Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc Direct writing of biocatalytic materials based on pens flled with high-tech enzymatic inks: “Do-it-Yourself” Soodabeh Hassanpour a , Arezoo Saadati a,1 , Mohammad Hasanzadeh b, ⁎ , Nasrin Shadjou c,d , Arezo Mirzaie a , Abolghasem Jouyban a a Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51664, Iran c Department of Nanochemistry, Nanotechnology Research Center, Urmia University, Urmia 57154, Iran d Department of Nano Technology, Faculty of Science, Urmia University, Urmia 57154, Iran ARTICLE INFO Keywords: Biocatalytic materials Nano-ink Advanced nanomaterial Biocompatible agents Enzymatic inks ABSTRACT An enzyme conductive ink was synthesized and applied for the sensitive detection of L-proline in human bio- fuids. The nano-ink is a mixture of materials graphite (Gr), intermediates Methylene blue (MB), binding agents (Polyethylene glycol and chitosan), and Proline dehydrogenase (PRODH). The morphology of enzymatic ink composition was evaluated by using high-resolution feld emission scanning electron microscope (FE-SEM). The results showed that the particle size of the proposed ink was below 100 nm. For electrochemical testing, the ink was fxed on the surface of copper electrode through utilization drop-casting technique via strong bonding between PEG and surface. The modifed electrode was used as an electrochemical biosensor for the determi- nation of L-proline in the alkaline solution. In optimal conditions, the concentration range and low limit of quantifcation (LLOQ) were 56 μM–1 mM and 56 μM, respectively. 1. Introduction Enzymes are broadly employed in sensing systems due to their high activity and specifcity [1–8]. Also, enzymes applied in bio-ink are se- lected based on the type of target analyte (e.g., proline). In enzyme- based electrochemical sensors, the electrochemical reaction of the en- zyme is the basis of the analyte determination. In the enzyme-based electrochemical sensor the existed enzyme in conductive ink is efec- tively immobilized on a substrate. In an efcient immobilization, while enzymes fxed on the substrate, they become capable of reacting with the target analyte and transferring electrons to conducting materials through the intermediary [1–8]. An enzyme-ink for use in electrochemical sensors should contain conductive materials, enzymes, intermediates and binding agents. Diferent varieties of conductive materials can be utilized in these inks, such as: carbon black, platinum carbon, gold particles, ruthenium particles, cerium particles, platinum/palladium alloy particles, pla- tinum particles, palladium and graphite [1]. Graphite as a conductive material is used in the construction of inks and coatings for a variety of electronic applications such as super ca- pacitors [2,3] and batteries [4], electrochemical sensors [5] and solar energy reaping [6]. Some advantages of graphite over other conductive materials include low cost, lack of insulation oxide layer, printing capability on temperature delicate substrates, non-toxicity, easy dis- persion in solution, fexibility, robustness, resistance to wrinkles and compatibility with all printing processes [7]. The intermediary in the ink should have the ability to connect to a substrate in an enzyme-based electrochemical sensor, while attached to the substrate, it can transfer electrons to the conductive material as a result of reaction with an enzyme. The binding agents used in enzy- matic inks include ethylene glycol, alcohol, methyl carbitol, vinyl monomers, etc.[1]. The conductive layer resulting from the coalescent dispersion of conductive materials, enzymes, intermediates and connectors increases the electron transfer between the conductive and intermediate material in comparison with the electron transfer between the layer of discrete conductive materials (such as common electrodes) and a separate in- termediate layer [1,9]. Drop-casting is a deposition method for making little-area flms, one of the advantages of this is simple and inexpensive [10]. Although the use of drop-casting is limited to flms and coatings with a little-area, this method relies on the release of big droplets with controlled size and https://doi.org/10.1016/j.microc.2018.10.050 Received 4 September 2018; Received in revised form 23 October 2018; Accepted 23 October 2018 ⁎ Corresponding author. 1 Co-frst author. E-mail addresses: hasanzadehm@tbzmed.ac.ir, mhmmd_hasanzadeh@yahoo.com (M. Hasanzadeh). Microchemical Journal 145 (2019) 266–272 Available online 25 October 2018 0026-265X/ © 2018 Published by Elsevier B.V. T