Analytical Biochemistry 413 (2011) 157–163 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Detection of inducible nitric oxide synthase using a suite of electrochemical, fluorescence, and surface plasmon resonance biosensors ⇑ Naumih M. Noah, Saamia Alam, Omowunmi A. Sadik Center for Advanced Sensors and Environmental Systems (CASE), Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA article info abstract Article history: A suite of biosensors for rapid detection of inducible nitric oxide synthase (iNOS) is described. First, a Received 17 December 2010 metal-enhanced electrochemical detection (MED) sensor, which relied on the redox properties of a silver Received in revised form 4 February 2011 monolayer, was developed. The linear detection range was between 8.64 Â 10À2 and 5.4 Â 101 ng/ml Accepted 7 February 2011 with a detection limit of 1.69 Â 10À4 ng/ml. This method was compared with surface plasmon resonance Available online 1 April 2011 (SPR) biosensors in which polyclonal mouse anti-iNOS was covalently immobilized onto a gold surface using an iNOS antigen. The linear detection range recorded was between 3.37 Â 101 and 5.4 Â 10À2 ng/ Keywords: ml with a detection limit of 2 Â 10À3 ng/ml. Finally, an ultrasensitive portable capillary (UPAC) fluores- Inducible nitric oxide synthase cence immunosensor, in which a mouse anti-iNOS antibody was covalently immobilized onto the inner Metal-enhanced detection Surface plasmon resonance surface of a capillary and a rabbit anti-iNOS antibody was employed as the secondary antibody, was Ultrasensitive portable capillary sensor developed. The resulting signals were found to be directly proportional to iNOS concentrations between 1 2 3 Pain biomarkers 1.52 Â 10À and 1.52 Â 10À ng/ml with a detection limit of 1.05 Â 10À ng/ml. These immunosensors exhibit low cross-reactivity toward potential interferents such as human serum albumin and ovalbumin. The SPR and UPAC biosensors were validated using simulated blood spiked with recombinant iNOS, resulting in recoveries of 85% and 88.5%, respectively. The research presented in this article could poten- tially provide new ways of detecting NO for diagnostic and biomarker purposes in medical research. Ó 2011 Elsevier Inc. All rights reserved. Nitric oxide (NO)1 is a highly reactive free radical and an impor- other hand, overproduction of NO often leads to its role as a major tant intra- and intercellular signaling molecule used for the regula- cytotoxic mediator in pathological processes, especially in inflamma- tion of diverse physiological and pathophysiological mechanisms tory disorders. Excess NO (>10 lM) interacts with oxygen radicals to such as cardiovascular, nervous, and immunological systems [1,2]. form highly reactive peroxynitrite, which in turn induces inflamma- Also, NO has contrasting roles in living organisms; it acts as a biolog- tory cellular cytokines and hence cytokine-induced cell death via ical mediator similar to neurotransmitters regulating the blood ves- apoptosis and necrosis [2,3]. sels as well as an important host defense effector in the immune NO is synthesized in vivo during the conversion of L-arginine to system. At small concentrations (62 lM), it acts as an intra- or inter- citrulline by nitric oxide synthase (NOS). It occurs in different iso- cellular second messenger, usually via guanylate cyclase [2]. On the forms with different properties depending on the role of NO to be synthesized. For example, endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutive enzymes and are constantly ex- ⇑ pressed, even at rest in the blood vessels and the brain neurons Corresponding author. Fax: +1 607 777 447. [4]. In contrast, inducible NOS (iNOS) is expressed in macrophages E-mail address: [email protected] (O.A. Sadik). and other tissues in response to infection or inflammation [5].Itis 1 Abbreviations used: NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible NOS; UPAC, ultrasensitive portable capillary; MED, metal-enhanced electrochemical expressed after an exposure to diverse stimuli such as the detection; BSA, bovine serum albumin; NaH2PO4, sodium dihydrogen phosphate; inflammatory cytokines and lipopolysaccharides (LPSs) [6]. Once Na2HPO4, disodium hydrogen phosphate; Na2CO3, sodium carbonate; NaHCO3, expressed, iNOS generates significantly large and sustained sodium hydrogen carbonate; IgG, immunoglobulin G; Tris–HCl, Tris–hydrochloric amounts of NO in the blood, resulting in pathological effects acid; HSA, human serum albumin; DDAO, 9H-(1,3-dichloro-9,9-dimethylacridin- 2-one-7-yl); PNPP, paranitrophenyl phosphate; GMBS, c-maleimido butyryloxy suc- [6,7]. The production of iNOS is a nonspecific event because it cinimide ester; NaCl, sodium chloride; H2SO4, sulfuric acid; NaOH, sodium hydroxide; can occur in a variety of cell types. For example, an increased pro- DMSO, dimethyl sulfoxide; HCl, hydrochloric acid; EDC, 1-ethyl-3-(3-dimethyl duction of NO has been observed during inflammation and arthri- aminopropyl) carbodiimide; NHS, N-hydroxysuccinimide; MgCl2, magnesium chlo- tis; hence, iNOS can be considered as a pain biomarker. It has also ride; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; DEA, been found to overexpress cyclooxygenase-2 (COX-2), which is an- diethanolamine; ELISA, enzyme-linked immunosorbent assay; PBS-T, PBS containing 0.05% Tween 20; OD, optical density; Ab–Ag, antibody–antigen; SPR, surface plasmon other pain biomarker. These two major pain biomarkers have been resonance; UPD, underpotential deposition; SAM, self-assembled monolayer. found to be expressed in cancer cells [8–10]. 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.02.010 158 Detection of inducible nitric oxide synthase / N.M. Noah et al. / Anal. Biochem. 413 (2011) 157–163 There are numerous approaches in the literature for studying and 1.0 mM ethylenediaminetetraacetic acid (EDTA), and PBSB NO. These include chemiluminescence, paramagnetic resonance consisted of PBS and 1% BSA. All PBS buffer solutions were adjusted spectrometry, resonance imaging, and bioassays. We previously re- to pH 7.6 using concentrated phosphoric acid or concentrated ported on the development of an inexpensive amperometric sensor NaOH. Tris–HCl buffer solution was prepared using 6.3509 g of for the determination of the inhibitory effects of NO on xanthine Tris–HCl and 7.2199 g of Tris base and the pH adjusted to 8.0 using oxidase, horseradish peroxidase, polyphenol oxidase, and glucose HCl or 10 M NaOH. Carbonate buffer (pH 9.6) was prepared using oxidase [11–13]. Recently, Njagi et al. [14] reported an electro- 0.1 M Na2CO3 and 0.1 M NaHCO3 and the pH adjusted to 9.6 by chemical sensor for monitoring NO in brain slices. Several micro- titrating the Na2CO3 with NaHCO3 via mixing. PNPP solution was electrodes based on either direct or catalytic oxidation of NO prepared by dissolving two PNPP tablets in 0.5 M diethanolamine have also been developed [15]. Other NO sensors developed have (DEA) buffer (pH 9.8) containing MgCl2. used metalloporphyrins and platinized platinum to provide an electrocatalytic oxidation of NO [16–18]. However, these methods Methods have been found to have poor sensitivity and selectivity and also lack stability in simulated biological samples [14,15,17]. In addi- ELISA tion, NO is highly reactive with a very short half-life (5 s), thereby The enzyme-linked immunosorbent assay (ELISA) protocol used making it difficult to be continuously monitored in real time and was based on a method described elsewhere [19]. Briefly, flat- in vivo. bottomed polystyrene 96-well microplates were coated overnight In this work, we propose to monitor iNOS as a viable surrogate at 4 °C with 100 ll/well of 1 lg/ml mouse anti-iNOS polyclonal for the short-lived species, NO. The goal of this study was to devel- antibody (prepared in carbonate buffer, pH 9.6) as the capture op and assess three different biosensors for rapid detection and antibody. The plate was washed three times with PBS containing characterization of iNOS. A suite of advanced biosensors that will 0.05% Tween 20 (PBS-T, pH 7.6), and the same washing procedure enable rapid, portable, autonomous, and real-time sampling of was followed at each subsequent stage of the assay. The plates iNOS detection capabilities is envisioned. Toward this end, we ini- were incubated overnight at 4 °C with 200 ll of 1 mg/ml BSA pre- tiated the development of portable immunosensors, ultrasensitive pared in PBS (pH 7.6). The plates were washed before incubating portable capillary (UPAC) and metal-enhanced electrochemical them at 4 °C with 100 ll of different concentrations of iNOS in detection (MED), for on-demand diagnostics. Comparative analysis PBS–BSA buffer overnight at 4 °C. After washing, 100 llof1lg/ of these biosensors is reported here. ml rabbit anti-iNOS polyclonal antibody was applied to the wells (except the blank). Following incubation for 2 h at ambient tem- peratures, the plates were washed and 100 ll of goat anti-rabbit Materials and methods IgG–alkaline phosphatase in Tris buffer (pH 8.0) was added (excluding the blanks), followed by an additional 1 h of incubation Materials at room temperature. After final rinsing with Tris buffer (pH 8.0), 100 ll of 1 mg/ml PNPP solution prepared in 10% DEA buffer was iNOS and rabbit anti-iNOS polyclonal antibody were purchased finally added and the plates were incubated at room temperature from Cayman Chemical (Ann Arbor, MI, USA) and stored at À20 °C. for 30 min. Optical densities (ODs) of the solutions were then mea- Mouse anti-iNOS polyclonal antibody was purchased from Abcam sured at 405 nm using a Synergy HTRDR multidetection microplate (Cambridge, MA, USA). Bovine serum albumin (BSA) was reader. purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). The following reagents were purchased from Sigma–Aldrich (St.