RESEARCH ARTICLE Quenching of graphene quantum dots fluorescence by alkaline phosphatase activity in the presence of hydroquinone diphosphate Marta Maria Pereira da Silva Neves | María Begoña González‐García | Alejandro Pérez‐Junquera | David Hernández‐Santos | Pablo Fanjul‐Bolado DropSens S.L, Ed. CEEI, Parque Tecnológico de Asturias, Llanera, Asturias, Spain Correspondence Marta Maria Pereira da Silva Neves and Pablo Fanjul‐Bolado, DropSens S.L, Ed. CEEI, Parque Tecnológico de Asturias, 33428 Llanera, Asturias, Spain. Email: mmpereira@dropsens.com; pfanjul@dropsens.com Funding information Spanish Ministry of Economy and Competi- tiveness, Grant/Award Number: Torres Quevedo grant (PTQ‐13‐05994) of the National Program for the Promotion of Talent and its Employability Abstract In this work, a turn‐off photoluminescent sensing proof‐of‐concept based on blue luminescent graphene quantum dots (GQDs) as the fluorescent probe was developed. For that purpose, GQDs optical response was related with the catalytic enzymatic activity of alkaline phosphatase (ALP), in the presence of hydroquinone diphosphate (HQDP). The hydrolysis of HQDP by ALP generated hydroquinone (HQ). The oxidation of HQ, enzymatically produced, to p‐benzoquinone (BQ) resulted in the quenching of GQDs fluorescence (FL). Therefore, the developed luminescent sens- ing mechanism allowed the FL quenching with ALP activity to be related and thus quantified the concentration of ALP down to 0.5 nM of enzyme. This innovative design principle appears as a promising tool for the development of enzymatic sensors based on ALP labeling with fluorescent detection or even for direct ALP luminescent quantification in an easy, fast and sensitive manner. KEYWORDS alkaline phosphatase, fluorescence quenching, graphene quantum dots, hydroquinone diphosphate, portable instrumentation 1 | INTRODUCTION Alkaline phosphatase (ALP) is an enzyme with high turnover number and broad substrate specificity that has been extensively used as an enzymatic label for bio‐assays development. [1–4] ALP is also an enzyme found in the human body, being widely distributed in the liver, bone, intestine, kidney, and placental tissues, [5–8] which makes ALP an analyte of high importance assayed in routine clinical analysis. Furthermore, ALP is also relevant for food analysis since it is an enzyme normally present in raw milk and its inactivation is an indica- tor of proper milk pasteurization process. [9,10] Consequently, due to the relevance of ALP in clinical diagnosis, food safety and also in bio- science there is a growing interest in the development of techniques for ALP detection in a sensitive, fast and easy manner. The com- monly reported monitoring techniques for ALP are based on the colorimetric, [10] chemiluminescent, [11] fluorometric, [12] surface‐ enhanced Raman spectroscopy [13] or electrochemical detection [14– 16] of the products of ALP reaction with specific substrates. How- ever, most of them, present several disadvantages since they are expensive, time‐consuming, or require bulky instrumentation. Bearing in mind that the (bio)analytical chemistry advances continuously demand the development of simple procedures employing easy‐to‐ use, low‐cost and portable detection devices, the combination of nanotechnology progresses with the electronics miniaturization is moving towards the achievement of exciting alternatives to tradi- tional analytical methods. Quantum dots (QDs) had been widely reported as fluorescent labels for biosensing developments and bio‐imaging. [17–19] More recently, new emerging nanoparticles such as graphene quantum dots (GQDs) appears as promising fluorescent probes for the development of luminescent sensing assays. [19,20] GQDs are a kind of zero‐ dimensional (0D) material that exhibit extraordinary optical and electronic properties due to their quantum confinement and edge effects. [21,22] GQDs are characterized by their exceptional features Abbreviations used: ALP, alkaline phosphatase; BQ, p‐benzoquinone; BSA, bovine serum albumin; FL, fluorescence; GOx, glucose oxidase; GQDs, graphene quantum dots; HQ, hydroquinone; HQDP, hydroquinone diphosphate; QDs, quantum dots. Received: 24 August 2017 Revised: 16 November 2017 Accepted: 20 November 2017 DOI: 10.1002/bio.3445 Luminescence. 2018;1–7. Copyright © 2018 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bio 1