1. Advanced Biosensors Based on Nano-scaled Arrays and Films of Electro- and Biocatalysts Arkady A. Karyakin Chemistry faculty of M.V. Lomonosov Moscow State University, 119991, Moscow, Russia

2. 2 Electroanalysis: widest dynamic range (down to a single molecule detection) less sensitive to matrix effects (insensitive to color, turbidity etc.) the cheapest equipment

3. 3 Scheme of biosensor action Transducer Biorecognition element Substrate recognition Coupling of biochemical and transducer reactions Signal processing

4. 4 Advanced biosensors: most efficient electrocatalyst (transducer) A.A.Karyakin, O.V.Gitelmacher, E.E.Karyakina. Analytical Chemistry 67 (1995) 2419 A.A.Karyakin, E.E.Karyakina, L.Gorton. Electrochem. Communications 1 (1999) 78. A.A.Karyakin, E.E.Karyakina, L.Gorton. Analytical Chemistry 72 (2000) 1720 A.A.Karyakin, E.A.Puganova, et al. Analytical Chemistry 76 (2004) 474. A.A.Karyakin, E.A.Puganova, I.A.Bolshakov, E.E.Karyakina, Angewandte Chemie-Int. Ed. 46 (2007) 7678. N.A.Sitnikova, A.V.Borisova, …, A.A.Karyakin Analytical Chemistry 83 (2011) 2359 improved enzyme immobilization protocol A. A. Karyakin, E. E. Karyakina, et. al. Analytical Chemistry 68 (1996) 4335. A.A. Karyakin, E. A. Kotel'nikova et. al. Analytical Chemistry 74 (2002) 1597. E.I. Yashina, A.V. Borisova,..., A.A. Karyakin. Analytical Chemistry 82 (2010) 1601 A.N. Sekretaryova, …, A.A. Karyakin Analytical Chemistry 84 (2012) 1220

5. 5 Oxidase based biosensors (> 90% of biosensors, kits, etc.) Monitoring of H 2 O 2 formation: Advantages: -low detection limit (<1  M). How to detect H 2 O 2 ? 1. Pt electrode, +0.6 V, Ag/AgCl. (G.G. Guilbault, et al.. Anal.Chem. 45 (1973)2255) 2. Peroxidase electrode. Disadvantages: reductants (ascorbate, paracetamol etc.) generate parasitic signal

6. 6 Prussian Blue Fe III 4 [Fe II (CN) 6 ] 3 : nano-scaled layer - advanced electrocatalyst ElectrocatalystSelectivity j H 2 O 2 /j O 2 Electrochem. constant, cm/s Pt0.14. 10 -6 Prussian Blue 400-6001. 10 -2 A.A.Karyakin, E.E.Karyakina, L.Gorton. Analytical Chemistry 72 (2000) 1720 thickness 40-80 nm

7. 7 Superstable H 2 O 2 transducer (continuous flow of 1 mM H 2 O 2 ) N.A. Sitnikova, A.V. Borisova, M.A. Komkova, A.A. Karyakin. Analytical Chemistry 83 (2011) 2359-63

8. 8 Nano-electrode arrays Nano-structuring of electrocatalyst onto inert support A.A. Karyakin, E.A. Puganova et.al. Analytical Chemistry, 2004, 76, 474-8 signal:noise ~ 1/r

9. 9 Electrochemical sensor with record performance characteristics A.A. Karyakin, E.A. Puganova, I.A. Bolshakov, E.E. Karyakina Angewandte Chemie 46 (2007) 7678-80.

10. 10 Improved enzyme immobilization protocol

11. 11 Immobilization from water- organic mixtures A.A.Karyakin, E.E.Karyakina, L.Gorton et.al. Anal. Chem. 68 (1996) 4335. Reproducibility Mixing enzymes with water- insoluble polymers Uniform membranes Deposition from real polymer solution Exposing enzymes to organic solvents

12. Combination of transducer and immobilization protocol – advanced biosensors glucose glutamate lactate A.A. Karyakin, E.E. Karyakina, et. al. Analytical Chemistry 68 (1996) 4335. A.A. Karyakin, E.E.Karyakina, L.Gorton. Analytical Chemistry 72 (2000) 1720 A.A. Karyakin, E.A. Kotel'nikova et. al. Analytical Chemistry 74 (2002) 1597. E.I. Yashina, A.V. Borisova,..., A.A. Karyakin. Analytical Chemistry 82 (2010) 1601 Sensitivity compared to Biosen C_Line 100 times improved Stability: > 500 detections without calibration 12

13. Lactate in blood: correlation with the standard method r > 0.99 27 sportsmens before and after exercise stress

14. 14 Non-invasive diagnostics Correlation in metabolite concentration with blood is required Suitable excretory liquids:  Exhaled breath condensate;  Sweat;  Tears.

15. 15 Non-invasive diagnostics of oxidative stress: condensate of exhaled breath

16. 16 Human stress control upon physical training Lactate detection in sweatLactate detection in sweat Member of national junior skiing team Universal sweat collector before training after training