2. OVERVIEW
Introduction
Antibiotic residue and development of resistance
Electrochemical Sensors
Working principle of Electrochemical Sensors
Use of EC Sensors in Antibiotic Residue
detection
Detection Based on Recognition Element
Detection Based on Electrodes
Use of Novel materials like Nanomaterial, CNT,
Graphene
Conclusion
Future Prospects
Q and A Session
3. INTRODUCTION
6% increase in global use of antibiotics(2010-2013) and
its developing resistance in microorganism is an
alarming fact.
Every year more than 4 Billion prescription are
distributed to Americans only, with variety of
pharmaceuticals
These drugs rarely get fully metabolized and are
excreted by body and often get discarded by patients.
In agriculture 90 % of drugs are used for prophylactic
and growth promoter.
These eventually make their way to the food and thus
resistance develops.
Many researches show antibiotic residue in meat, fish
and milk above MRL label specially in countries where
regulations are not well enforced.
4. ANTIBIOTIC RESIDUE IN FOOD AND RESISTANCE
MICROORGANISM
From 2010 to 2013, the ciprofloxacin resistant E. coli
has increased by 18% and resistance to
cephalosporin and gentamicin rose by 28 % and
27% respectively.Akbar, 2014 in poultry meat
• Salmonella species (5.26%)
• S. aureus (18.18%)
• Salmonella (72.72%) resistance to
tetracycline.
• Few of the Salmonella : MDR
• S. aureus (44.73%) : highly resistant
to tetracycline
Beef, camel, lamb and poultry in
Saudi Arab, Greeson et al., 2013
E. coli (72.2%), Enterococcus
(26.2%), S. aureus (24.6%) and
Salmonella (10.7%.) S. aureus
(79%) and Enterococcus (86%) to
Erythromycin E. coli to Ampicillin
(44%) Salmonella to Ceftiofur
(67%)
In near future we may face an immediate risk of entering to the post-antibiotic
period where the existing antibiotics cannot be used for medical application
5. ELECTROCHEMICAL SENSORS
Oxygen electrode was
invented on Oct 4 1954 by Dr.
Leland Clark on a closet shelf
in his home
1962 Clark reversed the polarity of the
same platinum electrode using the
same Glucose Oxidase enzyme to
measure glucose
Electrochemical detection of antibiotics
cover 21 % of detection technique
dominated by CV and SWV
Leland Clark jr with first enzyme
electrode, the glucose sensor
Global glucose monitoring devices market
is expected to exceed $ 18 billion in 2015
6. DEFINITION
Chemical sensor-A device that transforms chemical information
ranging from concentration of a specific sample component to total
composition analysis into an analytically useful signal.
Electrochemical devices transform the effect of the electrochemical
interaction of analyte - electrode into a useful signal. (IUPAC 1991)
Based on electrochemical species consumed or generated during
an interaction process. The signal of this interaction is measured by
electrochemical detector.
7. WORKING PRINCIPLE OF EC SENSORS
Consists of a transducer
element covered by a
recognition element
Recognition element interacts
with target analyte and signal
is generated
Electrochemical transducers
then transform the chemical
changes into electrical signals-
e.g. reduced faradic current
after antibiotics binding.
Produces an electrical signal
related to the concentration of
analyte
8. ADVANTAGES OF EC SENSOR OVER
CONVENTIONAL DETECTION TECHNIQUES
Convectional methods are time taking and less
sensitive.
ELISA is rather qualitative, requires specific antibodies,
sample treatment, long incubation and intermediate
preparations.
HPLC and other chromatographic method are
expensive and have higher limits of detection.
Electrochemical sensors provide all advantages over
these and provide a real time, easy and faster detection
of antibiotics to much lower detection level than MRL
allowed.
Since these are based on electrochemical approaches
very minute change in concentration can also be
detected and quantified.
9. ENZYME AS RECOGNITION ELEMENT
Polymeric films are used to immobilize enzymes on electrode
(Nafion, polypyrrole)
May be trapped by mixing with carbon paste, surface
adsorption or covanlent bonding.
Not many antibiotics detection using enzymes are done rather
they have wide application as carbohydrate, ethanol, starch
biosensors.
Rinken and Riik, 2006 developed lactate oxidase-based
amperometric biosensor for the determination of CAP ana
Penicillin residue in milk.
Setford et al., 1999 used Glucose oxidase for β-lactam
antibiotic residues in milk. Low sensitivity and concentration
determination were accounted with detection level in ppm
level.
10. USE OF ANTIBODIES AS RECOGNITION ELEMENT
Immunochemical sensors are used widely for detection of
antibiotics. ELISA have dominated till now.
Signal are generated by change in interfacial resistance
and/or capacitance between the electrode surface and
analyte once immobilized antibody react with antigen.
Chullasat et al., 2011 developed an impedometric
immunosensor for CAP in shrimps and LOD was 1.6 ng kg -1
Ionescu et al., immobilized ciprofloxacin on the polypyrrole
NHS layer covered by CF Ab. The resulting binding of CF in
solution was quantified using Impedance spectra.
Wu et al., 2012 –Neomycin determination in milk using
paper supported EC immunosensor. SWCNT were
impregnated with antibody against Neomycin, dip in
solution, the change in impedance with concentration of
Neomycin was noted. The LOD was 0.04ng/ml
11. USE OF APTAMERS AS RECOGNITION ELEMENT
Aptamers are ssDNA or RNA fragments used as sensing
probe.
Binding with an analyte occurs due to ionic interaction,
Vander-waals-force or hydrogen bonds leading to
detectable signal.
This used technology similar to DNA hybridization.
Their sensitivity is comparable to antibodies and are
chemically synthesized, thermal stable and easy to modify
and immobilize.
Zhang et al., Kim et al,. 2010 and Zhou L., et al., 2012 Used
DNA aptamers for the detection of Tetracycline and
Oxytetetracyline using EC approach.
Pilehevar et al., 2012 used aptamers for Chloramphenicol
detection, LOD was as low as 5µg/L
Zhu , Chandra et al., 2012 used DNA coupled with gold
nanoparticle for Kanamycin detection and LOD was
12. Tet binding ssDNA coupled
with Tet via EDC/NHS
chemistry on GCE.
Cyclic Volta grams of defined
Tet concentration were
recorded in the presence of
redox active K3Fe(CN)6
The relation between
increasing Tet conc. and
decreasing current was used.
The LOD was 10nM and used
in food, drinking water.
Zhou et al., 2012
13. MIP AS RECOGNITION ELEMENT
MIP (artificial antibodies or Plastic antibodies)- are polymers
containing template shaped cavities used for molecular
recognition.
Target analyte binds functional monomers by covalent or non-
covalent bonding in a polymerization process.
A core shell mMMIP was attached with mGCE and was used
for detection of MNZ in milk and honey samples. The LOD
was 1.6 × 10−8 M (Chen, Deng et al. 2013).
Redox active mMIP was synthesized and functionalized with
STR templates electrochemical detection of STR in food.
These synthesized Au-(III) promoted MIP nano-spheres thus
were capable of detecting as low as 10pg/ml (Liu, Tang et al.
2013)
Song et al., 2014 developed MIP based EC for detection of
ERY in honey, milk and milk powder. MIPs were used for
preparation of MIPs-modified carbon paste (MIP-CP)
electrode. Quantitative determination was done using DPV.
LOD was 1.9 × 10-8mol/L
14. Current responses for ERY and other two
structurally related antibiotics (oleandomycin
and tilmicosin) with a concentration of
2 × 10− 6 mol L− 1 at the MIPs and NIPs
based electrochemical sensor
(A) DPV of ERY in conc. range from
5.0 × 10− 8 to 1.0 × 10− 5 mol L− 1 (a–
h) at the MIPs based EC sensor at
the (B) Corresponding calibration
curve of ERY at the proposed
electrodes.
15. USE OF ELECTRODE SYSTEM
Various electrochemical approaches like potentiometric,
voltammetric (mostly CV and SWV) and amperometric are
have been used for detection of antibiotics.
In voltammetric detection a potential set by instrument
establishes conc. of reduced and oxidized species at
electrode based on Nernst Equation
A reduced faradic current is detected after antibiotic binding.
Apply
Potential
16. USE OF ELECTRODE SYSTEM CONTD…
Azithromycin was determined using a voltammetric
sensor based on MCNTs decorated with MgCr2O4
.DPV approach was used with LOD of 0.07µM/L
(Ensafi et al., 2013)
Fe/Zn-MMT catalyst on GCE was used for detection
of TC in meat and feedstuff. In the presence of SDS
the EC sensor showed LOD of 0.1 µM (Gan et al.,
2014)
An EC sensor developed by incorporating Au NPs
onto the poly-1-5-diaminonapthalene layer (pDAN)
coated pyrolytic graphite for the detection of
Cefpodoxime Proxetil gave excellent LOD of 39nM
(Yadav et al., 2013)
TC, CTC and OTC detection on screen printed gold
electrode was done using CV. LOD was 0.96, 0.58
and 0.35µmol/L, respectively. (Masawat and slater
17. Analytes
Linear
dynamic range
(μM)
Analytical sensitivity
(nA μM−1)
Intercept
(nA)
r2
Limit of detection
(μM)
Tetracycline (TC) 1–500 0.6098 6.5017 0.9968 0.96
Chlortetracyclin
e (CTC) 5–50 1.4932 2.3871 0.9999 0.58
Oxytetracycline
(OTC) 1–500 0.6983 12.064 0.9918 0.35
Table. Calibration characteristics of tetracycline, chlortetracycline and
oxytetracycline for the dc amperometry at SPGE flow-through cell
Masawat and slater 2007
18. USE OF REDOX TRACER CARBON NANOTUBE,
NANOPARTICLES
All antibiotics are not electrochemically active so require
electro active molecules to be added called as redox
tracers or electro catalyst.
Due to high surface area to volume ratio, good conductivity
and strength graphene and carbon nanotubes have
recently dragged a lot of attention for EC sensors
development.
Use of nanoparticles of gold, silicon, graphene, platinum
has also increased recently.
Various EC sensors have used the excellent properties of
CNT, AuNPs. Graphene among others for detection of
antibiotics successfully.
Use of ferrocene as redox tracer for detection of PAH was
reported by Dutduan et al. 2014.
Similar use of redox tags like hematein, methylene blue or
Fe 2+/Fe 3+ systems are often done.
19. CONCLUSION
The growing antibiotic residue and its associated
health hazards accounts for easy, fast and online
assessment of these drugs in food.
Electrochemical sensors prove best alternative to
conventional methods which are time consuming,
have limited application and costly.
The use of MIP, redox tracers have made
detection of virtually all antibiotics possible.
The high sensitivity of electrochemical sensors
allow very less limit of detection which may in turn
help for regulation of MRL of antibiotics in food.
20. FUTURE PROSPECTS
Recent trend has been focused on decreasing
detection time increased performance and sensitivity.
Hand held, wearable sensors are being developed.
Use of nanomaterial and other novel electrochemical
material in EC sensor for antibiotics detection will and
has to follow.
Electrochemical biosensors can also be useful in
elucidating the mode of action of antibiotics which will
help in studying resistance mechanism of antibiotics
Reusable and cheap sensors are to be focused with
food safety guidelines in consideration.
Online measurement of antibiotics residues in
processing lines should be focused.
Editor's Notes
Milk Samples from Kathmandu, Bhaktapur and Lalitpur in Nepal showed high residue of Penicillin, Amoxicillin , ampicillin , Chloramphenicol and streptomycin . 81 % samples shown positive for Amoxicillin 27 % for tetracycline 28% for tetracycline and all detected samples were above MRL
2. Meat sample in saudi Arabia
3.Feed in US National Swine survey. (25 % contains residue above MRL)
About 450000 new cases emerged in 2012 related with the multidrug-resistant tuberculosis
Microfluidics most promising technology.
Number of patents and resea
An electrode was modified with a self-assembled thiourea monolayer, gold anoparticles, mercapto succinic acid and antibody.
Fig 1. Schematic diagram of the screen-printed gold electrode (I) and the specially fabricated electrochemical wall-jet cell (II) used in the voltammetric and amperometric measurements.
Fig 2. Cyclic voltammograms at gold screen-printed sensors in 3 mM tetracycline, chlortetracycline, and oxytetracycline in 0.1 M potassium dihydrogen phosphate pH 2.0 (solid lines) and 0.1 M potassium dihydrogen phosphate pH 2.0 (dash lines). The scan rate was 10 mV s−1; area of gold working electrode, 0.5 mm2.