Recent advances on developing 3rd generation enzyme electrode for biosensor applications Priyanki Das a , Madhuri Das a , Somasekhar R. Chinnadayyala b , Irom Manoj Singha a , Pranab Goswami b,n a Centre For Energy, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India b Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India article info Article history: Received 16 September 2015 Received in revised form 17 December 2015 Accepted 18 December 2015 Available online 19 December 2015 Keywords: Direct electrochemistry Enzyme electrode 3rd generation biosensors Protein film voltammetry Cyclic voltammetry Electron transfer rate constant abstract The electrochemical biosensor with enzyme as biorecognition element is traditionally pursued as an attractive research topic owing to their high commercial perspective in healthcare and environmental sectors. The research interest on the subject is sharply increased since the beginning of 21st century primarily, due to the concomitant increase in knowledge in the field of material science. The remarkable effects of many advance materials such as, conductive polymers and nanomaterials, were acknowledged in the developing efficient 3rd generation enzyme bioelectrodes which offer superior selectivity, sensi- tivity, reagent less detection, and label free fabrication of biosensors. The present review article compiles the major knowledge surfaced on the subject since its inception incorporating the key review and ex- perimental papers published during the last decade which extensively cover the development on the redox enzyme based 3rd generation electrochemical biosensors. The tenet involved in the function of these direct electrochemistry based enzyme electrodes, their characterizations and various strategies reported so far for their development such as, nanofabrication, polymer based and reconstitution ap- proaches are elucidated. In addition, the possible challenges and the future prospects in the development of efficient biosensors following this direct electrochemistry based principle are discussed. A comparative account on the design strategies and critical performance factors involved in the 3rd generation bio- sensors among some selected prominent works published on the subject during last decade have also been included in a tabular form for ready reference to the readers. & 2015 Elsevier B.V. All rights reserved. Contents 1. Introduction ........................................................................................................ 386 2. Principles on 3rd generation biosensor .................................................................................. 387 3. Characterization of the 3rd generation biosensor .......................................................................... 388 4. Fabrication strategies and applications of the 3rd generation biosensor ........................................................ 389 4.1. Nanofabrication of electrode ..................................................................................... 389 4.2. Polymer based approach ........................................................................................ 391 4.3. Self-assembled approach ........................................................................................ 391 4.4. Reconstitution based approach ................................................................................... 392 5. Problems and challenges for developing 3rd generation biosensors............................................................ 393 6. Conclusion and future perspectives for developing 3rd generation biosensors ................................................... 396 Acknowledgments ....................................................................................................... 396 References ............................................................................................................. 396 Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics http://dx.doi.org/10.1016/j.bios.2015.12.055 0956-5663/& 2015 Elsevier B.V. All rights reserved. n Corresponding author. Tel.: þ91 361 2582202; fax: þ91 361 2582249. E-mail address: pgoswami@iitg.ernet.in (P. Goswami). Biosensors and Bioelectronics 79 (2016) 386–397