Ultrasensitive Detection of Parathyroid Hormone through Fast Silver Deposition Induced by Enzymatic Nitroso Reduction and Redox Cycling Aman Bhatia, † Ponnusamy Nandhakumar, † Gyeongho Kim, † Jihyeon Kim, † Nam-Sihk Lee, ‡ Young Ho Yoon, ‡ and Haesik Yang* ,† † Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, Korea ‡ EONE Laboratories, Incheon 22014, Korea * S Supporting Information ABSTRACT: Enzymatically induced silver deposition and subsequent electrochemical oxidation have been widely used in electrochemical biosensors. However, this method is ineffective for producing highly enhanced silver deposition for use in ultrasensitive detection. Herein, we report a fast silver deposition method that simultaneously uses three signal amplification processes: (i) enzymatic amplification, (ii) chemical-chemical (CC) redox cycling, and (iii) chemical-enzymatic (CN) redox cycling. DT-diaphorase (DT-D) is used for enzymatic amplification to convert a nitroso compound, a species incapable of directly reducing Ag + to an amine compound, which can directly reduce Ag + . NADH acts as a reducing agent for the indirect reduction of Ag + via the two redox cycling processes. 4-Nitroso-1-naphthol is converted to 4-amino-1-naphthol (NH 2 -N) in the presence of DT-D. NH 2 -N initiates two redox cycling processes: NH 2 -N, along with Ag + and NADH, are involved in the CC redox cycling, whereas NH 2 -N, along with Ag + , DT-D, and NADH, are involved in the CN redox cycling. Finally, the deposited silver is electrochemically oxidized to produce a signal. When this triple signal amplification strategy for fast silver deposition is applied to an electrochemical immunosensor for detecting parathyroid hormone (PTH), a detection limit as low as ∼100 fg/ mL is obtained. The concentrations of PTH in clinical serum determined using the developed immunosensor are found to agree with those measured using a commercial instrument. Thus, the use of this strategy for fast silver deposition is highly promising for ultrasensitive electrochemical detection and biosensing applications. KEYWORDS: immunosensor, silver deposition, redox cycling, parathyroid hormone, DT-diaphorase I mmunoassays have become the most useful tools for the detection of biological analytes as the binding between an antibody and an antigen is very strong and specific, allowing for the high sensitivity and specificity of these tests. 1-3 The most recent studies have focused on the generation of high signal amplification in order to lower the detection limits. Enhanced signal amplification can be achieved by incorporat- ing a catalytic label possessing a greater activity, such that more substrate can be converted to signaling species during a given incubation period. 1-3 In sensitive electrochemical detection, electrochemically active signaling species are generated near an immunosensing electrode and then either oxidized or reduced at the same electrode. However, during the incubation period, soluble signaling species can diffuse away from the electrode where the catalytic reaction occurs. As a consequence, not all the generated signaling species are converted to an electrochemical signal. This restricts the usefulness of sensitive electrochemical detection. To circumvent this limitation, silver deposition methods have been employed, in which the signaling species are deposited on the immunosensing electrode just after their generation. 4-9 Therefore, most of the generated signaling species can be converted to an electrochemical signal, as the deposited Ag atoms are electrochemically oxidized (stripped) to Ag + over a short period of time. Two methods are commonly used for achieving silver deposition in immunoassays. In the first approach, silver is deposited site-selectively on metal labels such as gold nanoparticles, using the catalytic reduction of Ag + by means of a reducing agent originally present in the solution. 10-13 In this case, the deposition rate is high, as the original concentration of the reducing agent is also high. However, noncatalytic (nonspecific) silver deposition can cause high and irreproducible background levels, since the direct reduction of Ag + by the reducing agent is unavoidable, although it is slow. In the second method, silver is deposited non-site-selectively, using direct reduction of Ag + by means of an enzymatically generated reducing agent. 4-9 This silver deposition method is used to circumvent the previously mentioned diffusion limitation of electrochemical detection. Enzyme labels such as alkaline phosphatase (ALP) can produce many reducing agents during the incubation period. However, this silver Received: March 6, 2019 Accepted: May 30, 2019 Article pubs.acs.org/acssensors Cite This: ACS Sens. XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acssensors.9b00456 ACS Sens. XXXX, XXX, XXX-XXX Downloaded by PUSAN NATL UNIV at 17:56:48:010 on June 12, 2019 from https://pubs.acs.org/doi/10.1021/acssensors.9b00456.