Open Access

Austin Journal of Biosensors &

Bioelectronics

Research Article

Advancement Towards Microfluidic Approach to Develop

Economical Disposable Optical Biosensor for Lead
Detection
Neelam Verma1*, Rajni Sharma1 and Sachin
Abstract
Kumar2
1
Department of Biotechnology, Punjabi University, India An economical single use optical biosensor has been developed for
2
Department of Biotechnology, JV College, India lead detection using a microfluidic approach. The present study represents
*Corresponding author: Neelam Verma, Biosensor the initiative step for amalgamation of microfluidic and advanced biosensor
Technology Laboratory, Department of Biotechnology, technologies which offers rapid analysis with lab-on-a-chip policy. Urease
Punjabi University, Patiala-147002, Punjab, India producing Bacillus sphaericus was co-immobilized with phenol red (pH indicator)
in the glass capillary which acted as a microchannel. For immobilization, a
Received: August 03, 2016; Accepted: September 08, combination of sol-gel approach and calcium alginate method cited first time
2016; Published: September 12, 2016 in literature was used which reduced the time of solidification to seconds as
compared to hours with sol-gel alone. Bioassay principle was based on urease
inhibition in the presence of lead. Fiber optic spectrophotometer was used as
transducer which measured the intensity of color change. Linear relationship
(10-1000 g/L) was observed between logarithmic concentration of lead and
absorbance. The study resulted in the development of cheap, miniaturized,
sensitive and reliable lead biosensor with requirement of small sample volume
(1 mL).
Keywords: Bacillus sphaericus; Microfluidic; Fiber optics; Lead; Sol-gel;
Biosensor

Introduction Differential Pulse Cathodic Stripping Voltametry (DPCSV) are

superseded by the use of biosensors [10,11].
Increased environmental awareness and stringent environmental
regulation led to the dire need of the techniques for the fast, easy and Biosensors are analytical tools capable of providing either
economic detection of various pollutants like heavy metals, pesticides qualitative or quantitative results, consisting of an immobilized
and toxic gases. Heavy metals are among the most hazardous biological recognition element such as an enzyme, antibody or cell
pollutants due to their ubiquitous presence in the biosphere, their receptor immobilized to a physicochemical transducer to create
bioavailability from both natural and anthropogenic sources as well a single unit. Now-a-days biosensor technology has emerged as
as their high toxicity even at trace level [1,2]. Lead is among the toxic the most promising tool for detection of heavy metals being cost-
heavy metals which harm the body when present above the threshold effective, fast, selective, sensitive, portable, easy to use and reliable
concentration [3]. The higher concentration of lead (>18 M) in the [12]. A number of lead biosensors have been developed using various
blood may cause coma and death [4]. It affects different parts of the transducers like conductometer [13-15], electrochemical [16-22] and
body particularly brain and central nervous system. Accumulation optical [3,23-27] based on whole cell, enzymes or DNAzyme.
of lead in the body produces damaging effects associated with The present study depicts the use of optical transducer along with
hematology, neurology and nephrology which include paralysis, microfluidic approach. Microfluidic system allows the controlled
mental retardation and neural deafness. The substitution of calcium flow of operations like fluid transport, valving, mixing, separation,
in the body with lead causes impairment in the development of bones concentration and detection in Lab-on-a-Chip field [28] which helps
and teeth [5]. Human exposure to lead occurs primarily through lead- to overcome the matrix effect, a major hindrance in the widespread
based paints, industrial waste, water from lead-laden pipes, soil and use of biosensors for extra laboratory testing and drug arrays. Fluids
dust generated from gasoline and food items like milk, dairy products are pumped into a particular channel known as microchannel at
and imported candies. The cattle grazing on the metal contaminated defined rate. Microfluidic is competent to analyze small sample
fields transmit lead contamination in the milk and dairy products volumes e.g. 10-9 10-18 L [29]. A few reports are available for use
[4,6,7]. A threshold lead limit of 10 g/L was estimated in food and of microfluidic approach in biosensor technology. Chang and his
water by the International Agency for Research on Cancer (IARC) workers [30] fabricated a microfluidic biosensor for lead detection
while 5 g/L in drinking water by Environmental Protection Agency using DNAzyme exhibiting linearity in the range of 0.1-100 M
(EPA) [8,9]. It is mandatory, therefore, to detect the lead in food (0.02-20.7 mg/L) concentration of lead with detection limit of 11 nM.
samples and drinking water to prevent the deleterious effects of The biosensor developed in this study demonstrated the linear range
lead. Conventional methods to detect heavy metals like differential of 10-100 g/L Pb2+ which is far lower than previously described
pulse polarography, Atomic Absorption Spectrophotometry (AAS), biosensor making it suitable for the application in milk, water and

Austin J Biosens & Bioelectron - Volume 2 Issue 2 - 2016 Citation: Verma N, Sharma R, Kumar S. Advancement Towards Microfluidic Approach to Develop Economical
ISSN : 2473-0629 | www.austinpublishinggroup.com Disposable Optical Biosensor for Lead Detection. Austin J Biosens & Bioelectron. 2016; 2(2): 1021.
Verma et al. All rights are reserved
Neelam Verma Austin Publishing Group

clinical samples. Moreover, use of whole cell for the fabrication of formation of ammonium ions. The absorbance was measured at
biosensor rather than DNAzymes help to make it economical. The particular wavelength (555 nm). Later on, urea solutions (2 M) spiked
present approach helps to realize the miniaturization of biosensors with different concentrations of analyte i.e. lead (10-1000 g/L) was
which provides the advantages of portability, reduced cost, increased allowed to pass through the microchannels at same flow rate and
analysis speed, reduction in sample and reagent consumption. time and the absorbance was measured. To check the reusability of
biosensor (microchannel with immobilized biocomponent), same
Materials and Methods microchannel was used to flow different solutions. The repeated use
All the chemicals and reagents used in the study were of analytical of microchannel required its regeneration which was performed with
grade. Glass capillaries (1mm) were used to prepare microchannels. 0.1M EDTA solution.
Fiber Optic Spectrophotometer (Ocean Optics, Maya 2000 series) has
Results and Discussion
been used as a transducer.
An optical biosensor was fabricated to detect the presence of the
Biocomponent
lead. The microfluidic approach allowed very confined space for the
Urease producing microbe Bacillus sphaericus MTCC 5100 (a immobilization of the biocomponent, flow of the analyte, reaction
novel soil isolate of Biosensor Technology Laboratory, Department between analyte and biocomponent as well as real time monitoring
of Biotechnology, Punjabi University, Patiala) was used as a of the reaction which ensured the miniaturization of the biosensor.
biocomponent in the development of biosensor. B. sphaericus was A novel immobilization strategy has been employed combining two
grown on the media containing urea (2.5%), beef extract (1%), different approaches for the immobilization of the biocomponent
peptone (1%), sodium chloride (0.5%) at 37C, pH 7.0 for 24 h under within the microchannel. The hydrosol gel immobilization method
aerobic conditions (200 rpm). Microbial cells were harvested from 24 was unified with calcium alginate method which has resulted in the
h grown culture by centrifugation at 5000 rpm for 10 min at 4C and drastic reduction in the solidification time from 90 -120 min to just
stored in 0.05M PBS buffer (pH 7.2). The urease activity of microbe few seconds. To the best of our knowledge, this is the first report of
was estimated by Nesslers method [31]. the blending two immobilization approaches which led to extreme
Immobilization strategy decline in the solidification time. Besides this, a small volume of the
The biocomponent was co-immobilized with phenol red indicator sample (1 mL) was required as compared to conventional methods.
using a hydrosol gel method [14]. TMOS (Tetramethylorthosilicate) The developed biosensor provided the quick and visible response
sol-gel stock mixture was prepared by mixing 20 L whole cells of B. with respect to analyte (lead).
sphaericus (U 197.44), 600 L ethanol, 100 L TMOS, 30 L of 5 mM The passage of standard urea (2 M) solution through the
NaOH, 80 L Phenol Red (5mg /4 ml of 50% Ethanol) and 70 L de- microchannel containing urease producing microbe B. sphaericus
ionised water. 100 L of 3% sodium alginate (dissolved in 0.05 M PBS resulted in the color change of the phenol red from yellow to pink.
buffer) was added in the stock mixture following incubation (1 hour) The color change was less intense (Figure 2A) with lead spiked
at 4C. Immediately 60-60 L of final mixture was dispensed into the solution as compared to standard urea solution (Figure 2B & C) due
glass capillaries in the centre and allowed to solidify. to the inhibition of urease enzyme with lead.
Fabrication of biosensor max was found to be 555 nm after scanning over 400 to 700 nm
The microchannel with immobilized biocomponent was fitted in (Figure 3). Therefore, the absorbance was noted down at 555 nm for
the hole of optic fiber dip probe (Figure 1A) which allowed the real all samples (Table 1). Figure 3 shows the overlay of spectrum obtained
time monitoring of the reaction taking place between the fluids passing after hydrolysis with urea and other synthetic solutions spiked with
through the microchannel and the immobilized biocomponent. The different concentrations of lead. The linear relation (R2 = 0.9834)
peristaltic pump was connected to adjust the flow rate. The whole was observed between logarithmic concentration of lead (g/L) and
assembly of the system is shown in the Figure 1B. The Spectra Suite absorbance at 555 nm (Figure 4). The detection range of lead (10-
software was used to measure the absorbance over the range of 400- 1000 g/L) with biosensor was found applicable for various water,
700 nm to determine max using fiber optic spectrophotometer. 2M
urea solution was passed through the microchannel at a rate of 0.33
ml min-1 (20 ml h-1) for 3 min to allow hydrolysis which resulted
in color change of phenol red from yellowish to pink due to the

Figure 1: (A) Fitting of microchannel within fiber optics dip probe (B) Figure 2: Color change in pH indicator, Phenol red (A) Fresh Microchannel
Assembly of the System. (B) After Urea hydrolysis (C) Urea hydrolysis in the presence of lead.

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Neelam Verma Austin Publishing Group

Table 1: Change in Absorbance (555 nm) with different concentration of lead

(Single use of developed biosensor).
Samples Absorbance (555nm)

Standard urea (2M) 0.977 0.0128

Urea (2M) spiked with 10 g/L of lead 0.812 0.0184

Urea (2M) spiked with 100 g/L of lead 0.509 0.0180

Urea (2M) spiked with 1000 g/L of lead 0.352 0.0130

Table 2: Change in Absorbance (555 nm) with different concentrations of lead

(Repeated use of developed biosensor).
Samples Absorbance (555nm)

Standard urea (2M) 0.922

Figure 3: Scanning of maximum wavelength of color substance produced Urea (2M) spiked with 10 g/L of lead 0.210
after urea hydrolysis (A) Standard Urea (B) Urea spiked with 100 g/L of lead
(C) Urea spiked with 1000 g/L of lead. Urea (2M) spiked with 100 g/L of lead 0.109

resulting in the production of less amount of NH4+, lesser change

1.2
in pH and color and consequently less absorbance as compared
y = -0.217x + 0.989
1 R = 0.983 to control. The greater the concentration of lead, the lesser will be
Absorbance at 555 nm

absorbance of the sample.

0.8
The single use of the developed biosensor (microchannel with
0.6 immobilized biocomponent) provided good linearity. To check the
multiple use of developed biosensor, same microchannel was used for
0.4
the analysis of different samples. As expected, absorbance decreases
0.2 with increase in concentration of lead (Table 2) even when the same
microchannel was used repeatedly. But the leaching of dye and wash-
0 out of biocomponent from the microchannel was also observed which
0 0.5 1 1.5 2 2.5 3 3.5 may lead to decrease in absorption rather than mere due to inhibition
Log of conc. of lead (g/L) of urease. This process may give false indication about concentration
of lead. Therefore, the single use of the developed biosensor was
Figure 4: Performance of optical biosensor with different concentration of
lead (10-1000 g/L) in the synthetic samples. preferable as compared to repeated use. The cost effective nature of
the developed biosensor surpasses the single use of the biosensor
sinceone microchannel enclosed only 60 l of immobilization
mixture containing biocomponent and pH indicator. However,
further improvement in immobilization method is recommended to
prevent leaching of dye to make the biosensor reusable.
Microscale fluid regulators (e.g. valves, mixers and pumps)
can be integrated on the lab-on-a-chip platform to increase the
analytical performance of biosensors [29]. A DNAzyme based
microfluidic biosensor was earlier developed for the lead detection
using a microfluidic device with multifaceted microfluidic channels
employing nanocapillaries to facilitate the efficient and expedient flow
in the device [30]. The use of sophisticated device made it costlier.
However, the microfluidic biosensor developed in the present study
is cheap, miniaturized, sensitive and reliable.
Figure 5: Bioassay Principle of the developed biosensor.
Conclusion
milk and clinical samples as the permissible limit of lead in milk and
An economical disposable optical biosensor was developed
drinking water is 10 g/L while Blood Lead Level (BLL) concentration
for lead detection in the synthetic media which can extend its
is 100 g/L [32].
application in different milk, water and clinical samples after minor
The developed biosensor is based on the enzyme inhibition (Figure optimizations. Urease producing Bacillus sphaericus was used in the
5); urease catalyses the hydrolysis of urea leading to the production of fabrication of biosensor employing the technique of microfluidic. The
NH4+ which results in the change of pH [10]. This change in the pH blend of microfluidic and advanced biosensor technologies offers new
can be indicated by color change in the pH dependent dye, phenol promises including high-throughput analysis and portability. Enzyme
red and absorbance can be measured with spectrophotometer. In the inhibition was used as a bioassay principle and inhibition of the
presence of lead (heavy metal as inhibitor), urease activity decreases enzyme with lead was measured with change in absorbance of color

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Neelam Verma Austin Publishing Group

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Austin J Biosens & Bioelectron - Volume 2 Issue 2 - 2016 Citation: Verma N, Sharma R, Kumar S. Advancement Towards Microfluidic Approach to Develop Economical
ISSN : 2473-0629 | www.austinpublishinggroup.com Disposable Optical Biosensor for Lead Detection. Austin J Biosens & Bioelectron. 2016; 2(2): 1021.
Verma et al. All rights are reserved

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