Analyst

PAPER

A new nano-optical sensor thin film cadmium

sulfide doped in sol–gel matrix for assessment
of a-amylase activity in human saliva
Cite this: Analyst, 2014, 139, 793

M. S. Attia,* H. Zoulghena and M. S. A. Abdel-Mottaleb

Received 2nd September 2013
Accepted 12th November 2013
DOI: 10.1039/c3an01645e
www.rsc.org/analyst
A novel, simple, sensitive and precise spectrofluorimetric method is developed for measuring the activity of
the a-amylase enzyme in human saliva. The remarkable quenching of the luminescence intensity at 634 nm
of nano CdS doped in a sol–gel matrix by various concentrations of maltose (produced from the reaction of
the enzyme with the starch substrate) was successfully used as an optical sensor for the assessment of
a-amylase activity. The calibration plot was achieved over the concentration range 4.8
1010 to 1.2

105 mol L1 maltose with a correlation coefficient of 0.999 and a detection limit of 5.7
1011 mol L1.
The method was used satisfactorily for assessment of the a-amylase activity in a number of human saliva
samples collected from various healthy volunteers.

enzymes are used to hydrolyze the resulting short-chain oligo-

1. Introduction
Amylase (1,4-a-D-glucanohydrolases, EC 3.2.1.1) is found in
saliva and it breaks starch into maltose and dextrin. It acts on
linear a(1,4) glycosidic linkages. It breaks large, insoluble starch
molecules into soluble oligosaccharides (amylodextrin, eryth-
rodextrin, and achrodextrin) and maltose.1–3
There are four common types of assays that are used to
determine the activity of a-amylase: (1) assays that measure the
increase in reducing power by measuring the reducing sugars
produced by the dinitrosalicylic acid (DNS) assay2,4–9 or the
Nelson–Somogyi1,10,11 method; (2) assays that measure the
stainability of residual starch by forming complexes with iodine,
this method was developed by Fuwa;9–16 (3) a colorimetric
method in which starch is covalently labeled with various dyes
and hydrolyzed by a-amylase to give soluble fragments that are
measured colorimetrically aer the removal of the unhydrolyzed
substrate;17,18 and (4) assays based on the decrease in viscosity of
starch solutions during enzymatic reaction.14 Most of these
methods are spectrophotometric, which is a technique that has
some disadvantages of its own: set up of the experiment is
required each time of measurement, the reaction of iodine with
starch is not 100% quantitatively, the sensor has a short life-time
and the method can be time consuming.
The recent method is based on the production of p-nitro-
phenol from a dened oligosaccharide substrate with blocking
groups attached to the terminal sugar. The action of the
a-amylase on the oligosaccharide yields a variety of chain
lengths aer hydrolysis. In this method a variety of coupling
Nanophotochemistry and Solarchemistry Lab, Department of Chemistry, Faculty of
Science, Ain Shams University, Abbassia, Cairo, Egypt. E-mail: Mohamed_sam@
yahoo.com; Tel: +20 44678582; +2 0122 9867311
saccharides to produce p-nitrophenol.19
Luminescent nano-optical sensors have gained a great
attention in the past decade.20–27
In comparison with organic dyes and uorescent proteins,
nano-optical sensors have a high quantum yield of uores-
cence, broad excitation spectrum, and narrow/symmetric
emission spectrum.28,29 In addition, nano-optical sensors have
been applied in the eld of high-sensitive analysis of protein
and deoxyribonucleic acid (DNA).30–33 CdS has been one of the
nano-optical sensors reported in the last decades because it has
a wide band gap. It is used in light emitting diodes,34 photo-
detectors,35 sensors,36 address decoders,37 and electrically-
driven lasers.38 The advantage of the present method with
respect to previous ones is the durability of the sensor: the thin
lm nano CdS optical sensor can provide a constant signal
response for 2 years which is a 24-fold better stability compared
to the life-time certied for chromatographic and colorimetric
methods.1–19 One lm is used for all measurements which
prevents a source of error in the measurement process and gives
low standard deviation values. The higher stability of the
present sensor can be attributed to the doping of the photo-
luminescent CdS in silica nanoparticles. In this paper, we report
on the determination of a-amylase enzyme activity in human
saliva by the quenching of the luminescence intensity of a nano
CdS optical sensor doped within a sol–gel by different maltose
concentrations in acetonitrile.
2. Experimental
2.1. Apparatus
All luminescence measurements were recorded using a Meslo-
PN (222-263000) Thermo Scientic lumina uorescence

This journal is © The Royal Society of Chemistry 2014 Analyst, 2014, 139, 793–800 | 793
Analyst
spectrometer in the range (190–900 nm). The absorption spectra
were recorded in the range of 220–750 nm with a ber optic
Ocean Optics USB4000 spectrophotometer using Spectra Suite
soware. The pH was measured using a pHs-JAN-WAY 3330
research pH meter. TEM images were obtained using a JEOL
JEM-1230 available at NRC, Dokki, Cairo. The JEOL JEM-1230
instrument is a high-performance, high-contrast, 40–120 kV
transmission electron microscope with excellent imaging
capabilities. Imaging modes include bright- and dark-eld and
electron diffraction. The electron gun is a standard tungsten
lament. The instrument is capable of magnications from 50

to 600 000
and the resolution at 120 kV is 0.2 nm.
2.2. Materials and methods
2.2.1. Materials. NaCl, KCl, CaCl2, starch, maltose, dextrin,
ascorbic acid, and DNSA (3,5-dinitrosalicylic acid) were
purchased from Stanbio and Fluka (Switzerland) and total
tissue protein Sigma-Aldrich (T5695, 0.5 mg). The a-amylase
(1,4-a-D-glucan-glucanohydrolase) Sigma-Aldrich (A4551) from
Bacillus licheniformis lyophilized powder. The enzyme solution
(1.0 unit mL1) was prepared as 0.5 g in 25 mL distilled water
and the pH was adjusted to 6.9 using phosphate buffer. One
unit (U) of enzyme activity is dened as the amount of enzyme
required to release 1 mmol of maltose per minute at 37  C
(calibrator).
Cadmium acetate, thiourea, tetraethoxyorthosilicate
(TEOS) were purchased from Sigma-Aldrich and diethoxy-
dimethylsilane (DEDMS) was purchased from Fluka. A stock
solution of maltose (1
103 mol L1) was prepared in aceto-
nitrile. The working standard solutions (1
104, 1
106, 1

108 mol L1) of maltose were freshly prepared by appropriate
dilution of the stock solution with acetonitrile. Phosphate
buffer of pH 6.9 was prepared from 100 mL of 0.1 mol L1
NaH2PO4 + 51.8 mL of 0.1 mol L1 NaOH. Dinitrosalicylic acid
color reagent, prepared by dissolving 1.0 g of 3,5-dinitrosalicylic
acid in 50 mL of water. 1% starch, prepared by dissolving 1.0 g
soluble starch in 100 mL of 0.1 mol L1 potassium phosphate
buffer, pH 6.9. The luminescence intensity was measured at
lex/lem ¼ 360/634 nm.
2.2.2. Materials synthesis
2.2.2.1. Synthesis of CdS nanoparticles. Nano CdS material
was prepared by a cadmium acetate–thiourea complex ther-
molysis route at 200  C for 3 h. The powder obtained was used
without further treatments.
2.2.2.2. Synthesis of CdS-doped TMOS–DEDMS thin lms.
CdS (0.12 g) was dissolved in 10 mL of ethanol. Part of this
solution (8 mL) was mixed with tetramethoxysilane (TMOS,
4 mL), diethoxydimethylsilane (DEDMS, 4 mL), and water
(2 mL). Glass vials (diameter 24 mm, height 48 mm) were lled
with 9 mL of this solution and were covered with Paralm. Aer
2 days, three small holes were pierced in the Paralm. Aer 6
days, the thin lm was prepared from the partly hydrolyzed and
condensed solution by spin-coating on a small quartz slide
(substrate) (width 8.5 mm; height 25 mm) to t in a cuvette for
measurement of the luminescence intensity. The substrate was
cleaned rst by putting it into distilled water with a surfactant.
794 | Analyst, 2014, 139, 793–800
Paper
In the next step, it was cleaned ultrasonically for 30 min in
distilled water with surfactant and then ultrasonically for 10
min in acetone, and nally it was boiled for 10 min in 2-prop-
anol. Before spin-coating, the substrate was rinsed with
2-propanol and spun dry. Then, the partially hydrolyzed and
condensed sol–gel solution was added dropwise onto the
substrate using a syringe through a 2 cm lter and spin-coated
at 2000 rpm for 30 s, Fig. 1.39,40
2.3. Sample preparation
Seven test foods were bought from local markets: cornour,
potato-baked, pasta-fresh, rice-white grain, potato, breadfruit
and wheat our. Foods were selected to span a range of glycemic
index (GI) values. Foods were prepared by boiling in an excess of
water.
2.4. Chewing – exposure to salivary amylase for designated
period
A series of pre-weighed 5.0 g samples of each of the test foods
(2.5 g for bread based on the high GI of bread) were chewed by
two volunteers with normal dentition until they reached the
urge to swallow. The samples were then expectorated and le to
sit as chewed boluses for 15 min. Prolonged bolus sitting
periods were included to reect the time taken in vivo to
swallow, and for food to become fully mixed with gastric
secretions, up to which time salivary a-amylase is likely still
digesting the starch. Between each sample, volunteers rinsed
their mouths thoroughly with distilled water.
All experiments were performed in compliance with the
relevant laws and institutional guidelines of the Egyptian
Ministry of Health and Population, and all volunteers were
consented to all guidelines of the Egyptian Ministry of Health
and population.
2.5. Proposed method
An appropriate amount (500 mL) of various standard concen-
trations of maltose in acetonitrile was mixed with the optical
sensor thin lm nano CdS doped in the sol–gel in the cell. The
Fig. 1 TEM image of thin film nano-optical sensor CdS.
This journal is © The Royal Society of Chemistry 2014
Paper
luminescence spectra were then recorded at the lex/lem ¼
360/634 nm. The optical sensor was rinsed with water and
acetonitrile aer each measurement and the calibration plot
was constructed by plotting the luminescence intensity at lem ¼
634 nm on the y-axis against the reciprocal of maltose concen-
tration on the x-axis. [The CdS nanoparticles doped in the sol–
gel matrix lm was rinsed using distilled water (because the
maltose is soluble in water and the adsorption of maltose on the
surface of the lm is physical adsorption) then by acetonitrile
aer each measurement and the uorescence intensity was
measured again and compared with the uorescence intensity
of the lm at zero maltose concentration. The standard devia-
tion was measured each time to verify the availability of the lm
for the measurement process.]
The a-amylase activity was determined by the proposed
method using an appropriate amount (1 mL) of (1% starch) that
was immediately mixed with an accurate volume (100 mL) of
a-amylase enzyme (0.5 g in 25 mL distilled water) and the pH
was adjusted to 6.9 by using the phosphate buffer, then the
solution was placed in an incubator at 37  C for 3 min.
a-Amylase activity was measured by withdrawing 50 mL of the
solution sealed in the incubator at different time intervals (every
20 s), made up to 3 mL with acetonitrile and then the emission
intensity of the optical sensor nano CdS thin lm was measured
at 634 nm.
2.6. Standard method
Enzyme assay. According to the method of Miller (1959),41
0.5 mL of a-amylase enzyme solution was pipetted into a test
tube (1). Another tube (2) contains a blank reagent with 0.5 mL
acetonitrile. The tubes are incubated at 20  C for 3–4 min to
achieve temperature equilibration before 0.5 mL starch (1%)
solution (at 20  C) was added to each tube. Withdrawal of a
50 mL sample from tube (1) was performed every 20 s; was mixed
with 1 mL dinitrosalicylic acid color reagent in another tube for
3 min time intervals. All tubes were incubated in a boiling water
bath for 5 min before being cooled to room temperature and
9 mL acetonitrile added. The contents were mixed well and the
absorbance measured at A560.
The a-amylase activity (U L1) in the saliva sample can be
calculated using the formula:
[Asample  Ablank/Acal  Aacetonitrile]
n
550 (U L1)
where Asample and Ablank are the absorbance of the sample and
blank at l ¼ 560 nm, respectively. Acal and Aacetonitrile are the
absorbance of the calibrator (solution containing 1 unit per mL
of a-amylase in 20  C puried water) and acetonitrile. The
parameter n is the dilution factor. The number “550” is the
equivalent activity (U L1) of the calibrator under the assay
conditions.
Note: if the calculated activity is higher than 300 U L1, the
sample is diluted with acetonitrile and the assay repeated. The
results are multiplied by the dilution factor (n). Unit denition:
one unit of enzyme catalyzes the production of 1.0 mmol of
maltose per minute under the assay conditions (pH 6.9).
This journal is © The Royal Society of Chemistry 2014
Analyst
3. Results and discussion
3.1. Spectral characteristics
3.1.1. Absorption spectra. The absorption spectrum of the
prepared thin lm doped with nano CdS (0.2 mm thickness)
shows the absorption peak position at the lower wavelength 320
nm which is attributed to the electron transition from the
valence band to conduction band of CdS. The electron transi-
tion is decreased by the addition of different maltose concen-
trations; this can be attributed to the physical adsorption of
maltose molecules on the surface of the CdS42, Fig. 2.
3.1.2. Emission spectra. The ideal photoluminescence
emission spectrum of the CdS nanoparticles shows ve broad
bands at 485, 510, 576, 634 and 702 nm. The bands corre-
sponding to 485 and 510 nm provide green emission while the
band at 576 nm gives yellow emission. The small bands at 634
and 702 nm were assigned to red emission. The green emission
bands were associated with the emission due to electronic
transitions from the conduction band to an acceptor level due
to interstitial sulfur (IS).43
The yellow emission was attributed to recombination via
surface localized states,44 the transition from interstitial
cadmium (ICd) to the valence band.45 The red emission was
observed due to the presence of sulfur vacancies.46 The emis-
sion bands at 485, 530 and 576 nm are unstable against the
ultraviolet radiation because the intensity of these bands will
decrease gradually aer exposure to UV light for long time.
On the other hand the band at 634 nm is stable against UV
radiation for a couple of reasons: (1) The generation of sulfur
vacancies at the surface of CdS nanoparticles due to the
oxidation of cadmium-bonded surface sulde ions,47,48 which
leads to the formation of defect-related lower energy states
between the valence- and conduction-bands (known as trap
states). This would decrease the actual band gap energy and
cause higher wavelength emission. (2) The partial loss of
Fig. 2 UV-visible absorption spectrum of nanosensor CdS doped in
the sol–gel matrix in the presence of different concentrations of
maltose in acetonitrile.
Analyst, 2014, 139, 793–800 | 795
Analyst
Fig. 3 Mechanism of the quenching of the luminescence intensity of
CdS by maltose.
crystalline features of the CdS nanoparticles leads to an
increased number of surface defects that are expected to be
present.49–51
The low-energy emission band at 634 nm is associated with
emissive surface-trap states, which could be highly affected by
the physicochemical environment of the nano CdS (Fig. 3),
therefore, the band at 634 nm could be highly affected by the
Fig. 4 Photoluminescence spectra of CdS nanoparticle doped in sol–
gel matrix in different concentrations of maltose [1
1010 to 1
104
mol L1] (1–0.12 g CdS doped in sol–gel matrix) in acetonitrile at lex ¼
360 nm.
796 | Analyst, 2014, 139, 793–800
Paper
maltose concentration. Fig. 4 shows that all of broad bands,
particularly at 634 nm, are decreased by the addition of
increasing maltose concentrations. The emission spectrum of
the nano CdS thin lm exhibited a well dened emission
spectrum similar to the spectrum of the nano CdS in solution.
Thus, in the subsequent experiment, the activity of the enzyme
a-amylase was determined through measuring the lumines-
cence intensity of the nano sensor CdS at 634 nm.
The inuence of solvents on the luminescence intensity of
the thin lm containing 0.12 g of nano sensor CdS was studied
under the established conditions. The quenching of the inten-
sity of the emission band of CdS at 634 nm is not observed in
the case of low-polarity solvents such as acetonitrile. This can be
explained through the low polarity solvents not being adsorbed
on the CdS surface.
The inuence of maltose concentration on the luminescence
intensity of the nano sensor CdS was studied under the
optimum experimental conditions. The results are shown in
Fig. 4. The luminescence intensity of nano sensor CdS thin lm
was quenched by increasing of maltose concentrations up to
1
104 mol L1, due to the maltose molecules being adsorbed
through the OH groups on the surface of CdS.
3.2. Effect of foreign species
The selectivity of the nano-optical sensor CdS to maltose was
tested by studying the inuence of a series of interfering
species, e.g. NaCl, KCl (1.0
103 mol L1), total protein
(0.001 g L1) and dextrin (0.28 g L1) on its luminescence
spectrum. The tolerable limit was dened as the concentration
of the individually added species causing a deviation of less
than 3% of the luminescence intensity of the nano CdS optical
sensor under the optimum conditions. The results indicated no
signicant change in the luminescence intensity of the nano
CdS thin lm optical sensor. Upon addition of 1
104 mol L1
of glucose to the nano CdS optical sensor, quenching of the CdS
luminescence intensity at 634 nm occurred by 5% compared
with the same concentration of maltose in acetonitrile. This can
be explained via the small size of glucose molecule and devia-
tion of the OH groups in glucose out of the plane of CdS due to
the repelling of the OH groups to each other.
3.3. Analytical performance
The validation of the proposed method for measuring the
a-amylase activity under the optimized experimental conditions
was determined via the limit of detection (LOD), limit of
quantication (LOQ), linear dynamic range, (Table 1), repeat-
ability and recovery (Table 2).
The luminescence intensity at 634 nm of the nano CdS
optical sensor was recorded at various concentrations of
maltose. The plot of the measured signal by the developed
procedure versus the reciprocal of the maltose concentration
was found to be linear over the concentration range from 4.8

1010 to 1.2
105 mol L1 with a correlation coefficient of
0.999 (Fig. 5) and the calibration plot has the following regres-
sion equation:
This journal is © The Royal Society of Chemistry 2014
Paper Analyst

Table 1 Sensitivity and regression parameters for optical sensor

Parameter

lem/nm 634
Linear range/mol L1 4.8
1010 to 1.2
105
Limit of detection (LOD)/mol L1 5.7
1011
Limit of quantication (LOQ)/mol L1 1.6
1010
Intercept (a) 0.18
Slope (b)
109 0.27
Standard deviation
103 4.5
Variance (S2)
105 2.0
Regression coefficient (r) 0.999

Luminescence intensity ¼ 2.7
109
1/concentration

(nmol L1) + 0.18.
According to IUPAC52 the value of LOD is calculated using
the formula LOD ¼ 3s/b where s is the standard deviation and b
is the slope of the calibration plot. The LOD value under the
established conditions was found to be 5.7
1011 mol L1.
The value of LOQ ¼ 10s/b was found to equal 1.6

1010 mol L1 (Table 1). The dynamic range and the LOD
obtained by the proposed method are better than or comparable
to most of the reported methods.2–19
To compute the accuracy and precision, the measurements
were repeated three times within the day to determine the
repeatability (intra-day precision) and three times on different
days to determine the intermediate precision (inter-day
Fig. 5 Calibration curve of the luminescence intensity of synthesized
CdS nanoparticle doped in sol–gel matrix versus 1/concentration of
maltose.
precision) of the method. These assays were repeated three
times for each sample (food samples contain % starch as
substrate, see Table 3). The results of this study are summarized
in Table 4.
The percentage relative standard deviation (%RSD) values
were in the range of #0.11–2.1% (intra-day) and #0.25–0.99%
(inter-day) for the seven samples. The inter-day values indicate
the high precision of the method. Accuracy was determined as
the percentage relative error (%RE) between the measured

Table 2 Comparison between standard (A) and the proposed (B) methods by (average, RSD and average recovery)

Proposed method (B)

Enzyme activity Average found,a Standard method (A)
Food sample (U L1) X (U L1) Average (U L1)  RSD (%) Student's t- and F-valuesb Average recovery  RSDb (%)

Cornour 201.1 201 202  0.28 t ¼ 4.7, F ¼ 1.63 99.5  0.12

200.6
201.3
Potato, baked 71.7 71.8 72  0.17 t ¼ 1.2, F ¼ 2 99.7  0.09
72.2
71.7
Pasta, fresh 90.1 90.4 90  0.30 t ¼ 2.1, F ¼ 1.8 100.4  0.10
90.7
90.6
Rice, white grain 245 245.1 246  0.33 t ¼ 3.7, F ¼ 1.4 99.6  0.13
245.5
244.7
Potato 23.5 23.9 23.1  0.28 t ¼ 2.7, F ¼ 1.2 103.4  0.16
23.9
24.5
Breadfruit 67.7 67.7 68  0.32 t ¼ 0.9, F ¼ 1.0 99.5  0.18
67.2
68.3
Wheat our 237 236.6 236  0.31 t ¼ 2.5, F ¼ 1.5 100.2  0.13
236.2
236.6
a
Each reading was repeated three times (X average was taking for three readings by three concentration of substrate). b Average of three
determinations, RSD is relative standard deviation (S/X)
100 and tabulated t value at the 95% condence level is 4.303, tabulated F value at
the 95% condence level is 19, F ¼ S12 (standard method)/S22(proposed method) where S1 $ S2 (S is standard deviation).

This journal is © The Royal Society of Chemistry 2014 Analyst, 2014, 139, 793–800 | 797
Analyst Paper

Table 3 Main characteristics of the food samples

Food % starch

Cornour 60
Potato, baked 17
Pasta, fresh 25
Rice, white grain 79
Potato 7
Breadfruit 16
Wheat our 76

mean activity aer the interaction of the enzyme in the saliva of

the healthy human with the real food samples and the taken
values aer incubation of the a-amylase enzyme with starch
extracted from each food sample at 20  C for 3 min. Bias {bias%
¼ [(enzyme activity in saliva  enzyme activity aer incubation)

100/(enzyme activity aer incubation)] was calculated for
each of the food samples and these results are also presented in
Table 4. Percentage relative error (%RE) values of #0.09–1.47
and 0.29–1.17% for all of the samples demonstrate the high
accuracy of the proposed method.
3.5. Analytical applications
The analytical utility of the proposed method was tested by
measuring the activity of the enzyme a-amylase for seven food
samples. The results obtained are summarized in Table 2. Good
agreement is seen between the average values obtained by the
developed procedure (23.9–245.1  0.09–0.18 U L1) and the
standard method (23.1–246  0.17–0.33 U L1) and no signi-
cant differences were found for the two methods. a-Amylase
activity was measured by withdrawing 50 mL of the solution,
which was sealed in the incubator (1 mL of 1% starch) and
immediately mixed with an accurate volume (100 mL) of
a-amylase at different time intervals (20 s) for both the proposed
and standard methods, Fig. 6 and 7.
The rate of the reaction of the a-amylase with the starch was
measured by rate of the change in concentration of maltose
relative to time, and the value was calculated as 1.5

107 mol L1 s1.
Fig. 6 Effect of the reaction time between enzyme and starch on the
luminescence intensity of synthesized CdS nanoparticle doped in sol–
gel matrix. (Incubation time, 20, 40, 60, 80, 100, 120, 140, 160, 180,
200 s.)
On the other hand, an excellent correlation between the
average values of the activity of the sample was measured by the
standard and the developed method (r2 ¼ 0.999, intercept ¼
4.18, slope ¼ 4.4 and p ¼ 0.0001) conrming the accuracy of the
developed procedure Fig. 8.
The correlation is positive and highly signicant in two
methods (r2  0.999, p < 0.0001) indicating that the proposed
method has high accuracy for measuring the a-amylase
activity. Moreover, the recovery, RSD% and F- and t-tests were
calculated for the proposed method in the case of cornour,
potato (baked), pasta (fresh), rice (white grain), potato, bread-
fruit and wheat our (Table 2). The average values obtained by
the standard and proposed methods were found to be in the
range of 23.1–246  0.17–0.33 and 23.9–245.1  0.09–0.18
U L1, respectively. The F- and t-tests at the 95% condence
levels did not exceed the theoretical value and no signicant
differences were observed between the developed and the
standard methods, conrming the accuracy of the developed
method.

Table 4 Evaluation of intra-day and inter-day accuracy and precision

Proposed method
Standard method
Intra-day accuracy and precision (n ¼ 3) Inter-day accuracy and precision (n ¼ 3)
1
Average ( U L ) Average found Average found
Food sample  RSD (%) ( U L1)  CLa %RE %RSD ( U L1)  CLa %RE %RSD

Cornour 202  0.28 202.2  0.19 0.09 0.20 203  1.39 0.49 0.77
Potato, baked 72  0.17 72.1  0.25 0.13 2.10 72.7  1.21 0.97 0.99
Pasta, fresh 90  0.30 90.1  1.3 0.11 0.18 90.5  1.5 0.55 0.33
Rice, white grain 246  0.33 245  0.19 0.40 0.11 247.2  1.74 0.48 0.29
Potato 23.1  0.28 23.0  0.2 0.43 1.20 23.3  1.48 0.86 0.87
Breadfruit 68  0.32 67  0.12 1.47 0.80 68.8  0.32 1.17 0.25
Wheat our 236  0.31 235  0.11 0.42 0.66 236.7  0.31 0.29 0.44
a
The average value for three concentration of substrate, % RE: percent relative error, % RSD: relative standard deviation and CL: condence limits
were calculated from: CL ¼ tS/On. (The tabulated value of t is 4.303, at the 95% condence level; S ¼ standard deviation and n ¼ number of
measurements).

798 | Analyst, 2014, 139, 793–800 This journal is © The Royal Society of Chemistry 2014
Paper
Fig. 7 Effect of the reaction time between enzyme and starch on the
absorbance of the reaction of maltose with dinitrosalicylic acid
(standard method). (Incubation time, 20, 40, 60, 80, 100, 120, 140, 160,
180, 200 s; p ¼ 0.0001, r2 ¼ 0.999.)
Fig. 8 Correlation between the standard method and the proposed
method (r2 ¼ 0.998, p ¼ 0.0001).
4. Conclusion
The developed method provides an excellent approach for
measuring the activity of a-amylase enzyme compared to other
reported methods. The method is sensitive and provides a wide
linear dynamic range of maltose concentrations with a detec-
tion limit of 5.7
1011 mol L1. The work is limited to the
acetonitrile as the solvent and the starch as the substrate.
References
1 M. F. NajaW and A. Kembhavi, Enzyme Microb. Technol.,
2005, 36, 535.
2 T. L. M. Stamford, N. P. Stamford, L. C. B. B. Coelho and
J. M. Araújo, Bioresour. Technol., 2002, 83, 105.
3 M. Coronadoa, C. Vargasa, J. Hofemeisterb, A. Ventosaa and
J. J. Nietoa, FEMS Microbiol. Lett., 2000, 183, 67.
This journal is © The Royal Society of Chemistry 2014
Analyst
4 Y. Marlida, N. Saari, Z. Hassan, S. Radu and J. Bakar, Food
Chem., 2000, 71, 221.
5 R. Gill and J. Kaur, J. Ind. Microbiol. Biotechnol., 2004, 31, 540.
6 P. Cornelis, C. DigneVe and K. Willemot, Mol. Gen. Genet.,
1982, 186, 507.
7 H. H. Hyun, G. J. Shen and J. G. Zeikus, J. Bacteriol., 1985,
164, 1153.
8 G. L. Miller, Anal. Chem., 1959, 31, 426.
9 D. O. Mountfort and R. A. Asher, Appl. Environ. Microbiol.,
1988, 54, 2293.
10 D. Primarini and O. Yoshiyuki, Starch, 2000, 52, 28.
11 M. Thomas, F. G. Priest and J. R. Stark, J. Gen. Microbiol.,
1980, 118, 67.
12 H. Fuwa, J. Biochem., 1954, 41, 583.
13 B. K. Gogoi, R. L. Bezbaruah, K. R. Pillai and J. N. Baruah,
J. Appl. Bacteriol., 1987, 63, 373.
14 C. F. González, J. I. Fariña and L. I. C. Figueroa, Enzyme
Microb. Technol., 2002, 30, 169.
15 H. K. Manonmani and A. A. M. Kunhi, World J. Microbiol.
Biotechnol., 1999, 15, 485.
16 R. A. Kaufman and N. W. Tietz, Clin. Chem., 1980, 26, 846.
17 A. Y. Foo and R. Bais, Clin. Chim. Acta, 1998, 272, 137.
18 D. Clem, J. Maidment and J. M. Ringham, Nutr. Food Sci.,
2001, 31, 141.
19 T. H. E. Blair, US Pat. no. 4,649, 1984, p. 108.
20 L. Wang, L. Y. Wang, C. Q. Zhu, X. W. Wei and X. W. Kan,
Anal. Chim. Acta, 2002, 468, 35.
21 Y. Tian, T. Tewton, N. A. Kotov, D. M. Guldi and J. H. Fendler,
J. Phys. Chem., 1996, 100, 8927.
22 M. A. Hines and P. J. H. Guyot-Sionnest, J. Phys. Chem., 1996,
100, 8468.
23 H. S. Zhou, I. Honma, H. Komiyama and J. W. Haus, J. Phys.
Chem., 1993, 97, 895.
24 T. P. Martin, U. Naher, H. Schaber and U. Zimmermann,
J. Phys. Chem., 1994, 100, 2322.
25 R. Rossetti, S. Nakahara and L. E. Brus, J. Phys. Chem., 1983,
79, 1086.
26 M. Xie, H. H. Liu, P. Chen, Z. L. Zhang, X. H. Wang, Z. X. Xie,
Y. M. Du, B. Q. Pan and D. W. Pang, Chem. Commun., 2005,
5518.
27 H. Y. Xie, J. G. Liang, Z. L. Zhang, Y. Liu, Z. K. He and
D. W. Pang, Spectrochim. Acta, Part A, 2004, 60, 2527.
28 A. M. Smith, X. H. Gao and S. M. Nie, Photochem. Photobiol.,
2004, 80, 377.
29 A. M. Smith and S. M. Nie, Analyst, 2004, 129, 672.
30 X. H. Gao, L. L. Yang, J. A. Petros, F. F. Marshal, J. W. Simons
and S. M. Nie, Curr. Opin. Biotechnol., 2005, 16, 63.
31 L. Y. Wang, Y. Y. Zhou, L. Wang, C. Q. Zhu, Y. X. Li and
F. Gao, Anal. Chim. Acta, 2002, 466, 87.
32 J. Wang, G. Liu, R. Polsky and A. Merkoc, Electrochem.
Commun., 2002, 4, 722.
33 Y. Y. Zhou and C. Q. Zhu, Chem. Res. Chin. Univ., 2003, 4,
612.
34 H. Murai, T. Abe, J. Matsuda, H. Sato, S. Chiba and
Y. Kashiwaba, Appl. Surf. Sci., 2005, 244, 351.
35 Y. Wang, S. Ramanathan, Q. Fan, F. Yun, H. Morkoe and
S. Bandyopadhyay, J. Nanosci. Nanotechnol., 2006, 6, 2077.
Analyst, 2014, 139, 793–800 | 799
Analyst
36 A. Ponzoni, E. Comini, G. Sbervcglieri, J. Zhou, S. Z. Deng,
N. S. Xu, Y. Ding and Z. L. Wang, Appl. Phys. Lett., 2006,
88, 101.
37 Z. H. Zhong, D. L. Wang, Y. Cui, M. W. Bockrath and
M. C. Lieber, Science, 2003, 302, 1377.
38 X. Duan, Y. Huang, R. Agarwal and C. M. Lieber, Nature,
2003, 421, 241.
39 B. Cao and C. Zhu, J. Non-Cryst. Solids, 1994, 178, 302.
40 P. A. Tanner, B. Yan and H. Zhang, J. Mater. Sci., 2000, 35, 4325.
41 G. L. Miller, Anal. Chem., 1959, 31, 426.
42 M. K. Farhan, M. S. Fouda, M. H. El-Noss, S. A. El-Sadany,
S. El-Sayed, R. Salah-El-Deen, D. A. Hamouda, E. Bakier
and M. S. Attia, Egyptian Journal of Pure and Applied
Science, 2011, 1, 31.
43 O. Vigil, J. Riech, M. G. Rocha and O. Z. Angl, J. Vac. Sci.
Technol., A, 1997, 15, 2282.
800 | Analyst, 2014, 139, 793–800
Paper
44 A. S. Okamoto, Y. Kanemitsu, H. Hosokawa, K. Murakoshi
and S. Yanagida, Solid state Comm., 1998, 105, 7.
45 C. T. Tsai, D. S. Chuu, G. L. Chen and S. L. yang, J. Appl. Phys.,
1996, 79, 9105.
46 B. A. Kulp and R. H. Kelley, J. Appl. Phys., 1960, 31, 1057.
47 A. E. Saunders, A. Ghezelbash, P. Sood and B. A. Korgel,
Langmuir, 2008, 24, 9043.
48 S. Bessor, T. Gacoin, C. Ricolleau, C. Jacquiod and
J. P. Boilot, Nano Lett., 2002, 2, 409.
49 H. Du, G. Q. Xu and W. S. Chin, Chem. Mater., 2002, 14,
4473.
50 N. Pradhan and S. Efrima, J. Am. Chem. Soc., 2003, 125, 2050.
51 X. Chen, A. C. S. Samia, L. Yongbing and C. Burda, J. Am.
Chem. Soc., 2005, 127, 4372.
52 J. C. Miller and J. N. Miller, Statistics for Analytical Chemistry,
Ellis-Howood, New York, 4th edn, 1994, p.115.
This journal is © The Royal Society of Chemistry 2014