Accepted Manuscript

Title: Smartphone based optical biosensor for the detection of

urea in saliva

Authors: Anuradha Soni, Sandeep Kumar Jha

PII: S0925-4005(18)30812-8
DOI: https://doi.org/10.1016/j.snb.2018.04.108
Reference: SNB 24584

To appear in: Sensors and Actuators B

Received date: 14-9-2017

Revised date: 16-4-2018
Accepted date: 21-4-2018

Please cite this article as: Anuradha Soni, Sandeep Kumar Jha, Smartphone based
optical biosensor for the detection of urea in saliva, Sensors and Actuators B:
Chemical https://doi.org/10.1016/j.snb.2018.04.108

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Smartphone based optical biosensor for the detection of urea
in saliva
Anuradha Soni1 and Sandeep Kumar Jha1,2*

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1
Centre for Biomedical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi,
India110016

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2
Department of Biomedical Engineering, All India Institute of Medical Sciences, New Delhi India
110029

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*
Corresponding author

Email: sandeepjha@cbme.iitd.ac.in
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Tel: +91-11-2659-1119, Fax: +91-11-2658-2037
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Highlights
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 Our present work is on the development of a noninvasive; smartphone based optical biosensor
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for the determination of urea levels in saliva, which is first such report on the use of smartphone
for salivary urea estimation
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 The sensor was fabricated using a simple methodology by direct immobilization of urease
enzyme along with a pH indicator on a filter paper based strip which changes color upon
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reaction with urea present in saliva. This color change can be used to deduce the urea
concentration present in the saliva sample using smartphone based application by reading RGB
levels.
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 Slope method (sensor response change per unit time) has been chosen instead of differential
method (difference in sensor response between two time intervals) to increase the sensitivity as
well as to eliminate interferences caused due to variation in ambient light conditions.

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 Clinical validation carried out on spiked saliva samples as well as clinical samples from healthy
volunteers indicate great possibility of using the biosensor for diagnosis of uremia especially in
subjects suffering from kidney disorders and other severe conditions.
 Use of a handheld gadget such as smartphone possess great opportunities for diagnosis where
the device can be operated even by a layman with limited training and saves time and cost as
compared to bulky spectroscopic procedures used worldwide for urea determination.

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Abstract

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In the present study, we have developed a smartphone based handheld optical biosensor for
determination of urea in saliva. A simple strategy was adopted by immobilization of urease enzyme

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along with a pH indicator on a filter paper based strip. The strip changed color upon the reaction
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with urea present in saliva and the color change can be estimated using our smartphone based
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application based on RGB profiling. Calibration of the biosensor was carried out using spiked saliva
samples and an exponentially decreasing calibration curve has been obtained for green pixel
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intensity in the broad range (10-1000 mgdL-1) with a linear detection range of 10-260 mgdL-1 and
a response time of 20 seconds. The sensitivity reported for the biosensor in the clinically significant
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range was -0.005 average pixels sec-1/mgdL-1 with a LOD of 10.4 mgdL-1. Studies carried out on
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spiked saliva samples showed a good correlation between salivary urea estimated using our
biosensor against phenol-hypochlorite based spectroscopic procedure. Development of a
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smartphone based biosensor for urea estimation eliminates the need for procuring a dedicated
instrument as well as trained technician for daily monitoring and saves time as compared to
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traditional laboratory methods of analysis.

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Keywords: Smartphone based biosensor; non-invasive biosensor; salivary urea; optical biosensor

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1. Introduction

Urea is the major end product of nitrogen metabolism in humans and is eliminated from the body
mainly by the kidneys through urine but is also secreted in body fluids such as blood and saliva.
Its level in urine ranges from 7-20 mgdL-1 which drastically rises under pathophysiological
conditions thus providing key information of renal function and in the diagnosis of various kidney

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and liver disorders [1]. Increase in urea levels in blood, also referred to as azotemia or uremia is
referred to as Chronic Kidney Disease (CKD) or End Stage Renal Disease (ESRD) and is generally
caused due to the progressive loss of kidney function. Normal glomerular filtration rate (GFR) lies

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between 100-120 ml/min. which begins to fall below 70 ml/min with the onset of azotemia or

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uremia. For subjects with kidney failure, GFR reaches around or even less than 15 ml/minute [2].
Apart from CKD, several other conditions such as heart failure, hypovolemic shock,
gastrointestinal bleeding, severe infections also leads to a rise in urea levels beyond normal [1].

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Diabetes and hypertension has been reported as the major risk factors for CKD in both developing
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as well as developed countries followed by glomerulonephritis and cardiovascular disease [3,
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4].According to National Kidney Foundation, CKD affects around 10% of world’s population [5]
and was ranked 18th among the various causes of deaths worldwide in 2010 with an annual death
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rate of 16.3 per 100000 [6] or over 1 million in total [3]. Therefore, diagnosis of kidney disease at
an early stage is important in order to prevent the development of drastic consequences.
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Kidney Function tests play an important role in the diagnosis of renal disorders at early stages.
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Several tests such as urinalysis, urine protein, creatinine clearance, serum creatinine, Blood Urea
Nitrogen (BUN), Glomerular Filtration Rate (GFR) etc. involving either urine or blood samples
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are commonly grouped under Kidney Function tests [7]. Most important of these tests are Blood
Urea Nitrogen (BUN) and serum creatinine which are frequently used in every diagnostic
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laboratory for estimation of renal function. These tests also form an essential part of radiological
screening procedures such as Magnetic Resonance Imaging (MRI) and Computed Tomography
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(CT) prior to the administration of radiological contrast agents such as iodinated contrast or
gadolinium based contrast agents so as to prevent complications such as contrast medium induced
nephropathy and nephrogenic systemic fibrosis [8].

Amongst several methods for the estimation of urea in body fluids, most are based on colorimetric
procedures. Some of these are nesslerization [9], phenol-hypochlorite or Berthelot method [10]

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and diacetyl monoxime method [11, 12]. Blood Urea Nitrogen test (BUN) is most commonly used
test for assessment of blood urea levels and the test is frequently combined along with a serum
creatinine test for the differential diagnosis of pre-renal, renal and post-renal hyperuremia. BUN
measures the amount of nitrogen present in a subject’s blood. The main drawback of this procedure
is that it is time-taking as well as involves blood extraction which is painful and inconvenient to
the subject undergoing the procedure; therefore use of alternate body fluids for urea estimation is

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of great importance. Among non-invasive body fluids such as urine, saliva, sweat and tears, saliva
is most easily accessible to the user due to ease in collection and its availability. Several research

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groups have also established a good correlation between salivary and blood urea levels using
conventional methods [13-17]. However, development of a biosensor for measuring urea levels in

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saliva samples is an immediate necessity.

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The first urea biosensor was reported by Guilbault and Montalvo by using a potentiometric urease
enzyme electrode [18]. Since then various biosensors have been reported till date for the estimation
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of urea in biological fluids employing both electrochemical [19-21] as well as optical methods [22-
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25] of detection. Various organic as well as inorganic matrices have been used for immobilization
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such as latex polymers [26], conducting polymers such as polypyrrole [27], metal nanoparticles
[28], metal oxides [29] and so on. Among non-invasive category, several reverse iontophoresis
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based biosensors have been developed for the determination of blood urea concentration using
potentiometric techniques [30, 31]. Chen et al developed a conductivity cell with 61 MHz surface
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acoustic wave resonator based measurement circuit for determination of urea in urine samples with
a LOD of 30 ngml-1 [32].A piezoelectric biosensor feasible for detecting urea in urine samples has
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been developed by Yang et al by immobilizing urease enzyme on nanoporous alumina membrane

[33]. Likewise, an amperometric urea biosensor capable of detecting urea in aqueous solutions as
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well as urine has been developed by covalent immobilization of urease enzyme on N2 incorporated
diamond nanowire film [34]. Another voltammetric sensor based on single walled carbon
nanotubes has been developed by Chen et al which can sense urea in urine samples [35].
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However, all these biosensors developed for urea estimation suffers from several drawbacks such
as use of complicated as well as costly methods in fabrication, requires trained personnel to gather
results and most of them are not portable in nature, and hence can’t be used for in situ monitoring
at hospitals or homes. One such portable clinical analyzer has been developed by Abott with

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commercial name ‘iStat portable clinical analyzer’ which can measure several parameters such as
urea and creatinine using few drops of blood through its disposable cartridges but the device is
costly as well as invasive in nature, therefore is not preferred by common people for routine
monitoring [36, 37]. One recent breakthrough in portable urea sensing was development of saliva
urea nitrogen dipstick test strips by Evans et al which was used to detect kidney disease in Malawi
but had several limitations such as being qualitative and lacking accuracy, as the change in urease

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based pH strip’s color was compared using naked eye [38].

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In this respect, for addressing all the drawbacks of these reported sensors, we have developed a
smartphone based optical biosensor for the determination of urea in saliva samples using a simple

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methodology by immobilization of urease enzyme along with pH responsive dye on a filter paper
based strip. The strip changed color due to the increase in pH upon the formation of ammonia as a

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result of urease enzyme reaction with urea present in saliva sample. The enzymatic reaction of
urea with urease enzyme can be described as Eq. (1)

CO(NH2)2+H2O
Urease
2NH3+CO2 Eq. (1)
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The higher the concentration of urea present in the sample, the more ammonia was liberated and
hence color change was more profound. This color change in the paper strip was then screened
through RGB profiling with the use of a smartphone based application developed in-house. The
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urea concentration was deduced using calibration curve equation fed into the smartphone
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application itself. In today’s digital era, smartphones have become ubiquitous and contains several
features such as high resolution camera, computational ability, and capability of integrating to
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several devices via in built Bluetooth and GPS systems, enabling researchers to use smartphone as
a diagnostic tool for the detection of various diseases, metabolites, biomarkers, pathogens etc. [39-
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43]. Following this trend, we have also used smartphone based platform to make the device more
user-friendly eliminating the need for procuring a dedicated instrument for analysis. Moreover,
slope based calculation of test results was followed to enhance the sensitivity of detection and
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reduce ambient light interferences. In our previous studies, we had developed a smartphone based
salivary glucose biosensor using a test strip and android app working on slope based calculation
method and validated it on real samples for mass diagnosis of diabetes [44]. The algorithm for
detection was altered in this app for sensing salivary urea level. For example, instead of cumulative

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calculation of sensor response in terms of Slope (R+G+B), in the present work we employed slope
of green pixel intensity. The test strip preparation was also altered to suit urea detection.

Few other groups have also demonstrated smartphone based detection of biomolecules [45, 46].
However their strategy involves more complex methods of fabrication of sensor or they have used
open source image processing softwares to deduce analyte concentration in mostly offline mode

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compared to our online sensing method. In this respect, our present work involves a simple
fabrication method where the strips are paper-based and hence biodegradable. Moreover, the

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analysis is done onsite with the help of developed smartphone app and hence can be used at such
places where healthcare facilities are not easily available.

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2. Materials and methods

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2.1. Materials

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Smartphone (Samsung Galaxy SIII) was of Samsung India Limited make; laminator (Model ECO
12) was from ExcelamTM. Filter paper (Whatman number 1), polyvinyl alcohol (Cat. No.563900),
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urease enzyme (Cat. No. U4002-20 KU), sodium phosphate dibasic (Cat. No.V800397), sodium
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phosphate monobasic (Cat. No. V800376), phenol (Cat. No. P4161), sodium hydroxide pellets (Cat.
No. 221465) were procured from Sigma Aldrich India; urea (crystalline, extrapure) was from Merck
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India Ltd.; phenol red indicator, sodium nitroprusside and sodium hypochlorite was procured from
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Fisher Scientific India. Lamination films, card stock sheets, double sided tape, nylon mesh and ear
buds were purchased from local market.
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2.2. Methods

2.2.1. Preparation of test strips and immobilization of urease

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The supporting material used for strip preparation was card stock sheet (credit card size) with
dimensions of 8.9 cm × 5 cm and thickness of about 0.35 micron. A 4.5 mm hole was created on
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the supporting layer at a distance of 1.5 cm from the top and the hole was laminated using a thin
lamination film. Circular filter paper with 5 mm diameter was then placed on top of the lamination
film covering the hole and was again covered with another lamination film containing a 4.5 mm
hole such that the filter paper gets embedded between the two films leaving a 4.5 mm detection
zone where enzyme immobilization as well as detection takes place. The detection zone was

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covered with 1.2 cm long and 0.8 cm wide nylon mesh with a pore size of 500 micron held on both
sides with the help of a double sided adhesive tape. The layered structure of the strip is depicted in
Supplement Fig. S1.

Immobilization of urease on the strips was carried out using simple entrapment method where a
solution of 13.5 mg urease along with 200 µL of dye (phenol red), 200 µL of phosphate buffer

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(1mM, pH 7.0) and 100 µL of 0.25% PVA was prepared for the immobilization of 100 test strips
and 5 µL of the above solution was immobilized per strip through the mesh with the help of a pipette

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such that 10 U enzyme loading was obtained per strip. The strips were then dried and stored
desiccated at 4°C until use. The enzyme activity of the strips was estimated using phenol

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hypochlorite method [10] and protein content was determined using Lowry’s method [47].

2.2.2. Development of android Smartphone application for RGB profiling

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For the estimation of urea concentration using RGB profiling, smartphone based android

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application (app) has been developed in-house incorporating the smartphone camera as well as
flashlight. The application could estimate urea levels with respect to pixel intensity using slope
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method.
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Slope method (change in sensor response divided by time interval) has been chosen against
differential method (change in sensor response between two time intervals) to increase the
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sensitivity as well as reduction of interference from fluctuations in flashlight intensity which can
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lead to errors in measurement. Moreover, no baseline correction was required in case of slope
method during calibration, as slope of sensor response curve remains almost constant within the
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dynamic range. Whereas, in case of differential method the sensor response changes with time,
requiring to deduct baseline value and in the event of a baseline shift the results are often erroneous.
The developed app was different from its earlier version [44] in a sense that it calculated Slope[G]
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instead of Slope [R+G+B] pixels.

For urea estimation using the developed application, saliva sample was applied on the strip which
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was placed inside a cardboard box (3 cm height) containing a hole so that the flashlight of the
camera illuminates the detection zone when smartphone is placed over it. The requirement of a box
was to minimize the problem associated with variations in distance from camera with the detection
zone, if one was to hold the test strip in hand or against a surface. The effect of height wise variation
on the strip under ambient light condition has been checked for printed strips (red, green and blue

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color) and slopes for red, blue and green pixel intensity were obtained within a fixed time interval
with varying height. Results are depicted in Supplement Fig. S2. After placing the test strip inside
the box, the app was initiated by adjusting the time interval for obtaining sensor response slope
(between 10-20 sec. in this case). The android app after initiation captures first image of detection
zone at 0 sec. Thereafter it calculates average G values for 20 random pixels within the detection
zone. Second image is captured after 10th sec in similar manner. The difference of average G values

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at two intervals is subtracted and divided by 10 sec to calculate slope of response curve. Hence, the
units of sensor response have been taken as average pixel / sec throughout the calculations. These

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software based calculations are not visible to the user and continues in background. If the test is
successful, it’ll show the slope value on screen, else, a prompt is shown stating that test was

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unsuccessful due to improper saliva volume. The change in slope (δpixel/10 sec for each color) is
then used for urea concentration calculation using calibration equation fed into the app (Supplement

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Fig. S3 and S4). Prior to that, to obtain calibration equation, raw slope data obtained from app is
plotted against known spiked urea concentrations. N
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2.2.3. Biosensor measurements
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In order to minimize variation in viscosity and other salivary parameters, calibration of the
biosensor was carried out using saliva samples of healthy individual spiked with 5% volume of
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known concentrations of synthetic urea prepared in phosphate buffer (1mM, pH 7.0) so as to

prepare concentration ranging from 0-1000 mgdL-1. Urea concentration already present in healthy
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individual’s saliva sample was estimated using phenol-hypochlorite method and the values
obtained were added to the known concentration so as to obtain actual concentration of urea
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present in the spiked sample. For biosensor measurements, 5 µL of the above sample was applied
on the strip through the mesh with the help of a pipette and color changes on the opposite site of
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the strip was screened using RGB profiling through our developed application as shown in Section
2.2.2. The reaction principle and schematics of salivary urea detection using the developed
biosensor and color changes in the strips with increasing glucose concentration are depicted in
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Fig.1 (A, B &C).The response time was fixed as 20 seconds where 10 seconds was the time
required for sample application and placing the strip inside the dark box and then the slope was
taken for remaining 10 seconds, altogether giving a response time of 20 seconds. Change in slope
for R, G and B pixels was obtained and calibration curves were plotted against urea concentration
v/s Slope (average pixels/sec) using Originlab 2017 software. For calibration, all the readings were
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obtained in triplicates and data plotted with standard deviation as error bars. Shelf life study on the
biosensor strips was also carried out for a period of 30 days.

2.2.4. Effect of chemical interferents on sensor response

Ascorbic acid, lactic acid and uric acid are the most common interferents present in human saliva
which can cause changes in sensor response. The concentration of these acids in human mouth

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range from 0-20 mgdL-1, which varies between individuals and at different times of the day
depending upon the food intake [48-50]. Ascorbic acid levels in human saliva increases with the

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intake of foods rich in vitamin C whereas presence of lactate is mainly administered with bacterial
fermentation process occurring in human mouth. To study the effects of these interferents on sensor

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response, 5 µL of saliva sample was added onto the strip with or without spiking with synthetic
urea. For carrying out these studies, saliva sample was spiked using 15 mgdL-1 urea and lactic or

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ascorbic acid concentrations in the range of 0-20 mgdL-1. The change in slope for green pixel

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intensity was obtained within a response time of 20 seconds for samples containing
ascorbate/lactate alone and for samples containing urea along with an interferent. pH changes on
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the strips upon addition of these samples was also recorded to predict the buffering action of saliva
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on addition of these acids. Interference studies with uric acid were also carried out in the range of
0-20 mgdL-1 uric acid. All the studies were carried out in triplicates and data plotted using standard
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deviation as error bars.

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2.2.5. Clinical validation of the biosensor with real samples

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For validation of the biosensor on real samples, saliva samples from few healthy donors was
obtained after obtaining their written consent as per the ethical guidelines, some of these samples
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were spiked with synthetic urea samples prepared in phosphate buffer so as to obtain higher
concentrations as found in subjects with impaired renal function. Fresh saliva was used for analysis
and the sample was obtained after rinsing of mouth with drinking water so as to nullify the effect
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of all possible interferences caused due to the presence of substances such as ascorbate, and lactate
commonly found in human mouth. The samples obtained were tested using our developed sensor
and also using the standard phenol-hypochlorite based spectrophotometric procedure and a

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correlation was established between both these methods using t-test performed using Microsoft
Excel software.

Correlation between blood and salivary urea concentration was carried out for 19 subjects enrolled
at IIT Delhi hospital with written consent obtained from them. Salivary urea concentration was
obtained using our developed biosensor and blood urea nitrogen readings were obtained from the

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pathology lab of IIT Delhi hospital where the blood urea concentration was estimated using
spectrophotometric method with the help of autoanalyzer (Cobas Integra). Correlation between

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blood and salivary urea concentration was established by t-test using Microsoft Excel. Tests for
repeatability and reproducibility were carried out by obtaining urea concentration using the

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developed sensor for same sample multiple times and for three different samples at three different
time intervals.

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3. Results and discussion

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3.1. Immobilization and characterization of urease on the strips
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Schematic of strip preparation and immobilization along with color change in the strips with respect
to increasing urea concentration is depicted in Fig. 1 (B&C respectively). The immobilized strips
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were stored desiccated at 4°C until use. Enzyme activity and protein content of the strips were
estimated in triplicates. The enzyme activity per strip calculated as per the phenol-hypochlorite or
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Berthelot assay was about 4.6 U (indicating ~46% yield of immobilization) whereas the protein
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content deduced from Lowry’s method was found to be around 225 µg in a detection zone of 0.635
cm2 circular area. Specific activity was therefore calculated as 20.5 U/mg protein. Shelf life studies
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carried out on the strips showed an exponential decay trend with around 50% loss of activity after
a period of 30 days (Supplement Fig. S5). This was obvious for the reason urease is not that stable
enzyme when compared to glucose oxidase. However, in entrapped condition, its activity decreases
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and saturates after few days of immobilization [51].

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3.2. Biosensor measurements

Calibration curves for the biosensor were obtained after calculating slope of response curves for
Red, Blue and Green pixel intensity v/s urea concentration within a response time of 20 seconds
(10 seconds for sample handling+10 seconds slope) using Originlab software. Within the broad

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range (10-1000 mgdL-1) an exponentially decreasing calibration curve has been obtained for Green
pixel intensity (Slope G). For Blue (Slope B) and Red pixels (Slope R) an exponentially increasing
as well as linear curves were obtained respectively, while showing negligible change in pixel
intensity with respect to red pixels (Fig.2A). The curves exhibit good linearity within the range of
10-260 mgdL-1 (Fig.2B). Highest sensitivity was obtained for Green pixel intensity (Slope G) as
compared to Blue and Red pixels within the clinically relevant range; therefore for clinical

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validation and other studies, calibration curve for Green pixels (Slope G) with respect to urea
concentration was chosen against Blue and Red pixels. The calibration curve within the broad range

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(10-1000 mgdL-1) for Green pixels (Slope G) can be depicted by equation (1) where X denotes
unknown urea concentration and Y denotes Slope (G)

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Y=1.42 × e(-X/137.33)-1.464 (1)

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The calibration curve for Slope (G) within the linear range of 10-260 mgdL-1 can be depicted by
equation (2) below with a sensitivity of -0.005 average pixels sec-1/mgdL-1.

Y= -0.005× X-0.09 (2)

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Limit of Detection (LOD) of a sensor can be practically calculated by measuring the pixel intensity
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change (Slope G) in samples devoid of urea. For estimation of LOD of the developed sensor, saliva
sample from a healthy donor was made to react with urease enzyme for 1 h till whole urea present
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in the sample gets consumed. The sensor noise level for this sample (n=4) was found to be -0.0565
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average pixels/second, which corresponded to 10.4 mgdL-1 as LOD. This was significant, as the
clinically relevant range for urea in serum starts at about 20 mgdL-1 concentration.
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3.3. Interference studies carried out on the biosensor

To estimate the effect of chemicals on sensor response, interference studies were carried out using
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ascorbic, lactic and uric acid concentrations within the range of 0-20 mgdL-1, which is a range in
excess of what usually found in saliva. It was observed that when saliva sample was spiked with
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ascorbic or lactic acid alone without the addition of urea, the slope of green pixel intensity showed
an increasing trend towards positive axis with increase in concentrations of these acids, which was
due to the decrease in overall pH of sample. However, in samples containing urea along with
interferent (ascorbic or lactic acid), saliva was found to act as a buffer at lower concentrations of
these acids [52, 53] and the pixel intensity was found to be almost constant for lower concentrations

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which then slowly started to increase with the increase in acid content in the saliva due to the
decrease in pH. The trend has been depicted in Fig. 3(A-D). Fasting lactic acid concentration present
in human saliva sample is around 1.8 mgdL-1 [49] and values for ascorbate concentration often
ranges in between 0-20 mgdL-1 [48]. Thus, for such subjects the sensor was found to be free of
these interferents within the clinically relevant range. The sensor was also found to be selective for
urea, with no interferences with uric acid in the clinically relevant range of uric acid present in

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human mouth (0-8 mgdL-1) (Fig. 3E). However, for subjects who have undertaken a vitamin C rich
diet or suffering from dental caries, slight interferences may result due to the presence of these acids

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in higher concentrations in mouth. Therefore, to obtain uniformity in sample collection and
eliminate all possible interferences, subjects were advised not to eat or drink anything especially

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vitamin C rich diet at least 2-3 hours before the test and were told to rinse their mouth with drinking
water prior to sample collection. Only fresh saliva accumulated in mouth was to be collected for

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measurements.

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3.4. Measurements with clinical samples
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Clinical validation of the biosensor was carried out using spiked saliva samples. Fresh saliva was
collected from 3 healthy donors after rinsing of mouth with drinking water without involvement of
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any filtration or centrifugation step. Saliva samples from these three subjects were then spiked with
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different concentrations of urea prepared in phosphate buffer. Nineteen different samples were thus
obtained (after simulated spiking). Salivary urea levels of all these samples were then obtained
using our developed sensor and at the same time phenol-hypochlorite method was also used to
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calculate urea concentration in conventional manner. Correlation between salivary urea

concentrations obtained using these two methods was then established by t-test using Microsoft
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Excel software. Further, for establishing correlation between blood urea level (using an
autoanalyzer) and salivary urea concentration (calculated using our biosensor), validation was
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carried out on 19 healthy subjects between the age group of 30-70 years and correlated was obtained
by t-test in similar manner.

R2 value of 0.93 (n=19, Slope=1.01, Pearson’s R=0.96) with P value of 0.16 was found between
salivary urea concentration in spiked samples obtained using our biosensor and spectrophotometric
procedure (phenol hypochlorite method), thus showing insignificant difference between the two

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methods (Fig. 4A). Thus a good correlation obtained with standard procedure in spiked samples
suggests that the developed biosensor can be used for urea estimation in saliva samples obtained
from healthy as well as uremic subjects for diagnosis of renal dysfunction. Similarly, when blood
urea concentrations in healthy volunteers obtained using an autoanalyzer was correlated with their
salivary urea concentration (obtained using our developed sensor), R2 value of 0.68, with Pearson’s
R value of 0.83 (n=19, Slope=0.8, P=1.33E-06) was obtained (Fig. 4B). In these volunteers, the

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blood urea levels were within the range of 10-40 mgdL-1. Such correlation was similar to that
previously reported in literature (Pearson’s R2 ~70) [54] and hence validated our device

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performance. In continuation of our studies, correlation tests with uremic subjects, particularly
those with CKD and other disorders shall be carried out in future after obtaining necessary ethical

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clearances for larger study samples. In the case of uremic subjects, significant correlation (R value
>/= 0.9) between blood and salivary urea concentration is expected. Higher correlation is also

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expected in case of subjects with hypertension or those suffering from diabetes, where more urea

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leaches out in saliva due to the damage of epithelial lining of salivary gland.
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Further, repeatability of sensor measurement was evaluated by obtaining urea concentration for a
healthy donor’s saliva sample concurrently for five times (n=5), the studies showed an average urea
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concentration of 18.17 mgdL-1 with a standard deviation of 1.8. Test for assessing the
reproducibility of the biosensor was also carried out. The sensor was found to be around 90%
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reproducible when readings were obtained for three different saliva samples at three different time
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intervals (Supplement Fig. S6).

Conclusion
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In the present study we have developed an optical urea biosensor using saliva sample and a
smartphone. The urease-pH indicator immobilized strips changed color with respect to urea
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concentration in saliva sample and the color changes were detected using smartphone based app
using RGB profiling and slope based calculation method. Calibration curve with green pixel
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intensity (Slope G) was found to be most sensitive with a sensitivity of -0.005 average pixels sec-
1
/mgdL-1 within the linear range of 10-260 mgdL-1 and a LOD of 10.4 mgdL-1. Salivary urea
determination using our developed biosensor shows good correlation with spectroscopic method
such as phenol-hypochlorite for spiked samples. Correlation of 0.83 was obtained between blood
and salivary concentration in case of healthy subjects; however more studies are required on CKD

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patients and those suffering from hypertension and diabetes for better standardization of the sensor.
The developed biosensor can be used by a layman with limited training against common laboratory
methods that can be performed only by trained professionals. Another benefit of the developed
sensor is that results can be obtained in less time (just 20 seconds) against time consuming
spectrophotometric procedures where a patient has to wait for report for a day or at least few hours.
Our developed sensor is fabricated using simple technique just by immobilizing urease along with

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pH responsive dye on a filter paper and is cost-effective. Moreover, the screening is done through
a smartphone which has become a common gadget nowadays among people, thus eliminating the

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need for procuring a dedicated instrument for analysis.

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Acknowledgement

The author is obliged for the intramural financial support provided by Indian Institute of

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Technology Delhi. The author Anuradha Soni thanks Indian Council of Medical Research, New
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Delhi for research fellowship. A part of this work has been filed for Indian Patent (1587/DEL/2015
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dated 1/6/2016).
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Author Biographies:

Dr. Sandeep K. Jha

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Dr. Sandeep K. Jha is a joint faculty at the Centre for Biomedical Engineering (CBME), Indian

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Institute of Technology Delhi and All India Institute of Medical Sciences New Delhi.
His areas of interest include Lab-on-a-chip and Microfluidics devices for biomedical applications;

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electrochemical and optical chemical and biosensors; bioinstrumentation & nanomaterials,
conducting and synthetic polymers based immobilization techniques.
Previously he served under various capacities at Banasthali University, India; Korea University,
South Korea; KIIT University, Bhubaneswar, India; Myongji University, South Korea; Indian

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Institute of Technology, Mumbai, India and Bhabha Atomic Research Center, Mumbai, India.

Ms. Anuradha Soni

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Anuradha Soni is a Ph.D. research scholar at Centre for Biomedical Engineering (CBME), Indian
Institute of Technology Delhi and has been working on development of clinical biosensors,
especially those which are non-invasive in nature. She has authored 2 manuscripts and has
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developed a smartphone based non-invasive biosensor for salivary glucose detection.

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Legends to Figures
Fig.1. (A) Reaction principle involved in the biosensor (B) Schematics of the biosensor showing
steps for urea detection and (C) Color change in the strips with increase in urea concentration (10-
1000 mgdL-1).

Fig.2. (A) Calibration curve plotted against urea concentration v/s change in slope for R (-□-), G

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(-○-) and B pixels (-Δ-) in the broad range (10-1000 mgdL-1). (B) Calibration curve for urea
concentration v/s change in Slope for R (-□-), G (-○-) and B (-Δ-) pixels within the linear range of

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10-260 mgdL-1. Maximum sensitivity has been reported for Slope (G) within this range.

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Fig.3. Interference studies using lactic and ascorbic acid. (A) Effect of ascorbic acid concentration
on sensor response. It has been observed that in the clinically relevant range of ascorbic acid found
in mouth, saliva was found to act as a buffer to some extent while with higher concentrations of

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ascorbic acid, sensor response increases with decrease in pH. (B) Study of buffering action of
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saliva upon addition of ascorbic acid. (C) Effect of lactic acid concentration on sensor response.
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Similar behavior has been noted as in the case of ascorbic acid with increasing concentration. (D)
Study of buffering capacity of saliva upon addition of lactic acid. (E) Effect of uric acid
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concentration on sensor response.

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Fig.4. (A) Correlation between salivary urea concentration obtained using phenol-hypochlorite
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method with salivary urea concentration deduced using our developed biosensor (-●-). Studies
were carried out by spiking saliva samples obtained from 3 healthy subjects with synthetic urea
solutions prepared in phosphate buffer (1mM, pH 7.0) (B) Correlation between blood urea
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concentration obtained using an autoanalyzer with salivary urea obtained using our developed
biosensor (-○-) for healthy subjects.
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Figures
Figure 1.

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Figure 2.

2.0
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1.5

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Slope(Av. pixels/sec)

1.0 y=1.61x1.92/(162.27)1.92+(x)1.92

0.5

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0.0
y=-0.000056x-0.0012

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-0.5
-1.0 y=1.42*exp(-x/137.33)-1.464

-1.5

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-2.0
0 200 400 N
600
Urea Concentration (mgdL-1)
800 1000
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1.5 B
Slope (Av. pixels/sec)

1.0
D

0.5
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y=0.004x-0.007

0.0
y=0.00016x-0.013
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-0.5
-1.0 y=-0.005x-0.09
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-1.5
0 50 100 150 200 250
A

Urea Concentration (mgdL-1)

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Figure 3.

0.05
A 7.1 B
0.00
Slope G(Av. pixels/sec)

7.0
-0.05
Ascorbate+saliva 6.9 Ascorbate+urea+saliva
-0.10

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6.8

pH
-0.15
6.7
-0.20
6.6 Ascorbate+saliva
-0.25 Ascorbate+urea+saliva

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6.5
-0.30
6.4
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35

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-1 -1
Ascorbic acid concentration (mgdL ) Ascorbic acid concentration (mgdL )
0.05
C
6.90 D
Slope G(Av. pixels/sec)

0.00
6.85

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-0.05
Lactate+saliva 6.80 Lactate+urea+saliva
pH

-0.10

-0.15
Lactate+urea+saliva
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6.75

6.70
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-0.20 6.65
Lactate+saliva
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-0.25 6.60
0 5 10 15 20 0 5 10 15 20
-1 -1
Lactic acid concentration (mgdL ) Lactic acid concentration (mgdL )
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-0.14 E
Slope G (Av. pixels/sec)

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-0.12 Uric acid+urea+saliva

-0.10
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-0.08

-0.06
0 5 10 15
A

Uric acid concentration (mgdL-1)

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Figure 4.
Salivary urea (Biosensor) (mgdL-1)

80
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70 n=19

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R2= 0.93
60 r= 0.96
50 Slope= 1.01

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40

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30
20

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10
0 10 20 30 40 50 60
Salivary urea (Phenol hypochlorite) (mgdL-1)
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A
Salivary Urea Concentration (mgdL-1)

30 B n=19
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r = 0.83
25 R2= 0.68
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Slope= 0.8
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15
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10
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5
10 15 20 25 30 35 40
A

Blood Urea Concentration (mgdL-1)

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