Measurement 44 (2011) 312–319

Contents lists available at ScienceDirect

Measurement
journal homepage: www.elsevier.com/locate/measurement

Prediction of calcium concentration in human blood serum using an

artificial neural network
P. Neelamegam a,⇑, A. Jamaludeen b, A. Rajendran c
a
Department of Electronics & Instrumentation Engineering, SASTRA University, Thanjavur, Tamil Nadu 613 402, India
b
PG Department of Electronics, St. Joseph’s College (Autonomous), Tiruchirappalli, Tamil Nadu 620 002, India
c
PG and Research Department of Physics, Nehru Memorial College (Autonomous), Puthanampatti, Tiruchirappalli, Tamil Nadu 621 007, India

a r t i c l e i n f o a b s t r a c t

Article history: A predictive method, based on artificial neural network (ANN) has been developed to study
Received 3 December 2009 absorbance and pH effects on the equilibrium of blood serum. This strategy has been used
Received in revised form 11 September to analyze serum samples and to predict the calcium concentration in blood serum. A ded-
2010
icated data acquisition system is designed and fabricated using a LPC2106 microcontroller
Accepted 16 September 2010
Available online 29 September 2010
with light emitting diode (LED) as source and photodiode as sensor to measure absorbance
and to calculate the calcium concentration. A multilayer neural network with back propa-
gation (BP) training algorithm is used to simulate different concentration of calcium (Ca2+)
Keywords:
Artificial neural network
as a function of absorbance and pH, to correlate and predict calcium concentration. The
Back propagation algorithm computed calcium concentration by neural network is quite satisfactory with correlations
Blood serum R2 = 0.998 and 0.995, standard errors of 0.0127 and 0.0122 in validation and testing stages
Calcium respectively. Statistical analysis are carried out to check the accuracy and precision of the
Microcontroller proposed ANN model and validation of results produce a relative error of about 3%. These
LED results suggest that ANN can be efficiently applied and is in good agreement with values
Photo diode obtained with the current clinical spectrophotometric methods. Hence, ANN can be used
as a complementary tool for studying metal ion complexion, with special attention to
the blood serum analysis.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction called ‘perceptron’. In 1957, Frank Rosenblatt introduced a

Artificial neural network (ANN) is a promising area of
research for prediction and forecasting in the field of
instrumentation and measurement. ANNs are biologically
inspired computational methods, which can be used to
capture complex and non-linear relationships between
data [1]. The ANNs basically consist of several non-linear
processing units, called neurons or nodes and they are
connected in a massive parallel architecture [2]. The con-
cept of artificial neurons is first introduced in 1943 [3].
Rosenblatt and Block et al. proposed a neuron like element
⇑ Corresponding author.
E-mail addresses: neelkeer1@yahoo.com (P. Neelamegam), jam80i-
n@yahoo.com (A. Jamaludeen), neelrajnmc@gmail.com (A. Rajendran).
training algorithm that provided the first procedure to
train a simple ANN: a perceptron [4]. It consists of a single
neuron with adjustable synaptic weights and a hard lim-
iter. In 1986, Rumelhart et al. presented the concept of
back propagation training algorithm [5]. ANN acquires
the knowledge through a learning process and stores the
knowledge in inter-neuron connection strengths known
as synaptic weight. Multilayer perception (MLP) has been
applied successfully by training it in a supervised manner
with a generalized steepest descent algorithm called back
propagation (BP) algorithm. This algorithm works on the
weight error correction rules. A data acquisition system
has been designed using LPC2106 microcontroller, that
facilitated with all essential peripherals in a single-chip
provides a highly versatile platform for the determination
of calcium concentration.

0263-2241/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.measurement.2010.09.035
 P. Neelamegam et al. / Measurement 44 (2011) 312–319
Calcium is the most prevalent cation, which plays
important role in skeletal mineralization, blood coagula-
tion and neuromuscular conduction. Estimation of calcium
concentration in blood serum plays a vital role in identifi-
cation of many deficiencies such as risk of hypertension,
arteriosclerosis, alzheimers [6], colon cancer and premen-
strual syndrome. Serum calcium level gives indication
about thyroid diseases [7] and serves as indicator for vita-
mins A and D deficiency. There are many methods for the
determination of calcium ions, which involve atomic
absorption spectrometry, potentiometry with ion selective
electrodes, EDTA titration and spectrophotometry with
variety of reagents. Among these methods, spectropho-
tometry is well-suited because of its simplicity and cost
effective [8]. The basic principle of the spectrophotometry
is the transmittance measurement which relies on Beer’s
law. For spectrophotometric determination of calcium,
arsenazo III, a metallochromic indicator used as selective
ligand and it forms a colored complex with calcium. Arse-
nazo III has high sensitivity and selectivity for calcium ions,
forms stable complexes over a wide pH range [9]. A study
is made to find the effect of magnesium ion in absorbance,
relative to Ca2+ ion concentration. When, Mg2+ ion concen-
tration is less than or equal to Ca2+ ion concentration, no
change in absorbance is observed. When, Mg2+ ion concen-
tration is 5-fold or above relative to Ca2+ ion concentration,
increase in absorbance is observed. Hence, lower concen-
tration of Mg2+ ion concentration does not interfere with
Fig. 1. The block diagram of microcontroller based calcium measurement system.
313
calcium determination [10]. The instrumentation system
has been developed based on LPC2106 microcontroller, to
measure the absorbance, calculate the concentration and
to store the test result for future reference. The system is
programmed to share the data with external peripherals
like PC.
2. Experimental
2.1. System hardware
The block diagram of microcontroller based calcium
measurement system is presented in Fig. 1. The system
hardware consists of light intensity measurement unit
with photodiode as sensor, temperature measurement
unit, an analog multiplexer to select light intensity or tem-
perature, a 12-bit analog to digital converter and LPC2106
microcontroller. A liquid crystal display (LCD), keyboard,
temperature control circuit and MAX232 are also inter-
faced with the microcontroller.
The hardware is built with LPC2106 RISC microcontrol-
ler from Philips Semiconductor. It is a 32-bit ARM7TDMI-S
processor with 128 Kbytes on-chip flash program memory
and 64 Kbytes on-chip static RAM. This device operates
with a clock frequency of 60 MHz with two UART units
one with full modem interface, PWM unit, two 32-bit tim-
ers, real time clock and watchdog timer [11]. LEDs are an
314 P. Neelamegam et al. / Measurement 44 (2011) 312–319
attractive alternative to conventional white-light sources
in calcium analysis because of reduced power consump-
tion and increased flexibility. The LED is used as a source
to emit 650 nm wavelength of light. A photodiode
(GASPG1124) is used as sensor to detect the amount of
light and it generates current in an external circuit propor-
tional to the incident light. The photodiode exhibits 85% of
quantum efficiency at 650 nm of wavelength with very low
dark current. A black protective casing ensures isolation
form the ambient light. The LF357 operational amplifier
from National Semiconductor is designed for high slew
rate, low current noise and low 1/f noise corner, make this
well suited for photocell applications. The output current
of photodiode is converted to voltage using successive sig-
nal conditioning circuit made up of LF357. For better re-
agent and sample contact, the solutions are left to stand
for 5 min at a controlled incubation temperature of 37 °C.
An AD590 (Analog Devices) is used as temperature sensor
and LF357 amplifies the voltage generated from the tem-
perature sensor (1 mV/K). The heater is controlled using
solidstate on/off power controller made up of optocoupler
MCT 2E (Vishay), UJT 2N2646 (Philips Semiconductor) and
TRIAC BT 136 (Philips Semiconductor). An 8-channel ana-
log multiplexer IC 4051 from Philips semiconductor having
three address selection inputs are used to select light
intensity or temperature by channel selection command
form the microcontroller. The analog light intensity and
temperature voltages are converted into digital bits using
an analog to digital converter ICL 7109 (Maximum Inte-
grated product) which is also interfaced with microcon-
troller. It is a low power integrated device providing high
accuracy, low drift and dual slope integrator with 12 bits
parallel outputs. The need of the MAX232 voltage level
converter circuit arises, because of the output signal gener-
ated by the microcontroller are not compatible with the
RS232 protocol. The MAX232 meets the TIA/EIA-232 spec-
ification to transmit and receive the output signals gener-
ated by the microcontroller with the personal computer
(PC). Two rows and sixteen characters wide (2  16) dot
matrix display from Hitachi, which can show 160 different
numeric and alphanumeric characters is interfaced with
microcontroller to display the absorbance, concentration
of calcium in sample and the incubation temperature.
The user enters experimental variables and modes of oper-
ation into the system through a keypad, which is interfaced
parallel with the microcontroller.
2.2. Software
Software is developed in C language, to reset all register
values for port orientation and it includes channel selec-
tion of light intensity or temperature using multiplexer,
measurement and controlling of temperature, reading dig-
ital bits form ADC, selection of UART mode with 4800 baud
rate, initialization of LCD display and COM1 port in PC to
transfer and receive data with microcontroller. Software
is also developed to train the neural network using data
and to predict the calcium concentration of the sample at
a desired pH and absorbance using the trained neural
network.
2.3. Sample preparation and measurement
To measure absorption of calcium in the serum, sam-
ples are prepared using the arsenazo III reagent. All the re-
agent used are of analytical grade and all solutions are
prepared with distilled and deionized water. Reagent Kit
for calcium determination is purchased from BioSystems,
Spain. The normal reference value of calcium present in a
serum is 2.15–2.58 mM L1 [12]. The Ca2+-arsenazo III
complex formation occurred at wide pH range of 3.0–
12.0. However, the formation of complex is detectable with
a maximum absorbance at pH 9.0. A 1000 lL of arsenazo III
solution is mixed with each 15 lL aliquot of serum, cal-
cium standard of 15 lL and 15 lL of nanopure distilled
water for the preparations of sample, standard and blank
solutions respectively. The same preparation procedure is
followed for all samples. For the conventional system (Stat
Fax Ò 2000), the reagent and sample preparation proce-
dures are same. The prepared solutions are used to mea-
sure the absorbance and to compute the concentration.
By placing the prepared blank, standard and sample solu-
tions in the sample holder, the voltages Vblank, Vstd and
Vsample are measured by the microcontroller and concen-
tration of the calcium is computed using the relation,
Concentration of the sample ðmg=dlÞ
lnðVblank =Vsample Þ
¼  Concentration of standard
lnðVblank =Vstd Þ
2.4. Implementation of neural network
An ANN is formed by interconnections of simple pro-
cessing elements or neurons with adjustable weights,
which constitute the neural structure and are organized
in layers. The behavior of the overall ANN depends upon
the operations of the artificial neurons, the learning rule
and architecture of the network. Each neuron has many in-
puts and outputs. The output path of a neuron is connected
to the input path of another neuron through connection
weights [13]. In this study, the feed forward network
termed as the multilayer perception (MLP) is used to de-
velop a non-linear ANN model of a system [14]. The
back-propagation (BP) learning rule is used to train MLP
consist of input layer, one or more hidden layer with a pre-
defined number of neurons and output layer. Fig. 2 shows
the representation of the multilayer ANN used to analyze
the influence of pH and absorbance on calcium concentra-
tion. The three layered neural network having one input
layer with two neurons, one hidden layer with five neurons
and one output layer with one neuron are used for training
of the network.
2.4.1. Training of the neural network
The measured data obtained from developed instru-
ment are used for training and validation. The test param-
eters used in the neural network model for this study is
given in Table 1. Logistic sigmoid function produces output
that varies continually with the activity level and it is used
as transfer function with momentum 1. The training
pattern for the inputs are pH, absorbance and output is
 P. Neelamegam et al. / Measurement 44 (2011) 312–319 315

Fig. 2. ANN structure to analyze the effects of pH and absorbance on calcium concentration.

Table 1
Test parameter used in the neural network. Table 2
Results of experiments for calcium concentration in different pH and
No of samples 39 absorbance (test numbers 2, 7, 11, 14, 18, 21, 23, 27, 33 and 37 are used for
No of inputs (total) 78 validation and others for training).
Learning rate 0.1
Activation function logistic Sigmoid function Test number pH Absorbance Concentration
Learning error limit 0.001 of calcium (mM L1)
Momentum 1 1 8 0.67 1.86
Training algorithm Back propagation 2 8.05 0.78 1.93
No of hidden 5 3 8.15 0.92 2.105
Neurons no of iterations 24,800 4 8.04 0.77 1.925
Actual error 0.0019 5 8.14 0.912 2.1
6 8.07 0.82 1.98
7 8.08 0.83 2
8 8.09 0.84 2.02
concentration of calcium (Table 2). The data sets are taken 9 8.03 0.75 1.91
such a way that, pH ranges from 8.0 to 10.0 and absorbance 10 8.1 0.87 2.04
are measured from 600 nm to 700 nm of wavelength. Dur- 11 8.13 0.91 2.08
12 8.01 0.72 1.9
ing training (learning), the weights between the neurons
13 8.11 0.89 2.05
are adjusted according to the mean square error between 14 9.01 0.825 2.19
the target output and the measured value for all the train- 15 9.2 0.965 2.58
ing set falls below a predetermined threshold or the max- 16 9.09 0.91 2.41
imum allowable number of epochs is reached [15]. The 17 9.14 0.935 2.5
18 9.04 0.855 2.25
neurons in the input layer acts as buffer for distributing 19 9.07 0.9 2.37
the input signals xi to neurons in the hidden layer [13]. 20 9.18 0.96 2.56
Each neuron j in the hidden layer sums up its input signals 21 9.08 0.9 2.38
xj, after them with the strengths of the respective connec- 22 9.02 0.84 2.23
23 9.15 0.95 2.53
tions Wij from the input layer and computes its output yj
24 9.11 0.93 2.46
as a function of the sum, 25 9.06 0.89 2.35
26 10.1 0.745 1.903
1 27 10.08 0.712 1.89
yj ¼ f ðIjÞ ¼ ð1Þ
1 þ eðIjÞ 28 10.11 0.78 1.908
29 10.22 0.91 2.15
where f is one of the activation function used in ANN archi- 30 10.17 0.89 2.05
tecture. A learning algorithm gives DWij(t) weight connec- 31 10.05 0.7 1.88
tion between neurons i and j at time t. The weights are 32 10.12 0.81 1.91
33 10.14 0.86 1.98
updated according to the formula: 34 10.09 0.723 1.9
35 10.21 0.908 2.14
Wjiðt þ 1Þ ¼ WjiðtÞ þ DWjiðt þ 1Þ ð2Þ 36 10.15 0.88 1.99
37 10.2 0.901 2.1
Under supervised learning, both input and output data are 38 10.13 0.83 1.95
given for the training. The training cycle is continued until 39 10.18 0.9 2.09
the network achieves desired level of tolerance. Training of
316 P. Neelamegam et al. / Measurement 44 (2011) 312–319

the neural network is terminated, when the network has
learnt to generalize the underlying trends or relationships
exemplified by the data. Generalization implies that, the
neural network can interpolate sensibly at points not con-
tained in its training sets, as indicated in Fig. 3.
2.4.2. Validation and testing pattern
The neural network learning ability is typically assessed
by means of cross-validation, where the performance of
the network is evaluated against a novel set of test data,
not used during training. Optimal number of hidden neu-
rons and number of layers as well as number of training
epochs are found by experimental cross-validation. The
cross-validation process is used to accomplish a good gen-
eralization ability, prevents from over fitting and structure
the ANN as an effective prediction model. Random test
numbers from training set (Table 2) are taken and they
are used for the validation process. It is found that, the val-
idation results agree well with the results of experimental
values, which show the good learning efficiency of the neu-
ral network model. In testing process, the efficiency of
ANNs in estimating the concentration of calcium using
the previous observed data during the training stages is
investigated. New data set are taken for the testing, to
check the ANNs prediction ability. Table 3 gives the con-
centration of calcium using the developed instrument, con-
ventional instrument and ANN prediction. From the table,
the developed prediction model is well suited for the cal-
cium measurement and the prediction accuracy is proved
with the help of statistical studies.
3. Results and discussion
In order to evaluate the efficiency of the developed
instrument, preliminary analyses are performed. All the in-
puts and outputs data are scaled before feeding into the
network so that, they are changed in the range between
0 and 1 (logistic sigmoid function) using the mean and
standard deviation,
AP  mean APS
NP ¼ ð3Þ
STD AP
where AP is actual parameter, mean APS is mean of actual
parameters, STD AP is standard deviation of actual param-
eter and NP is normalized input parameter [14]. The mean
and standard deviation for pro-processing of input and
output data are given in Table 4. From the table, it is ob-
served that, at pH 9.0, maximum absorbance is recorded
and the maximum concentration for calcium as compared
to other pH ranges like 8.0 and 10.0. The minimum size of
the network is figured out first without affecting its learn-
ing ability, including the number of neurons in the input,
output and hidden layers [16].
Optimum numbers of neuron in the hidden layer play a
vital role in the root mean square error (RMSE) [17]. Selec-
tion of neuron number based on the criteria of a minimum
divergence of training and test data during training curves
and the lowest value for RMS error of the test data. The
RMS error for the neurons number in the hidden layer is
plotted in Fig. 4. From the figure, low RMS errors are ob-
served if the neuron numbers are five in the hidden layer.
When, this number exceeds, high RMS errors are observed.
More the RMS error, the predicted outputs are far away

Table 4
Pre-processing parameters for ANN prediction.

Variables pH Absorbance Concentration of calcium

(mM L1)
Mean 8.076 0.821 1.992
9.095 0.905 2.400
10.139 0.824 1.989
Standard 0.048 0.079 0.081
deviation 0.061 0.046 0.130
0.051 0.080 0.099
Fig. 3. Comparison of generalization by a neural network.

Table 3
Comparison of calcium concentration determined using the developed instrument, conventional instrument and ANN prediction.

Test number pH Absorbance Concentration of calcium (mM L1)

Conventional Developed ANN predicted
1 8.06 0.8 1.9 1.918 1.893
2 8.02 0.73 1.98 1.937 1.965
3 9.16 0.96 2.56 2.538 2.57
4 8.12 0.9 2.04 2.06 2.064
5 10.16 0.885 2.01 2.02 2.009
6 9.05 0.865 2.285 2.31 2.278
7 9 0.81 2.15 2.15 2.142
8 9.21 0.97 2.554 2.59 2.581
9 10.19 0.899 2.09 2.1 2.074
10 9.1 0.92 2.43 2.45 2.464
11 10.07 0.717 1.87 1.885 1.868
 P. Neelamegam et al. / Measurement 44 (2011) 312–319 317

Fig. 4. RMSE value for 1–10 neurons number in hidden layer.

from the true or measured value. Hence, five numbers of
neurons are chosen in the hidden layer to reduce the
RMS error. The monitoring plots are drawn for the training
and testing data sets with RMS error as a function of the
number of iterations. The number of iterations that pro-
duced the minimum RMS error in the test data is used to
predict the concentration of the unknown sample. The
optimal network topology obtained has five hidden neu-
rons, input scaling between 1.0 and 0.0 and learning rate
of 0.1. The algorithm has been trained with 25,000 itera-
tions. Determining of the number of iterations for the
training and test sets is shown in Fig. 5. The figure reveals
that, the first value of the error is around 6.0 and 8.4 for the
training and test data sets respectively. It is also observed
that, RMS error decreases as the iterations increases. This
network needs 24,800 iterations to reach RMS errors of
0.0012 of the training data and 0.0017 of the test data sets
respectively. Training data sets helps the network to learn
the relationship between the input and output. During the
training, MLP is repeatedly presented with training data
and weights in network are adjusted until desired input–
output mapping occurs. After the sufficient learning by
the network, the network gains the knowledge to predict
the output for the unknown input. Calcium concentration
prediction by neural network in validation process versus
measured values by the developed instrument is shown
in Fig. 6. The correlation coefficient (R2) for the validation
set is 0.998 with standard error of about 0.0127. Statistical
analyses are carried out and it is found that for the valida-
tion results produced a relative error of about 3%. Testing
set which actually tests how good the model predict the
output unknown calcium concentrations. Fig. 7 shows the
comparison of results between developed and estimated
by ANN in testing process. The R2 value for testing sets is
0.995 i.e., almost equal to the one with standard error of
about 0.0122. The strength of the linear association be-
tween two variables is quantified by the correlation coeffi-
cient. The R2 value reveals that all the points would lie on
the straight line. From the figure, it is observed that, there
is a liner line between measured and predicted values.
Hence, careful selection of algorithm, network parameters
and proper trained network, tend to give reasonable an-
swers when presented with inputs that the network has
never seen before. The serum samples used in the conven-
tional instrument is also used in the microcontroller based
instrument to check the accuracy of the instrument. The

Fig. 5. Determination of the number of iterations for the training and test data sets.
318 P. Neelamegam et al. / Measurement 44 (2011) 312–319

Fig. 6. Calcium concentration prediction by neural network in validation process versus measured values by the developed instrument.

Fig. 7. Comparison of calcium concentration between ANN and developed system in testing process.

Fig. 8. Graph of calcium concentration measured using conventional, developed and predicted by ANN.

conventional and developed instruments are calibrated Repeatability and precision of the developed instrument is
with standard calcium stock solutions and calibration checked and it is observed that, the developed instrument
graphs are drawn to check the linearity of the instrument. reflects about 98% of reproducibility. Table 3 compare the
 P. Neelamegam et al. / Measurement 44 (2011) 312–319
results between conventional, developed and ANN pre-
dicted. This table presents that, the developed and pre-
dicted results are almost close to the reference or
conventional instrument results. Average differences be-
tween these three methods are very small and no precision
differences are observed between the methods by the F-
test. The measured results between conventional method,
developed Instrument and predicted by ANN are shown
in Fig. 8. The figure shows that, prediction by ANN is very
closer with the results of conventional and developed
instruments. Hence, the MLP with back propagation algo-
rithm complies with all statistical approaches with 95%
of confidence level and maintains the overall measurement
errors within the limit.
4. Conclusion
This work has dealt with the analysis of effects of pH
and absorbance on the complex formation of calcium in a
human blood serum. The ANN is used as a method to sim-
ulate and predict the behavior of calcium as a function of
pH and absorbance variations. A popular and well known
MLP with back propagation algorithm is used to train
ANN to model this complex system. The LPC2106 micro-
controller based data acquisition system is developed for
calcium determination in serum samples. The developed
instrument with a LED source is very attractive, cost effec-
tive, simple and user friendly. The developed design is en-
ough sensitive for use in the spectrophotometric
determination of calcium using arsenazo III as a complex-
ation reagent. With the availability of user-friendly soft-
ware, optimum selection of ANN structure and
parameters, it is possible to design a network depending
upon the field requirement. For this study, two neurons
in the input layer, one neuron in the output layer and five
neurons in the hidden layer are chosen to reduce the RMS
error. This network is scaled and properly trained to pre-
dict the unknown calcium concentration. In validation
and testing stages, the correlation coefficients are 0.998
and 0.995 respectively, which are almost equal to one. This
means that, all the measured and predicted point fall in the
straight line. The accuracy of the results suggested that
ANN approach can be used as a complementary tool for
helping in diagnosis related to calcium, where patient data
can be simultaneously analyzed. The predicted results by
ANN show a good level of accuracy and precision compare
with those obtained by conventional and developed instru-
ments. No significant differences are observed between the
results and validation results produced a relative error of
about 3%. In all approaches ANN results achieved excellent
319
analytical goals and proved that the suggest network is
ideal for the prediction of calcium concentration in serum
samples. This technique has a potential future in the field
of instrumentation and measurement.
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