ELECTROCHEMICAL SENSORS

1. Electrochemical sensors for detection of antibiotics in Food Lavaraj Devkota Id No: 116494 Special Study, June 2015

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 9.4±0.4nM

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 K 3 Fe(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 -8 mol/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 MgCr 2 O 4 .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 a l., 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 2007).

17. Analytes Linear dynamic range ( μ M) Analytical sensitivity ( nA μ M−1) Intercept ( nA ) r2 Limit of detection ( μ M) Tetracycline (TC) 1–500 0.6098 6.5017 0.9968 0.96 Chlortetracycline (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 an t ibiotics 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.

21. Thank You !! Question Time !!