Sensors and Actuators B 240 (2017) 248254

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Development of cadmium specic recombinant biosensor and its

application in milk samples
Sachin Kumar a,b , Neelam Verma a, , Ashish Kumar Singh a
a
Biosensor Technology Laboratory, Department of Biotechnology, Punjabi University, Patiala, India
b
Department of Biotechnology, JV College, Baraut (Baghpat), Uttar Pradesh, India

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

Article history: A novel and highly specic recombinant biosensor has been investigated for detection of highly toxic
Received 11 April 2016 metal ion cadmium (Cd(II)) based on Escherichia coli DH5 (pNV12). The working principle of biosensor
Received in revised form 25 August 2016 is based on the expression of gfp gene under the control of cad promoter and the cadC gene of Staphylo-
Accepted 28 August 2016
coccus aureus plasmid pI258. Escherichia coli DH5 (pNV12) was tested for its response in the presence of
Available online 29 August 2016
different combinations of Pb(II), Cd(II) and Zn(II) ions. The investigation resulted in the development of
a cadmium specic recombinant whole cell biosensor with a response time as low as 15 min. A good lin-
Keywords:
ear range for cadmium ion concentrations of 1050 g/l with R2 0.9946 was obtained and the detection
Cadmium
Biosensor limit of 10 g/l. Further, we reduced the detection limit up to 5 g/l by increasing the incubation time
Fluorescence from 15 to 30 min. High throughput microarray techniques for testing of 48 samples simultaneously has
Gene also been developed with reaction volume miniaturized from 2000 l to 200 l. The developed biosen-
Heavy metal sor was applied to detect cadmium ion concentration in milk samples collected from different areas of
Food sample etc Punjab, India. Two samples collected from industrial areas, shown to have cadmium concentration above
permissible limits (10 g/l).
2016 Published by Elsevier B.V.

1. Introduction A whole-cell bioassay has been developed for total toxicity

Some heavy metals nickel, cobalt and zinc are essential in trace
amounts for the growth of living beings, whereas others for exam-
ple cadmium and lead which have no known benecial roles and
are toxic at higher concentrations [1]. There are certain microbes
that have developed different mechanisms e.g. precipitation, metal
efux, suppression of inux and enzyme detoxication etc. to
deal with metal concentrations and grow in metal contaminated
zone [2]. For conventional detection of cadmium metal ion atomic
absorption (AA) spectroscopy, inductively coupled plasma (ICP)
optical emission spectrometry and inductively coupled plasma
mass spectrometry (ICPMS) are widely used [3]. Although, these
methods are sensitive and allow discrimination of different metal
ions however, they require tedious sample pretreatments (e.g.,
desalting, ltration and concentration) and are time consuming.
Therefore, it is necessary to develop a rapid, simple and specic
detection method to assured the presence of metal ions.
Corresponding author at: Department of Biotechnology, Punjabi University,
Patiala, 147 002, Punjab, India.
E-mail address: verma.neelam2@gmail.com (N. Verma).
testing of liquid samples. The method is based on the induction
of the bioluminescent activity of genetically manipulated mam-
malian cells. For that purpose, a DNA sensing element that responds
to chemical stress agents (heavy metals, genotoxic agents, and
endocrine-disrupting chemicals) was transfected in HeLa cells.
Such element was designed to direct the expression of a report-
ing gene (rey luciferase) through the activation of Drosophila
melanogaster hsp22 promoter [4]. Although, there is many other
stress causing reasons such as genotoxicity, endocrine disruption
etc. to induce the luc gene expression, therefore this approach is
not suitable in the term of specic detection of cadmium metal ion.
A new type of membrane containing immobilized whole cell
microalgae was used for development of cadmium biosensor.
In this the Chlorella vulgaris alkaline phosphatase activity (APA)
was inhibited in the presence of cadmium ion and the detection
limit achieved was 1 ppb [5]. Silwana et al. [23] was reported
an amperometric biosensor based on enzyme horseradish paroxi-
dase immobilized in polymeric material on platinum electrode. The
enzyme showed inhibition in the presence of cadmium, lead and
mercury metal ions with detection limit 8 104 ppb for cadmium
metal ion. Qu et al. [24] have used ssDNA/Au probe for the detec-
tion of various heavy metal ion including cadmium. Although they
achieved a very low detection limit up to 0.3 103 ppb, but selec-

http://dx.doi.org/10.1016/j.snb.2016.08.160
0925-4005/ 2016 Published by Elsevier B.V.
 S. Kumar et al. / Sensors and Actuators B 240 (2017) 248254
tivity for cadmium metal ion has not been quoted clearly. Present
study is an effort to develop a cadmium specic biosensor with a
lower detection limit by expressing GFP variant under the control
of cadmium specic promoter.
Recently, Matsuura and coworkers [6] have developed a recom-
binant biosensor for the detection of cadmium and other toxic
metals in environmental samples. They targeted the gene which
expresses enzyme phytochelatine synthase (PCS) for which heavy
metals act as cofactor. They have engineered PCS gene into yeast cell
and the whole recombinant cell were used to construct a recom-
binant biosensor for the detection of cadmium. In the same way,
a specic and sensitive biosensor for cadmium ion was developed
[7]. In this approach two promoter sequences CadR-TC10 and CadR-
TC21 were redesigned by truncating 10 and 21 amino acids from the
C-terminal extension of CadR, respectively. Genes of CadR, CadR-
TC10 and CadRTC21were used to construct the sensing element of
the GFP based E. coli biosensors. A comparison of various biosen-
sors for the detection of cadmium metal is listed in Table 2. Most of
the developed biosensors are not specic for cadmium and have an
interference of other metals e.g. Pb(II), Zn(II) etc. hence the study
was carried out to develop a biosensor with specicity towards
cadmium and application of the same to detect cadmium in milk.
Some of mechanisms are very specic and precisely regulated by
genetically encoded resistance. The resistance mechanism against
cadmium in Staphylococcus aureus due to plasmid pI258 was iden-
tied by Novick & Roth [8], and Endo & Silver [9], cadA operon from
Staphylococcus aureus plasmid pI258 consist of two genes i.e. cadA
and cadC in which cadA gene encodes for an energy-dependent ion
pump for cadmium efux [10] while cadC gene encodes for the
regulatory protein. Contamination of milk with toxic heavy metals
ions is considered most alarming as it is largely being consumed
by infant and children [27]. The main source of cadmium in milk
is through food chain, as plant and grasses grown in contaminated
soil are fed by cattle [28,29]. Many studies have been carried out for
estimation of cadmium in milk samples [3032]. This sort of studies
give an alarm that milk can serve as a potential source of cadmium
for human community, and could be more toxic even at lower con-
centration in many cases. As it has also been reported that cadmium
absorption increases with increase in fat and protein contents and
milk is rich in fat and protein [33].
In the present study cad promoter and cadC gene was ampli-
ed from plasmid pI258 and ligated to the upstream to a modied
promoter-less gene encoding a variant of green uorescent protein
in pAD123. A new plasmid pNV12 has been constructed, and used
as sensing element for cadmium metal. The specicity and sensi-
tivity of the developed biosensor was also evaluated. The system
tested in real and spiked milk sample and validation of the sensor
response was done using F-AAS.
2. Experimental
2.1. Materials
Tryptone, Yeast Extract, ampicillin, chloramphenicol, C-TAB,
Tris-HCL, Na2 EDTA, LiCl, NaCl, CdCl2 , ZnCl2 and Pb(NO3 )2 were
procured from Himedia Labs Pvt. Ltd. Mumbai, India. Chloroform,
Isoamyl alcohol and Isopropanol were procured from Qualigen ne
chemicals Mumbai, India. Restriction enzymes EcoRI and BamHI,
DNA-polymerase, primers used for PCR amplications were pro-
cured from B. Genei. MSB Vario Cleanup PCR purication Kit (Cat.
No. 10202302) was procured from Invitek, Germany while Hipura
Agarose gel DNA extraction kit (Cat. No. MB-511-50PR) was pur-
chased from Himedia. Skim milk powder, BCR certied reference
material grade was procured from Sigma-Aldrich, Mumbai, India.
249
2.2. Bacterial strains and plasmids
Bacterial strains and plasmids used in the study are Staphylo-
coccus aureus (pI258), Escherichia coli (pAD123) and Escherichia coli
DH5. Staphylococcus aureus (pI258)NCTC 50581 was procured
from National Collection of Type Cultures, Colindale, London, UK.
Escherichia coli (pAD123) and Escherichia coli DH5 were provided
as a generous gift by Bacillus Genetic Stock Centre (BGSC), Depart-
ment of Biochemistry, Ohio State University. Plasmids containing
bacterial strains were maintained on L-agar (LA) plates.
2.3. Construction of plasmid pNV12
The strategy for the construction of recombinant plasmid
pNV12 has been depicted in Fig. 1. cad promoter and cadC of pI258
were amplied through PCR and cloned into pAD123 upstream
to promoter-less gene encoding a variant of green uorescent
protein in a promoter-trap plasmid, pAD123 [11]. Plasmid pNV12
was constructed employing standard recombinant-DNA tech-
niques [12]. Plasmid pI258 was isolated from S. aureus (NCTC
50581) following Atashpaz et al. [13] with some modications
and used as a template for polymerase chain reaction (PCR) using
primers 5 -ATATGAATTCGTGTATTTTTTAATAAATTATTTTACTT-3
at the beginning of the cadC gene and 5 -
TTAAGGATCCCCTTTCAGACATTGACCTTCAC-3 at the end of the
cadC gene [14] to generate a fragment containing cadC gene and
promoter/operator of the cadA operon with BamHI and EcoRI
restriction sites at ends. In Primer sequence restriction sites of
EcoRI and BamHI have been bolded and bases in italics correspond
to the cadC gene. DNA was amplied using RT-PCR miniopticon
(Bio-Rad) with an initial Denaturation step of 3 min, followed by
20 cycles of denaturation at 95 C for 1 min, annealing at 50 C for
1 min and extension at 72 C for 1 min. After 20 cycles, sample was
kept for 5 min at 72 C for a nal extension followed by incubation
at 4 C for 5 min.
The resulting PCR product of 572 bp was puried with MSB Vario
cleanup PCR purication kit (Invitek Germany) and digested with
EcoRI and BamHI and repuried from an agarose gel by Hipura
agarose Gel DNA extraction kit (Himedia). The resulted fragment
was ligated to EcoRI and BamHI sites of double digested plasmid
pAD123 with the same restriction enzymes, thus generated plas-
mid was named pNV12 was transformed into Escherichia coli DH5
through competent cell method [15].
2.4. Cultivation of microbes and uorescence measurement
Staphylococcus aureus (pI258) was cultured in LB medium
containing erythromycin (50 g/ml) at 37 C and 200 rpm while
Escherichia coli (pAD123) and Escherichia coli (pNV12) were cul-
tured in LB-medium (10 g Tryptone, 5 g Yeast extract, 5 g NaCl,
and Distilled water 1 L, pH 7.0) supplemented with ampicillin
(100 g/ml) and chloramphenicol (10 g/ml) at 37 C and 200 rpm.
For the assay a single colony of E. coli harboring pNV12
was grown overnight in LB media supplemented with ampicillin,
chloramphenicol at a concentration of 100 g/ml and 10 g/ml
respectively at 37 C, 200 rpm. Next day the overnight culture was
diluted 100-fold in fresh LB medium supplemented with ampicillin,
chloramphenicol and incubated at 37 C, 200 rpm until the optical
density at 600 nm (OD600) reached 0.6. Thus prepared culture was
used for the bioassay.
2.5. Biosensor assay route
For biosensor assay, 400 l of diluted milk sample (200 l ster-
ile DI water + 200 l milk (from reference material)) was added
to 1.6 ml of fresh culture of E. coli harboring pNV12 (OD = 0.6 at
250 S. Kumar et al. / Sensors and Actuators B 240 (2017) 248254

Fig. 1. Schematic representation of the construction of biosensor plasmid pNV12. Plasmid pAD123 was digested with BamHI and EcoRI and ligated with 572 bp fragment of
PCR amplied product carrying CadC promoter, plasmid harbors genes required for replication (rep); Abbreviation: cat, gene encoding chloramphenicol resistance; bla, gene
encoding for ampicillin resistance; GFP, green uorescent protein. The diagram is not drawn to scale. (For interpretation of the references to colour in this gure legend, the
reader is referred to the web version of this article.).

600 nm) and incubated for 1hr at 37 C, similarly samples with
different cadmium concentration were tested for induction of
uorescence by adding sterile solution of different cadmium con-
centrations (1.0, 10, 20, 50 and 100 g/l) in place of sterile DI water.
Fluorescence of the samples was checked by ber optic spectrou-
orometer (Maya 2000, Ocean Optics, FL, USA). Specic Fluorescence
Index was calculated by dividing the uorescent count with OD of
the sample at 600 nm.
with 50 l of sterile DI water was added to 100 l aliquots of
biosensor cultures prearranged as mentioned above. Testing &
Validation of the performance of developed biosensor was done
through Flame-Atomic Absorption Spectrophotometer, Model SL-
173, Elico Limited. Milk samples were acid digested and 10 times
concentrated for the analysis. Wavelength used for the AAS study
was 228.8 nm that have an optimum working range of 0.2 g/l to
1.8 g/l with sensitivity up to 0.009 g/l (Cook Book, Elico Ltd.).

2.6. Microarray based detection 3. Results and discussion

For the experiment 100 l aliquots of recombinant bacterial cul-
ture were placed in a 48 well microarray plate, thereafter 50 l
of various concentrations of Cd (II) with 50 l of sterile DI water
was added to 100 l aliquots of bacterial cultures and incubated for
15 min at 37 C. Six independent experiments were performed for
each concentration of metal ion. The uorescence of GFP-producing
cells that were grown in culture was measured using miniopticon
(Bio-Rad, USA). Miniopticon was set with a 490 5 nm excitation
lter and a 510 5 nm emission lter to measure uorescence.
100 l of sterile DI water was added to 100 l aliquots of bacterial
cultures of rst six wells and used as the baseline/blank sample.
The interference in the induction of the sensing system by other
metal ions i.e. Pb(II) & Zn(II) reported in literature [14,16] was also
studied. To study the specicity of induction, 50 l of different con-
centration of Cd(II) (10, 50 and 100 g/l), Pb(II) (100 g/l) and Zn(II)
(100 g/l) separately as well as in combination was added to 100 l
aliquots of bacterial cultures.
2.7. Application of biosensor in natural milk samples and
validation
Developed biosensor was applied to various milk samples col-
lected from different places including rural, urban, industrial areas
of Punjab, India (Table 1). For the experiment 50 l of sample
3.1. Preparation of recombinant plasmid pNV12 and
transformation
Different steps of plasmid pNV12 preparation and growth of
transformed colonies was compared to native E. coli DH5 in pres-
ence of antibiotics (Fig. 2). Amplication of cad promoter and cadC
gene resulted in the product of about 572 bp (Fig. 2a). Fig. 2b illus-
trates isolated pAD123 used as expression vector. In Fig. 2c lane 2 is
presenting pNV12 while in lane 3 sample of pNV12 double digested
with EcoRI and BamHI is shown which conrms integration of Cad
promoter. Growth of E. coli DH5 transformed with pNV12 against
native E. coli DH5 is demonstrated in Fig. 2d.
3.2. Conrmation of bioassay principle
In presence of cadmium the uorescence count has upturn with
increasing cadmium ion concentration (10100 g/l). Fig. 3. dis-
plays the change in uorescent count with Cd(II) concentration. We
found good linearity from the range of 1050 g/l of the cadmium
concentration with coefcient of correlation R2 0.9946. It is to note
here that concentration of cadmium indicated in all the gures cor-
responds to the concentration of the samples/standards that was
added to the assay and not the nal concentration of Cd(II) in the
assay.
 S. Kumar et al. / Sensors and Actuators B 240 (2017) 248254 251

Table 1
Comparative results of cadmium concentration detected by recombinant biosensor and FAAS.

Natural Milk Samples (From Fluorescent Count (standard Cadmium Concentration (g/l)
Different places) error) by Recombinant Biosensor
Recombinant Biosensor FAASa % Error (Accuracy)

LD-1 (LudhianaOutskirt) 0.197 0.011 <10 BDLb

LD-2 (Ludhiana-City) 0.199 0.011 <10 BDL
LD-3 (Village-Tharika) 0.260 0.010 12 11.3 6.2 (93.8)
GB-1(Gobindgarh-outskirt) 0.265 0.006 18 16.5 9.1 (90.9)
GB-2 (Gobindgarh-City) 0.212 0.014 <10 BDL
GB-3 (Gobindgarh-Mandi) 0.205 0.008 <10 BDL
B-1 (Bathinda-City) 0.207 0.011 <10 BDL
B-2 (Bathinda- outskirt) 0.204 0.011 <10 BDL
K-1 (Khanna-City) 0.206 0.010 <10 BDL
K-2 (Khanna- outskirt) 0.210 0.006 <10 BDL
K-3 (Khanna-Main) 0.201 0.014 <10 BDL
P-1 (Patiala-City) 0.201 0.008 <10 BDL
P-2 (urban estate Patiala) 0.204 0.014 <10 BDL
P-3 (Patiala- outskirt) 0.211 0.014 <10 BDL
a
Flame Atomic Absorption spectroscopy. b Bellow detection limit, (n = 5).

Fig. 2. (a) Gel showing PCR product (Cad promoter and CadC gene) compared with 1 kb DNA ladder, (b) pAD123 (c) recombinant plasmid pNV12 in lane 2, ligation conrmed
with double digestion in lane 3 (d) Petri plates (containing ampicillin and chloramphenicol in nutrient agar base) showing growth of transformed colonies as compared to
native E. coli DH5.

3.3. Microarray based biosensor assay on synthetic system
Following the biosensor assay the reaction volume was minia-
turized to 200 l and this was performed in micro well plate to
develop microarray system. In micro well plate six replicates of
each experiment were done at a time simultaneously to nullify the
dispersion, use of multichannel auto-pipette nullied the handling
error. A linear increase in uorescence was found with increas-
ing Cd(II) ion concentration up to 10200 g/l, after that there
was a gradual decrease which can be explained in terms of cyto-
toxic effect of cadmium metal ion on the bacterial cells (Fig. 4).
The toxic effect of Cd(II) has been reported to cause single-strand
breakage in chromosomal DNA of E. coli [25]. To overcome this
problem, there is need to improve the genetic basis of bacterial cells
and also optimization of inuential factor like pH, media composi-
tion and culture conditions [26]. To overcome the readout problem
when Cd(II) concentration is higher than 200 g/l there is need
to apply serial dilution of the sample so that the reading com-
prises within the range 10200 g/l. The lowest detection limit was
adopted as the concentration that caused a noticeable induction
(background + 3 standard deviation), the detection limit for cad-
mium achieved in the present investigation is 5 g/l, which is lower
than most of the earlier reported recombinant cadmium biosen-
sors (Table 2). Although the detection limit is much higher than
252 S. Kumar et al. / Sensors and Actuators B 240 (2017) 248254

Present
work
[14]

[21]

[22]
[20]
Ref.

[4]
[5]
[6]

[7]
Not specic
Not specic

Not specic
Not specic

Not specic
Specicity

Specic

Specic
specic

Reaction time

120180 min
Incubation/

240 min

Fig. 3. Performance of recombinant biosensor in synthetic system with different

15 min
30 min

60 min

30 min
cadmium concentrations (10100 g/l). The inset shows the good linear range from
24 h

10 to 50 g/l. Data represent the means standard deviations of six replicates (n = 6).

0.7
Reaction
volume

200 l

0.6

Flourescence Count (A.U.)

0.5
1. A Cd responsive promoter from E. coli 2. Alakaline Phosphatase 3. Green orescence protein, * for different promoters, # Units are standardized to g/l.

0.4
110, 150 & 170* g/l

0.3
0.2
0.1
22.5 g/l

112 g/l
0.4 g/l

3.0 g/l
10 g/l

10 g/l

0
5 g/l
LOD#

0 100 200 300 400 500 600 700 800 900 1000

Concentration of cadmium (g/l)

Fig. 4. Performance of recombinant biosensor in synthetic system. Data represent

the means standard deviations of six replicates (n = 6).
1012 102 g/l
22511250 g/l

1121120 g/l

1121120 g/l

the detection limit reported by Tauriainen et al. [14], but the time
10100 g/l
0.412 g/l

6120 g/l
Detection

of incubation is too high 60 min as our developed system required

112 g/l
range#

only 15 min while.

3.4. Selectivity study

Interference of other toxic metals like Pb(II) and Zn(II) towards

Electrochemical
Conduct metric

the induction of GFP was studied and it is found that Pb(II) and
Transducers

Zn(II) do not interfere with cadmium. In the presence of cadmium

(100 g/l) only, uorescent count was upturn to 0.329 0.007 in
Optical

Optical

Optical
Optical

Optical

Optical

Optical

comparison with control (0.210 0.008, p < 0.005) while in pres-

ence of Pb(II) and Zn(II) both at a concentration of 100 g/l the
uorescent count was 0.221 0.004 (p < 0.05) that is close to
Comparison of developed biosensors for the detection of cadmium.

control, no signicant increase in uorescence was observed. Com-

Promoter/Reporter

binations of these metals (Pb(II) and Zn(II)) were also checked

with cadmium (100 g/l) and no signicant change in uores-
Phytochelatin
combination

HpSEO1 gfp3
hsp22-lucFF

cence statistically not signicant was observed with comparison

P0659-lacZ
zntA1 -lacZ
Cad-lucFF
synthase
Gene gfp

to the uorescence count observed in only presence of cadmium

Cad-gfp

(100 g/l) (Fig. 5). Tao et al. [7] has developed a recombinant
APA2

biosensor using similar strategy, in which three kinds of con-

structed gene CadR, Cad-TC-10 and Cad TC-21 were used as sensing
element but didnt respond specically against cadmium. In the
S. cerevisiae BY4741

present study a single promoter is employed that showed high

Chlorella vulgaris

Rec E. coli DH5

Bio-component

specicity for cadmium and hence is the one of the great advan-
S. aureus & B.

Deinococcus

tage of our developed recombinant biosensor against earlier work

radiodurans

polymorpha
Hansenula
Rec E. coli

Rec E. coli
HeLa cell

done so for.
subtilis

3.5. Analysis of natural milk samples by developed recombinant

biosensor
Table 2

S.No.

Developed biosensor was applied to detect cadmium concen-

1.
2.
3.

4.
5.

6.
7.

8.

9.

tration in milk sample collected from different areas of Punjab.

 S. Kumar et al. / Sensors and Actuators B 240 (2017) 248254 253

0.4 3.6. Validation of developed recombinant biosensor with FAAS

0.35 ***
***
Flourescence (A.U.)

0.3 *** Concentration of cadmium in the milk samples analyzed with

0.25 ** recombinant biosensor was cross-checked with FAAS. Cd(II) lev-
0.2 els detected by FAAS were found in comparison with the results of
0.15 developed biosensor. In the samples collected from Ludhiana and
0.1 Gobindgarh biosensor detected a Cd(II) ion concentration of 12 g/l
0.05 and 18 g/l respectively; the same were analyzed with FAAS to
0 be 11.3 g/l and 16.5 g/l respectively. Hence an accuracy of more
than 90% is achieved (Table 1).
In the recent past, recombinant DNA technology has been
used to construct genetically engineered microbial biosensors
frequently by joining the natural analyte regulatory genes that
encodes a transcriptional regulator plus a promoter/operator to
a reporter gene e.g. lux/luc, gfp and lacZ, recombinant genes then
Fig. 5. Performance of recombinant biosensor against milk samples spiked with
transformed into microorganisms as a plasmid or integrated into
different metals, showing specicity towards cadmium. Data represent the the chromosome [1719]. In present study cad promoter has been
means standard deviations of six replicates (n = 6), ***p < 0.005, **p < 0.05. attached with gfp to develop a biosensor for cadmium. A compar-
ative statistics between the present biosensor and the biosensors
developed in the past has been presented in Table 2. Developed
0.4
biosensor demonstrated specicity for Cd(II) in presence of Pb(II)
***
0.35
*** and Zn(II) as well that have been reported to interfere with Cd(II)
0.3 * [20]. Joe at al. [21] constructed a whole cell biosensor using the
*** 393-bp deletion fragment (P0659-1) of the P0569 promoter and
0.25
lacZ as a reporter was by the detection range achieved was from 1
A.U.

0.2 to 10 mM of Cd(II). For macroscopic detection, the sensor plasmid

0.15 (pRADI-P0659-1) containing crtI as a reporter gene under the con-
trol of P0659-1 was introduced into a crtI-deleted mutant strain of
0.1 D. radiodurans (KDH018). Color change by the red pigment synthe-
0.05 sis could be recognized in a day (24 h) with the naked eye and the
detection range was from 50 nM to 1 mM of Cd(II).
0
0 10 20 30 40 50 60 70 80 90 100 In comparisons between recombinant whole cell cadmium
Concentration of cadmium (g/l) biosensors in the past, present biosensor has several relative advan-
tages in terms of rapidity, specicity and limit of detection etc.
Fig. 6. Fluorescent count of recombinant biosensor against milk samples spiked Biosensor BR151 (pTOO24) developed by Tauriainen et al. [14],
with different Cd concentrations, (n = 6) ***p < 0.001 and *p < 0.1. showed a cadmium detection limit of 0.3 g/l (3.3 nM), with induc-
tion coefcients up to 5. Though the detection limit of biosensor
0.3 developed in present study is 10 g/l (55 nM), with an induc-
tion coefcient up to 2, present biosensor is found relatively rapid
0.25 in detection as well as showed specicity towards cadmium. Fur-
ther, we found if we increase the incubation time up to 30 min the
Flourescence count (AU)

0.2 detection limit achieved up to 5 g/l (27.5 nM).

0.15
4. Conclusions
0.1
The developed biosensor for the estimation of the bioavailable
0.05
concentration of cadmium in milk has been proved to be a specic
and rapid option with response time as low as 15 min. The linear
0 range of detection is 1050 g/l with R2 0.994. Using a microarray
Blank 10g/l B1 K2 LD3 GB1 GB2 P3 approach maximum 48 samples could be analyzed simultaneously.
Milk samples from different rural and cities area of Pun jab, Ind ia
The sample volume has been miniaturized up to 200 l. The devel-
Fig. 7. Fluorescent count of milk samples compared with blank (sample with-
oped biosensor is sensitive, easy to handle, fast, and do not require
out cadmium) and sample spiked with cadmium (10 g/l). Data represent the any pre-treatment of samples.
means standard deviations of six replicates (n = 6).
Acknowledgements

Concentration was calculated on the basis of uorescent count The authors are thankful to ICAR (NAIP), New Delhi, India for the
employing Fig. 6 as standard. Statistical comparison between research grant C4/C10125/2008 supporting this work.
spiked milk samples with diffenert Cd(II) ion concentrations were
obtained statistically signicant p < 0.001. In most of the samples
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Biographies
Dr. Sachin Kumar obtained his PhD degree in Biotechnology from Punjabi university
Patiala in 2014. He is currently working as Assistant Professor in Dept. of Biotech-
nology, JV college, Baraut, India. His research interests are Biosensor Technology,
analytical chemistry, analysis of heavy metal ions, bioremediation and molecular
biology.
Dr. Neelam Verma obtained her PhD degree in chemistry from Punjabi univer-
sity Patiala in 1986. She is currently a Professor (Former HeadMarch 12, 2005
to March 11, 2008), Dept. of Biotechnology, Punjabi University, Patiala, Punjab,
India. Her research interests are Biosensor Technology, Environmental Biotech-
nology/Biochemistry, Medical Biotechnology, Biophysics, Molecular modeling &
Protein Engineering.
Ashish Kumar Singh obtained his M.Sc. in Biotechnology from Punjabi university
Patiala in 2010. He is currently doing doctoral research under the supervision of
Dr. Neelam Verma and Co-supervision of Dr. Minni Singh in Dept. of Biotechnology,
Punjabi University, Patiala. His topic of research is biosensor development for the
analysis of different analytes in food and clinical samples.