Author’s Accepted Manuscript A polyaniline based ultrasensitive potentiometric immunosensor for cardiac troponin complex detection Qi Zhang, Alok Prabhu, Avdar San, Jafar F. AlSharab, Kalle Levon www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(15)30085-3 http://dx.doi.org/10.1016/j.bios.2015.04.084 BIOS7652

To appear in: Biosensors and Bioelectronic Received date: 30 January 2015 Revised date: 15 April 2015 Accepted date: 25 April 2015 Cite this article as: Qi Zhang, Alok Prabhu, Avdar San, Jafar F. Al-Sharab and Kalle Levon, A polyaniline based ultrasensitive potentiometric immunosensor for cardiac troponin complex detection, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.04.084 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A polyaniline based ultrasensitive potentiometric immunosensor for cardiac troponin complex detection Qi Zhang a,*, Alok Prabhu a, Avdar San a, Jafar F. Al-Sharab b and Kalle Levon a a

Department of Chemical and Biomolecular Engineering, New York University, Six MetroTech Center, Brooklyn, NY, 11201, United States b

Department of Materials Science and Engineering, Rutgers University, 607 Taylor Road, Piscataway, NJ, 08854, United States

*Corresponding Author: Tel: 718-260-3664; Fax: 718-260-3136; E-mail address: [email protected] (Q.Zhang).

Abstract An ultrasensitive immunosensor based on potentiometric ELISA for the detection of a cardiac biomarker, troponin I-T-C (Tn I-T-C) complex, was developed. The sensor fabrication involves typical sandwich ELISA procedures, while the final signal readout was achieved using open circuit potentiometry (OCP). Glassy carbon (GC) working

electrodes

were

first

coated

with

emulsion-polymerized

polyaniline/dinonylnaphthalenesulfonic acid (PANI/DNNSA) and the coated surface was utilized as a transducer layer on which sandwich ELISA incubation steps were performed. An enzymatic reaction between o-phenylenediamine (OPD) and hydrogen peroxide (H2O2) was catalyzed by horseradish peroxidase (HRP) labeled on the

secondary antibodies. The polymer transducer charged state was mediated through electron (e-) and charge transfers between the transducer and charged species generated by the same enzymatic reaction. Such a change in the polymer transducer led to potential variations against an Ag/AgCl reference electrode as a function of Tn I-T-C complex concentration during incubations. The sequence of OPD and H2O2 additions, electrochemical properties of the PANI/DNNSA layer and non-specific binding prevention were all crucial factors for the assay performance. Under optimized conditions, the assay has a low limit of detection (LOD) (< 5 pg/mL or 56 fM), a wide dynamic range (> 6 orders of magnitude), high repeatability (coefficient of variance < 8% for all concentrations higher than 5 pg/mL) and a short detection time (< 10 min).

Keywords ELISA,

troponin

I-T-C,

potentiometry,

polyaniline,

ultrasensitive

immunosensor 1. Introduction Cardiovascular diseases (CVDs), such as acute myocardial infarction (AMI), are currently among the leading causes of human mortality (WHO, 2014). The development of rapid, sensitive and low-cost diagnostic approaches for such diseases is a crucial step towards therapeutic interventions (Yang, Z and Zhou, D. M., 2006). At elevated levels in the blood of AMI patients, cardiac biomarkers such as C-reactive protein (CRP), troponin I (cTnI), troponin T (cTnT), myoglobin and the MB isoenzyme of creatine kinase (CKMB) are major indicators in AMI diagnosis (Ellenius et al., 1997; Panteghini et al., 1999; Hasdai et al., 2003; Qureshi et al., 2012). Enzyme-linked immunosorbent assay (ELISA), based on specific antibodyantigen interactions, provides the possibility of quantifying a wide range of cardiac biomarkers in biological fluids at good sensitivity and selectivity (Engvall, E. and Perlman, P., 1971; Voller et al., 1978; Lequin R. M., 2005). A conventional ELISA relies on optical density readouts as chromogenic reactions are triggered by the enzymatic

reporters tethered to the secondary antibodies. More recent signal readout methods involve the use of fluorogenic (Levine, M. N. and Raines, R. T., 2011), electrochemiluminescent (Phillips, R. W and Abbott, D., 2008), and real-time PCR (Tahk et al., 2011) reporters. Electrochemical ELISAs, where the detections are realized by electrochemical readouts rather than optical measurements, have emerged as alternative assays. They exhibit immense potential for increased sensitivity, simplicity of instrumentation, rapidity of testing and low cost compared to optical ELISAs (Piermarinia et al., 2007; Wei et al., 2009; Ricci et al., 2012; Rusling, et al., 2012). Additionally, miniaturization, microfluidics and multiplexed detection can easily be integrated in such systems (Rusling, et al., 2013; Lin et al., 2010; Kirby et al., 2004). Electrochemical ELISAs that leverage amperometric, capacitive and potentiometric measurements were developed in the recent past (Akanda et al., 2011; Bhimji et al., 2013; Purvis et al., 2003; Labib et al., 2009; Tang et al., 2004; Yuan et al., 2004). Potentiometric ELISA is the most straightforward method among those, as no external excitement such as current and voltage is required during the measurement. However, in label-free potentiometric immunosensors, the formation of electrical double layer (EDL) may occlude signals derived from biological bindings and lead to lower sensitivity (Tang et al., 2004; Yuan et al., 2004). Such problems can be largely circumvented if the signal results from redox reaction occurring in close proximity to the electrode surface. Polypyrrole-based potentiometric ELISA exploiting this concept were previously reported (Purvis et al., 2003; Laczka et al., 2013). Sandwich ELISA was first performed on the polymer transducer layer to form HRP labeled immune-complexes. With the oxidation state change of polypyrrole layer upon the transfer of electrons and charged species facilitated by HRP catalyzed reaction, the resultant surface potential variation was amplified and used to quantify the biomarkers at low detection limits. Nonetheless, the understanding of this technique is still preliminary and more research is required for it to advance towards clinical applications. We studied this technique in greater depth in this contribution. A novel polyaniline (PANI) based potentiometric ELISA for the detection of troponin I-T-C (Tn I-T-C), a complex of the three distinct single-chain troponin subunits, is presented. Since

the stability of cTnI, the most commonly used cardiac biomarker, was proved to be significantly higher in such ternary complexes, Tn I-T-C samples are considered as ideal candidates for AMI diagnosis (Cui et al., 2012). PANI, a conductive polymer widely used in immunoassays (Cui et al., 2012; Laczka et al., 2013; Filatov et al., 1999), was selected as the transducer layer because of its exceptional pH and redox sensitivity, environmental stability and versatility in bio-immobilization compared to other conjugated conductive polymers (Dhand et al., 2011). The assay was accomplished with a conventional two-electrode open circuit potentiometry (OCP) system, suitable for multiplexing. Drop-casted PANI/DNNSA layer was successfully used as a transducer, which broadens the range of immunosensor applications on different types of substrates. We also observed that the optimization of transducer electroactivity contributed to an increase in sensitivity by multiple orders of magnitude. The blocking step was confirmed to be effective on the transducer layer as the potential response due to non-specific bindings in the blank test was brought down to ca. 2mV in 1% BSA/PBS environment and 13mV in neat human serum environment, resulting in high signal to noise (S/N) ratios. To avoid compromised assay sensitivity incurred by the direct reaction between PANI/DNNSA and OPD, a novel two-step sequential addition of OPD and H2O2 was proposed to amplify the signal from HRP catalyzed enzymatic reaction. The optimized assay was demonstrated to be ultrasensitive, rapid, highly repeatable and wide in dynamic range. A much more comprehensive understanding of the underlying potentiometric ELISA achieved in this paper could also expand its applications in ion-modulated biosensing systems such as cutting-edge ion sensitive field effect transistor sensor arrays (Shen et al., 2003; Barbaro et al., 2006; Zhang et al., 2015). 2. Materials and methods 2.1 Materials Hydrogen peroxide (H2O2), citric acid, sodium phosphate dibasic (Na2HPO4), glutaraldehyde (GA), o-phenylenediamine (OPD) tablets, PBST tablets (0.05% w/v Tween) and horseradish peroxidase (HRP) were all purchased from Sigma Aldrich. Ethanol

(EtOH)

was

purchased

from

VWR

International.

Polyaniline/dinonylnaphthalenesulfonic acid (PANI/DNNSA) mixture was obtained from Crosslink Inc. This mixture with high solubility in a variety of organic solvents was synthesized through emulsion polymerization (Kinlen et al., 1998). Phosphate buffered saline (PBS) solution (Sigma Aldrich) used in this study (pH 7.4) contains 10mM phosphate, 2.7mM KCl and 137mM NaCl. Citric phosphate (CP) buffer (pH 5.0) was prepared by mixing 0.2M of 25.7mL Na2HPO4 and 0.1M of 24.3mL citric acid aqueous solutions. ELISA grade bovine serum albumin (BSA) (Sigma Aldrich), casein blocking buffer (1% w/v) (Thermo Scientific), skim milk powder (BD Difco) and superblock buffer (Pierce) were used as blocking reagents. Cardiac troponin I (cTnI) free human serum, monoclonal mouse anti-cardiac troponin I (cTnI, MAb 810), human cardiac Tn IT-C complex (apparent Mw of 39, 29 and 18 kDa for cTnT, cTnI and TnC) and two types of HRP conjugated monoclonal mouse anti-cardiac troponin I (MAb MF4 and MAb 19C7) were all purchased from HyTest Ltd. 2.2 Polymer coating on working electrodes Glassy carbon (GC) electrodes with 3 mm disc dia. (BASi) were polished with 1 μm and 0.3 μm Al2O3 powders followed by cyclic voltammetry (CV) treatment in 0.5 M H2SO4 solution to remove any residues and contaminants on electrode surface. The electrodes were then sonicated in Milli-Q deionized water (18 ΩM) and dried with a stream of nitrogen. The freshly prepared GC electrodes were each coated with 8μL of 1.5 wt% PANI/DNNSA mixture in CHCl3 and dried in an oven at 60°C for 2 hours. 2.3 Ethanol treatment Ethanol (EtOH) treatment of the PANI/DNNSA layers was used to adjust its DNNSA/PANI doping ratio for better electroactivity. The PANI/DNNSA coated GC electrodes were treated by dipping them into 100% EtOH for 60 seconds followed by thorough DI-water rinse and nitrogen drying. 2.4 Energy-dispersive X-ray Spectroscopy (EDS) Quantitative chemical analyses were conducted using Oxford INCA PentaFETx3 EDS system (Model 8100) attached to a field emission scanning electron microscopy (FE-SEM). The EDS system provides fully digital image collection, transfer and

analysis. Standard calibration was conducted on the EDS system prior spectral collection for accurate quantitative analysis with an estimated error of 6 orders of magnitude). The assay with samples spiked in human serum exhibited a higher LOD, by an increase of roughly 1 order of magnitude. The unwanted background and loss in signal might be attributed to unrelated proteins and other biomolecules in human serum as discussed. Though approaching the detection limit, the assay using human serum still had ca. 2mV of potential increase over the baseline at 5 pg/mL Tn I-T-C level.

The sensitivity and repeatability of our assay exhibited great promise compared to both preclinical and clinical cardiac troponin detection assays previously reported (Fathil et al., 2015). The low LOD is especially important when cTnI as low as 0.01 ng/mL can still be related to heart failure, despite the clinical threshold of 100 ng/mL for large myocardial infarction (Agewall et al., 2011). Due to the elimination of excessive dopants in PANI/DNNSA, 2-3 orders of magnitude increase in sensitivity were achieved through EtOH treatment [Fig. 6(d)]. Without the treatment, the electroactivity of PANI/DNNSA layer was largely compromised and the transducer layer became more inert towards the enzyme reaction [Fig. 6(c)]. The understanding of such a phenomenon is crucial for potentiometric immunosensors when conductive polymers are integrated components.

4. Conclusion In this paper, we developed a novel PANI based immunosensor using the concept of potentiometric ELISA, for the detection of cardiac troponin complex. This assay is very promising compared to existing methods in terms of sensitivity, repeatability, detection range and rapidity. We also demonstrated the necessity of sequential addition for chromogenic compounds in the ELISA detection step for the best sensitivity. The electrochemical properties of PANI/DNNSA transducer layer and non-specific binding prevention approaches were also optimized to improve the assay performance. Our future study will focus on assay time optimization, assay automation and multiplexing as well as other potential applications of the assay. Due to the simplicity, efficiency and ion-modulating nature of the assay, we also look forward to implement it in cutting-edge sensing platforms such as ion sensitive floating gate field effect transistor (ISFGFET) sensing arrays.

Acknowledgement The authors wish to thank Professor Tung-Tien Sun at NYU Langone medical center for useful discussions and comments.

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An ultrasensitive immunosensor based on potentiometric ELISA was developed to detect troponin I-T-C. Emulsion polymerized polyaniline/DNNSA was employed as the transducer. The optimized sensor is ultrasensitive, rapid, highly repeatable and wide in dynamic range. The sensing platform is flexible and can be applicable to a wide spectrum of analytes.