Short communication Development of microfluidic-based assays to estimate the binding between osteocalcin (BGLAP) and fluorescent antibodies Hector Carmona a , Hector Valadez a , Yeoheung Yun b , Jagannathan Sankar b , Lissette Estala a , Frank A. Gomez a,n a Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA b Department of Bioengineering, North Carolina Agricultural & Technical State University, 1601 E. Market Street, Greensboro, NC 27411, United States article info Article history: Received 15 July 2014 Received in revised form 12 October 2014 Accepted 13 October 2014 Available online 22 October 2014 Keywords: Osteocalcin (BGLAP) Binding assay Fluorescent antibodies abstract Osteocalcin (bone gamma-carboxyglutamate protein; BGLAP) is a highly conserved molecule associated with mineralization of bone matrix. It regulates the dynamics of new bone formation and bone resorption. The synthesis of osteocalcin by osteoblasts is regulated by the active form of vitamin D. In this paper, we report the use of two fluorescent-based assays, one bead-based and a second involving an amino-silane surface pre-treatment, to obtain binding constants of 3.53  10 6 M 1 and 3.19  10 6 M 1 , respectively, for the binding of BGLAP to fluorescently labeled monoclonal-anti BGLAP-clone 2D5. These simple microfluidic techniques demonstrate the feasibility of developing microsphere-based and surface electrostatically-attached binding assays to study peptide–antibody interactions. Both techniques utilize sub-microliter volumes of material an important consideration when studying bone turnover markers (BTMs) like BGLAP. & 2014 Published by Elsevier B.V. 1. Introduction The bone is a metabolically dynamic tissue that undergoes continuous remodeling throughout its lifetime. Bone remodeling serves to adjust bone architecture to meet changing mechanical needs and it helps to repair micro-damages in bone matrix prevent- ing the accumulation of old bone [1]. Imbalances between osteoclast- mediated bone resorption and new bone formation causes metabolic bone disorders such as postmenopausal osteoporosis and osteope- trosis [2,3]. In osteoporosis, bone resorption outpaces bone formation resulting in ever porous bones. It is often treated by inhibition of bone resorption; however, bone formation is unfortunately also suppressed by this type of treatment illustrating the tightly coupled processes involved in bone remodeling. In osteopetrosis, the bones become denser due to an imbalance of bone formation over bone resorption [4]. The ability to detect and monitor the enzymes and proteins called bone turnover markers (BTMs), excreted biochemical products formed during bone metabolism would allow better under- standing and earlier diagnosis of bone remodeling imbalance [5–7]. Osteocalcin, also known as bone gamma-carboxyglutamate (gla) protein (BGLAP), is a BTM produced primarily by osteoblasts during bone formation [8]. Because it is the most abundant of the non- collagenous proteins in bone it is widely studied as information can be garnered regarding bone formation and resorption. While BGLAP can be measured by a number of immunoassays they are hampered by the minute quantities of the protein in serum, lack of experi- mental protocols, and degradation in serum limiting analytical approaches to its study [9,10]. There are a myriad of studies espousing the use of bead-based techniques to probe the chemistry of biological systems [11–14]. Specifically, microsphere-based analyses enable facile separation of bound complexes from unbound species without the use of complex techniques and instrumentation. The limits of detection (LOD) are naturally decreased since the use of microspheres increases the surface area-to-volume ratio of the medium allowing for sample concentration. The plethora of microspheres commer- cially available come in a variety of diameters and surface termini thereby allowing for a paucity of chemistries to be conducted and heretofore immobilization of a wide range of biological species. Poly(dimethylsiloxane) (PDMS) has for years been the material of choice in many microfluidic applications due to its elastomeric properties, ease of use and cost just to name a few reasons [15]. Unfortunately, unmodified PDMS is hydrophobic and is difficult to wet with aqueous solvents hindering many biochemical applica- tions. In the important area of cell adhesion research, the use of amino-silane and homobifunctional aldehyde reagents has allowed for the covalent modification of PDMS and subsequent immobiliza- tion of proteins, antibodies, and other biological species. In this paper we report the development of two fluorescence- based techniques to analyze the binding of BGLAP to fluorescently Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta http://dx.doi.org/10.1016/j.talanta.2014.10.025 0039-9140/& 2014 Published by Elsevier B.V. n Corresponding author. Tel.: þ1 323 343 2368; fax: þ1 323 343 6490. E-mail address: fgomez2@calstatela.edu (F.A. Gomez). Talanta 132 (2015) 676–679