the images of the paper strip after the addition of serum spiked with different concentrations of ALP.

Figure 9(A): depicts a plot of absorbance vs. varying serum concentrations of ALP on PAD. The violet channel intensity of the sample and blank was used for calculating absorbance.

Figure 9(B): depicts a plot of absorbance vs concentrations of total protein on PAD. The orange channel intensity of the sample and blank was used for calculating absorbance.

Figure 10: depicts the images of the PAD obtained after the colourimetric detection of serum protein (0 – 12.6 g/dL) in serum.

Figure 11(A): depicts a bar graph of repeatability studies carried out for ALP on PAD by plotting the relative intensity of reflected light captured in the violet channel of the spectral engine.

Figure 11(B): depicts the relative intensity of reflected light captured in the orange channel from ten different serum samples of total protein with a concentration of 4.6 g/dL.

Figure 12(A): depicts the Bar chart showing the reflected light intensity captured in the violet channel (A) and the orange channel.
Figure 12(B): depicts the Bar chart of the colourimeter for various biomolecules and ions

DETAILED DESCRIPTION OF THE INVENTION:
Accordingly, the present invention discloses the design and fabrication of an indigenous, lightweight, hand held pocket-sized, accurate low-power electronic colourimeter for analyte quantification on colourimetric paper analytical devices (PADs). The device works on the principle of light reflection from the sample and can quantify as well as display the concentration of the analyte on an organic light-emitting-diode (OLED) display. The accuracy of the colourimeter is unaffected by ambient light conditions, which is enabled by the deployment of spectral sensors. The colourimeter facilitates quick sample analysis by enabling data acquisition and its processing and transmission to a smartphone using Bluetooth, in less than 7 seconds.

Fabrication of the Paper Analytical Device (PAD):
Leak-proof Paper analytical device (PAD) for the colourimeter was developed by wax printing, comprising a circular reagent zone of 8mm diameter surrounded by a hydrophobic barrier, created using Clewin software. The design was wax patterned onto Whatman chromatographic paper, followed by heating at 120 °C for 20 minutes to form the hydrophobic barrier. An adhesive tape was applied to the underside of the wax-patterned paper to prevent leakage of analyte and reagent. Finally, the respective reagent was drop-cast onto the reagent zone utilizing a micropipette.

Design and Fabrication of the Colourimeter:
Fig. 1 depicts the cross-sectional view of the developed handheld colourimeter depicting the different components. Broadly, the colourimeter comprises of a 3D printed platform (5) for optical arrangement, highly integrated six-channel spectral sensor chip (4), LEDs (3) to illuminate the sample and OLED display (1) for displaying the analyte concentration. The figure represents the colourimeter placed on a PAD (6) having a reagent zone (7). A low-power microcontroller is the heart of the device as it accesses the controls and spectral data of the device. The microcontroller also communicates with the Bluetooth module that helps transmit data to a suitable smartphone as well as to an OLED screen that displays the analyte concentration. These design aspects help overcome the challenges associated with variations in ambient light settings, which is crucial for accurate quantitative analysis.

Working principle of the Colorimeter:
The working principle of the colourimeter is shown in Fig 1. Briefly, two white LEDs illuminate the paper substrate, which is kept at 1 cm from the spectral sensor. The two white LEDs are placed on either side of the spectral sensor such that the angle between the paper substrate and the sensor chip is 0º while the angle between the LED and the substrate is 45°. Thus, by creating an optical setup with a 0°/45° configuration, the developed device can eliminate interference from the specular reflection component of light reflected from the surface of the PADs. As shown in Fig. 1, the surface area of the cone defines the angle of incident light striking the analytical strip, while the spectral sensor positioned along the cone’s axis captures the reflected light. The paper analytical strips are placed at the tip of the conical structure of the 3D-printed enclosure with an apex angle of 45º. An integration time of 120 ms is provided to collect the reflected data before transferring it to the analog to digital circuit (ADC) for conversion to digital data, which is then moved to the appropriate data registers in the microcontroller.

Eventually, the colourimetric data is expressed in terms of absorbance values that are directly related to the concentration of the analyte mimicking commercial spectrophotometric data. This is achieved by calibrating the reflectance of known sample concentrations using Beer-Lambert’s law. However, Beer-Lambert law applies only to radiation of a single wavelength. Normally, employing LEDs as a light source causes non-linearity in absorbance values. This non-linearity can be minimized by using LEDs with a uniform spectral distribution in the absorbance region of the analyte. Keeping this in mind, the design employed the use of white LEDs that have spectral emissions of 100% at 430 nm (violet region) and 90% at 625 nm (orange region). The data from the channel complementary to the colour formed on the paper substrate is processed, and the analyte concentration is displayed on the OLED screen (Fig. 1C). The acquired data can be processed and transmitted to a nearby smartphone using the low-power Bluetooth module under 7 seconds. A pictorial representation of the fabricated circuit board is shown in Fig 1 D & E. To prevent reflections from the side walls, black coloured onyx material was used for 3D printing the enclosure and optical setup of the device. The device has dimensions of 6 × 6.6 × 3.7 cm and weighs 43 g.

To express analyte concentration in absorbance units (AU), the logarithm of the intensity obtained from the paper substrate without the colourimetric reagent (I0) is divided by the intensity of the colour (I) produced after the reagent reacts with the analyte as in equation (1).

Absorbance=log(I_o/I) ………………. (1)

To obtain the colour intensity of the paper substrate without the colourimetric reagent, two pieces (8×9 cm) of Whatman chromatography paper were placed as at the base of the analysis zone to reduce the absorption of light by the 3D-printed platform. The thickness of two sheets of chromatographic paper was found to be adequate to provide consistent results for a reagent-less paper strip.

Design of the Electronic Circuit of the Colourimeter:
The schematics of the electronic module of the colourimeter is shown in Fig. 2. The fabricated handheld colourimeter works at a low voltage of 3.3 V. and employs a highly integrated six-channel spectral chip (AS7262) capable of sensing violet, blue, green, yellow, orange, and red (from 430 to 670 nm), with a full-width half maximum (FWHM) of 40 nm and a resolution of 16 bits. A major advantage of this spectral chip is its capability to measure light intensity linearly across its spectral range, indicating that the measured intensities are proportional to the actual light intensity in that spectral band. The low-power microcontroller PIC16LF1788 accesses control and spectral data through I2C. Illumination of the sample is achieved using white LEDs. The LEDs in the circuit are powered using a constant current of 50 mA throughout the illumination time of 5 s using the in-built programmable LED driver circuit. The electronic circuit was designed and developed using Autodesk Eagle. The electronic components were soldered on the two-layer PCB while the microcontroller was programmed using embedded C language with MPLABX software and XC8 compiler and flashed using Pickit3. A 3D platform for the optical arrangement was designed using Autodesk Inventor Professional and 3D printed using onyx material.

Validation of reproducibility of the developed Colorimeter:
In order to evaluate the reproducibility of the developed Colorimeter and to understand the stability of the signal and components, studies were performed using commercial Pantone colour papers. The Pantone Uncoated Colour Bridge offers the benefit of having a texture that resembles the Whatman chromatography paper, which is the substrate for the colourimetric assays using the fabricated colourimeter of the present invention. Additionally, it provides consistent colour stability over time, making it an ideal choice. The intensity of the colour papers in CMYK standard (cyan, magenta, yellow and key (black) standard) was tested continuously for 10 days at room temperature under ambient environmental conditions. The average intensities for the different Pantone colour papers with CMYK values of (0,0,78,0), (0,0,66,0), and (0,0,44,0) showed a minimal variation with RSD values of 0.49%, 0.38% and 0.38%, respectively (n = 5). These results indicate that the device performed with high reproducibility, as shown in Fig. 3. This device was then used to detect reflected light from different colourimetric assays.

UV - Visible Diffused Reflection Spectroscopic (UV-DRS) studies of the PADs:
It is known in the state-of-the-art that the activity of acid phosphatase is measured by an enzymatic reaction that converts para-nitrophenyl phosphate (PNPP) to para-nitrophenol (PNP), liberating phosphate. The product, PNP, absorbs light of the wavelength 400 nm at extremely alkaline pH.

The colour reaction of Alkaline Phosphatase (ALP) with P-nitrophenyl phosphate (PNPP) was studied initially using a UV-Vis spectrophotometer. P-nitrophenyl phosphate (PNPP), a colourless reagent, was utilized for the detection of ALP. As depicted in Fig. 4A, a distinct absorption peak was observed at 320 nm for the PNPP. Upon the addition of ALP, the intensity of the peak observed at 320 nm decreased, and a new absorption peak appeared at 405 nm, which is characteristic of p-nitrophenol (PNP). The intensity of the peak was found to increase exponentially with an increase in the concentration of ALP from 10 to 400 U/L.

The above test was translated to paper analytical devices (PADs). Initially, the PADs incorporated with PNPP were colourless, whereas a distinct yellow colour was observed in the presence of ALP. Colourimetric detection of ALP was carried out on the PAD by introducing different concentrations of ALP onto the different reagent zones with PNPP. 6 µL of PNPP was introduced into the reagent zone of the PADs and dried at room temperature for 30 minutes. Further, it was observed that the intensity of the yellow colour increased with increasing concentrations of ALP which is attributed to the increased formation of PNP. Fig. 4 shows the images of the paper strip obtained with an increase in ALP concentration from 20 to 1000 U/L, while Fig. 4B depicts the colour band obtained. As is clear from the figure, the increase in concentration of ALP exhibits an increase in the colour intensity on the paper strips.

These paper strips were further analysed using UV-visible diffused reflection spectroscopy (UV-DRS) to understand the reflectance spectra of PNP from the paper substrate. As observed from Fig. 5A, PNP has a maximum absorption at 410 nm, which corresponds to the violet region of the visible spectrum, while the paper substrate without any colourimetric reagent showed uniform spectral reflectance of 75% from 400 to 750 nm. Hence, the developed colourimeter utilised the colour channel corresponding to violet, which is complementary to the yellow colour formed on PADs.

Similarly, UV-Vis spectroscopic studies were carried out for total protein detection. The colourimetric reagent TBPB is originally yellow and shows absorbance at 436 nm. When mixed with serum proteins, hydrophobic and electrostatic interactions form a stable blue complex, causing a bathochromic shift to 626 nm. Moreover, it was observed that the absorbance increases with an increase in total protein concentration (Fig. 5B).

Colourimetric assays to estimate the concentration of total protein were developed by drop casting 5 µL of the reagent prepared using 12 mM of tetra bromophenol blue (TBPB) solution in 4% ethanol, 250 mM of citrate buffer of pH 1.8, and Triton-X 100 into the reagent zone of the PADs followed by drying at room temperature for 20 minutes. The calibration equation was deduced using total protein concentrations ranging from 1 to 12 g/dL. Fig. 6A depicts the paper strip for four replicate measurements of protein solutions of concentrations ranging from 1 to 12 g/dL. As the intensity of colour changed with concentrations of serum protein, a colour band was made for visual interpretation by trimming the centre portion of the coloured image in the presence of total protein, the yellow-coloured TBPB reagent incorporated PAD turned bluish green with an increase in blue colour intensity as the total protein concentration increased. Therefore, the orange channel of the spectral sensor, which is complementary to the blue colour, was used to quantify total protein in the developed handheld device.

Testing and validation of the developed device:
To validate the performance of the developed device towards ALP analytical paper strip, experiments were conducted using ALP concentrations of 20, 60, 100, 140, 180, 300, 400, 500, 600, 700, 800 and 900 U/L. Out of the available six colour channels, the violet channel showed maximum sensitivity, which agrees with the obtained UV-DRS results. Since the violet colour channel is complementary to yellow, an increase in yellow colour on the PAD with increasing ALP concentration results in an exponential decrease in violet reflection (Fig. 7A). The graph displayed in Fig. 7B shows a linear correlation up to 160 U/L of ALP. The figure illustrates the linear fit achieved by plotting the logarithmic concentrations against the reflected light intensity, which is extended to 600 U/L of ALP concentrations. From the graph, it is observed that the developed colourimetric device was capable of linearly measuring ALP up to 600 U/L with a linear relation, Y = (-0.5285 ± 0.0547) + (0.3993 ± 0.0226 (log C)), a regression coefficient of 0.975 and the number of trials (n) = 4.

To study the response of the colourimetric device towards total protein, experiments were conducted using protein concentrations in the range of 1 to 12 g/dL. As deciphered from the UV-Vis DRS analysis, the orange channel of the spectral sensor, complementary to the blue colour, was used for total protein quantification. This channel showed maximum sensitivity and exhibited an exponential fit for increasing concentrations of total proteins, as shown in Fig. 7C. The reflectance of the orange spectrum (complementary to blue) was further converted into absorbance values, and a linear fit was obtained, which is depicted in Fig. 7D. The indigenously developed device responds linearly to the total protein concentration range from 1 to 12 g/dL, with a regression equation of Y = (0.3522 ± 0.0076) + (0.2439 ± 0.0093(log C)), R2=0.984 and the number of trials (n) = 4.

Real sample analysis using the indigenously developed colourimetric device:
To understand the response of the developed device towards physiological samples, serum containing a known ALP concentration of 48 U/L was used as the analyte and spiking this serum to obtain serum ALP concentrations of 68, 88, 108, 128, 148, 248, 348, 448, 548, 648, 748, 848, and 948 U/L. Upon the addition of serum and spiked serum samples on the PAD, the colour of the detection zone changes to yellow, and the intensity increases with increasing spiked concentrations of ALP. The change in colour is depicted in Fig. 8A.

The developed colorimeter exhibited a linear response in absorbance towards increasing concentrations of ALP in the serum with a regression equation Y = (-0.5494 ± 0.0507) + (0.4198 ± 0.0224 (log C)) and an R2 value of 0.975 when N = 10 and the number of trials (n) = 4 (Fig. 9A). The change in intensity of the yellow colour of ALP on PAD showed a sensitivity of 0.4198 AU/U/L. The limit of detection (LOD) and limit of quantification (LOQ) were calculated, and the results obtained are 1.1 and 3.89 U/L as shown in Table 1. The repeatability studies were conducted by quantifying ten serum samples with ALP concentration of 48 U/L.

Table 1. Comparison of ALP detection on PAD with recent reports.
Technique Detector
LOD
(U/L) LOQ
(U/L) Linear range
(U/L) User Quantitative Portability
CL Xi’an Remex IFFS–A 0.05 - 1-70 Highly trained Yes No
Colour Visual perception 19.7 - 0-400 Minimally trained No Yes
FL Hitachi F- 7.4 - 0.01-8 Highly trained Yes No
2700
Colour Hitachi U3310 9.2 - 10-200 Highly trained Yes No
FL Visual perception 1.1 - 5-1000 Minimally trained No Yes
Colour Colourimeter 1.1 3.89 0-600 Minimally trained Yes Yes

The mean and the standard deviation were calculated for four trials for each concentration. The absorbance values were calculated by the logarithmic ratio of the reflected violet intensity of the sample (I) to the blank (I0), and error bars were represented by the standard deviation.

Further, the analysis of total protein was also carried out on paper with a human serum sample having a total protein concentration of 4.6 g/dL spiked with protein to obtain concentrations of 5.6, 6.6, 7.6, 8.6, 9.6, 10.6, 11.6 and 12.6 g/dL (Fig. 10). It was observed that the intensity of the blue colour increased with an increase in the concentration of total protein in the serum. The mean and the standard deviation were calculated from five trials per concentration. The absorbance values obtained from the colourimeter for total protein spiked serum were plotted against the logarithm of concentrations (Fig. 9B) and exhibited the relation Y = (0.284 ± 0.0185) + (0.3147 ± 0.02 ((log C) and R2 = 0.97 for the number of trials (n) = 4. The sensitivity of the colourimetric quantification of total protein was calculated to be 0.3147 AU/g/dL. The LOD and LOQ values were calculated from the calibration equations and the obtained values are shown in Table 2.

Table 2. Comparison of total protein detection on PAD with recent reports.
Technique Detector
LOD
(U/L) LOQ
(U/L) Linear range
(U/L) User Quantitative Portability
Colour Smartphone, ImageJ 0.17 g/L - 0-10 g/L Highly trained Yes No
Colour Smartphone, ImageJ 0.9 mg/dL 2.9 mg/mL 0.5–6 mg/mL (TBPB) Highly trained Yes No
Colour Smartphone, ImageJ 1.33 mg/mL 4.91 mg/mL 0.5–6 mg/mL (TBPB) Highly trained Yes No
Colour Hach, DR5000 5 µM - 10 to 300 µM Highly trained Yes No
Colour Colourimeter 0.037 g/dL 0.12 g/dL 1-12.6 g/dL Minimally trained Yes Yes

Reproducibility studies:
Repeatability studies were carried out with the designed caolorimeter by analyzing ten serum samples with an ALP concentration of 48 U/L. The data depicted in Figure 11A indicates the PAD's consistency with minimal variability. Similarly, the repeatability studies for total protein involved analyzing ten serum samples with a concentration of 4.6 g/dL, and the outcome shown in Figure 11B demonstrates the PAD's consistency.

Solutions of glucose (50 mM), uric acid (200 µM), ascorbic acid (200 µM), dopamine (50 µM), creatinine (200 µM), NaCl (40 mM), KCl (40 mM), ALT (100 U/L), AST (100 U/L), serum protein (1 g/dL), bilirubin (3 µM) and ALP (100 U/L) were tested on the PAD to prove the selectivity of the assay. The violet channel reflected light intensity was found to be around 32600 a.u for all the ions and other biomolecules (Fig. 12A). When the solution containing ALP was spiked and introduced onto the PAD, it turned into a yellow colour and the reflected light intensity of the violet channel reduced to 19208. Then, the PAD was tested with a solution containing all the biomolecules, ions and ALP, the yellow colour was very similar to that observed only with ALP solution. The violet channel reflected intensity was found to be 18976 a.u, which is 99% of the intensity obtained only with ALP. This shows that the PAD and the device developed were highly selective to ALP even in the presence of other biomolecules.

For total protein selectivity studies, the colourimetric analysis was carried out with the same samples and serum protein of 5 g/dL on the PAD. The yellow colour of the TBPB remained constant unless altered to a blue colour by the presence of serum protein. The intensity of the reflected orange colour channel of the colourimeter was found to be around 28500 a.u for the ions and other biomolecules whereas it is 9516 a.u for protein (Fig. 12B). The reflected intensity was 9311 a.u was obtained for the solution of all the ions, biomolecules and serum protein. This reflected colour intensity was about 98% of that obtained only with protein, indicating the excellent selectivity of the PAD and the device.

Clinical validation of the developed device:
To explore the applicability of the developed device towards testing clinical samples, ALP and total protein concentrations were measured from the blood samples of 3 volunteers and compared to results obtained from the colourimetric test in a clinical laboratory. ALP present in the serum sample reacted with the PNPP on the paper and formed yellow colour. The intensity of the yellow colour was captured using the developed meter and quantified. The results obtained were found to be in good correlation with the clinical results and exhibited a relative error of less than 10% (Table 3). Similarly, the colour changes from yellow to blue on the PAD in the presence of total protein was captured using the meter and quantified. As is clear from the Table, the relative error calculated by comparing the results obtained for total protein using the developed colourimetric meter and a clinical laboratory technique was found to be less than 8%. The data from these tests clearly demonstrate that the developed meter is highly suitable for use with clinical samples.

Table 3. Comparison of ALP and protein concentrations in blood samples tested on PADs using the developed electronic meter

Clinical sample Code ALP concentration (U/L) The relative error (%) Standard deviation
(U/L) Protein concentration (g/dL) The relative error (%) Standard deviation
(U/L)
Clinical method PAD and meter Clinical method PAD and meter
A 65.9 59.75 9.32 3 6.8 6.91 1.6 0.202
B 190.5 172.64 9.56 3.06 7.1 7.62 7.2 0.241
C 261.6 277.85 6.21 16.41 7.9 8.34 5.56 0.351

The handheld colourimeter developed in this study is a very promising tool for point-of-care colourimetric quantification as the absorbance values generated correspond to the analyte concentration and can be easily compared to results generated from a clinical laboratory as observed from real sample analysis. With an electronic design that employs commercially available components and added advantages of ease of use, portability, and elimination of interference from ambient light conditions, together with the possibility of measuring different analytes on PADs, the developed device is poised to find commercial success.

,CLAIMS:We Claim:
1. An indigenous lightweight, handheld, pocket-sized, low-power electronic meter for analyte quantification on colourimetric Paper Analytical Devices comprising:
- a 3D printed platform for optical arrangement,
- a six-channel spectral sensor chip,
- LEDs to illuminate the sample,
- an OLED display for displaying analyte concentration,
- a low-power microcontroller for accessing the controls and spectral data,
- a Bluetooth module for transmitting the data to a suitable smartphone as well as to an OLED for display
wherein the colorimeter performs point-of-care colourimetric quantification of analytes from real life samples and achieves data acquisition, processing, and transmission of the data to a smartphone expeditiously.
2. The handheld colourimeter as claimed in claim 1 wherein the data acquisition, processing, display and wireless transfer of data to is achieved in less than 7 seconds.
3. The handheld colourimeter as claimed in claim 1 wherein the LEDs have spectral emissions of 100% at 430 nm (violet region) and 90% at 625 nm (orange region).
4. The handheld colourimeter as claimed in claim 1 wherein the accuracy of the colorimetric assessment is unaffected by ambient light conditions.
5. The handheld colourimeter as claimed in claim 1 wherein the analyte concentration is displayed on an organic light-emitting-diode (OLED) display.
6. The handheld colourimeter as claimed in claim 1 wherein said colorimeter works at a voltage ranging from 1.0 to 3.3 V.
7. The handheld colourimeter as claimed in claim 1 wherein the microcontroller is a low-power 8-bit microcontroller.
8. The handheld colourimeter as claimed in claim 1 wherein the sensor used for six-channel multi-spectral sensing in the visible range from 340 to 670 nm with a full-width half maximum (FWHM) of 40 nm.
9. The handheld colourimeter as claimed in claim 1 wherein the control and spectral data access on the sensor are implemented through the I2C registers.
10. The handheld colourimeter as claimed in claim 1 wherein an OLED display SSD1306 is used via I2C communication by programmed C language.
11. The handheld colourimeter as claimed in claim 1 wherein the spectral sensor placed at a viewing angle of 0° captures the light intensity values in six different colour channels.
12. The handheld colourimeter as claimed in claim 1 wherein the linearity exhibited with R² values is 0.98 for ALP and 0.97 for total protein.
13. The handheld colourimeter as claimed in claim 1 wherein the LOD and LOQ obtained for ALP and total protein are 1.1, 3.89 U/L and 0.037, 0.12 g/dL, respectively.
14. The handheld colourimeter as claimed in claim 1 wherein the validation of the colourimeter was carried out with the clinical samples in which a relative error of less than 10% is obtained for both the analytes.
15. A method for quantitative analysis of the analyte by the electronic meter as claimed in claim 1, comprising the steps of:
- introducing the sample to be analyzed is introduced on fabricated paper analytical strips,
- placing the loaded analytical strip on the custom 3D printed platform housing the indigenously developed colourimeter,
- illuminating the analytical strip using a 0°/45° optical configuration,
- capturing the light intensity values in six different colour channels by the spectral sensor placed at a viewing angle of 0°,
- processing the intensity values by a low-power microcontroller into absorbance values,
- correlating the absorbance values to the corresponding analyte concentration using a calibration equation,
- displaying the analyte concentration on the OLED screen.
- communication and data transfer with a smartphone via Bluetooth and a custom Android application.