These promising sensors are easily fabricated at relatively low cost, and could be mass-produced with commercial inkjetters

These promising sensors are easily fabricated at relatively low cost, and could be mass-produced with commercial inkjetters. in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection 360A limit of 20 pg mL?1 in calf serum, sensitivity of 11.4 nA pg?1 cm?2, and a linear dynamic range of 20C400 Ly6a pg mL?1. 1. Introduction Electrochemical detection combined with nanoparticle amplification offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins that have yet to be fully realized. Amperometric sensors, field effect transistors, and impedance methods are among the approaches being explored.1C5 The sensitivity of an electrochemical sensor can be improved by using nanostructured electrodes, such as those based on carbon nanotubes6,7 or gold nanoparticles.8,9 In the electrochemical detection of proteins on these high specific area electrode surfaces by immunoassay protocols, appropriate functional groups on the nanoparticle facilitate high concentrations of chemically linked capture antibodies.10 In this approach, antibodies on the electrode capture analyte proteins from the sample, then the 360A surface can be treated with an enzyme-labeled secondary antibody, and the enzyme label is detected electrochemically.5 It is important to detect multiple proteins for accurate medical diagnostic predictions.5 Electrochemical detection formats can measure multiple proteins on a multi-electrode microelectronic chip11C15 and can be coupled with bioconjugated enzyme-antibody particles with large numbers of enzyme labels for further amplification as required for the target protein.9 Microelectronic arrays for this purpose should have 360A high surface area to enhance sensitivity, be easy to produce, and be inexpensive enough for single use application to avoid contamination and regeneration of the sensing surface. Electrochemical protein arrays have been developed by Wilson and Nie for up to seven tumor biomarkers using chips based on the porous iridium oxide electrode.11,12 Microelectronic arrays for detecting salivary biomarkers such as IL-8 and thioredoxin have been developed by Wong high temperature, laser pulsing, or microwave irradiation.16,21C24 Protein biomarkers are up-regulated and down-regulated in blood serum due to disease. 25 Determination of concentrations of these proteins in serum is an emerging tool for detecting and monitoring cancers.5,26,27 Reliance on the concentration of a single biomarker such as prostate specific antigen (PSA) can result in significant numbers of false positives and false negatives.28,29 However, measurement of panels of protein biomarkers increases the statistical accuracy of prediction and can overcome problems associated with single biomarkers.5 Interleukin-6 (IL-6) is a promising early indicator of several serious medical conditions.30 Elevated IL-6 serum levels are associated with development and/or progression of breast, cervical, oral, and colorectal cancers.31C34 IL-6 has also been reported as 360A an early marker for inflammation and post-operative infections.35,36 Additionally, recent studies suggest that traumatic brain injury patients with elevated serum concentrations of IL-6 are more likely to develop life-threatening symptoms.37 Concentrations of IL-6 in healthy adults are less than 6 pg mL?1, but elevate to above 80 pg mL?1 in patients with cancer or abnormal inflammation.31C34 The low concentrations of IL-6 in serum present a challenging target for immunoassays, and we recently reported nanostructured single-electrode immunosensors featuring carbon nanotube forests or 5 nm AuNP layers for detection of IL-6 in the pg mL?1 range and below.38,39 Immunosensing of IL-6 was chosen in the present work as our proof-of-concept application target. In this paper, we report the first direct inkjet fabrication and characterization of electrochemical arrays using 4 nm gold nanoparticles and poly(amic acid) inks on heat-resistant Kapton plastic, and subsequent application to an immunosensor array demonstrated by the detection of IL-6 in serum in the pg mL?1 range. 2. Experimental 2.1 Chemicals and materials A Kapton FPC film 127 m thick was purchased from American Durafilm. These large polymer sheets were washed with water and ethanol prior to use. Lyophilized 99% bovine serum albumin (BSA), sterile-filtered bovine calf serum, gold(III) chloride trihydrate, 1-dodecanethiol, tetraoctylammonium bromide, sodium borohydride, a common saturated calomel reference (SCE) electrode, and cells employed a common platinum auxiliary electrode. A Dimatix Materials Printer (ModelDMP-2800, FUJIFILM Dimatix, Inc. Santa Clara, CA) was used for inkjet printing. Dimatix 10 pL, liquid crystal polymer printer cartridges were used for all printed inks (Model DMCLCP-11610). Printing patterns were made utilizing the Dimatix materials printer software. All printing patterns were developed using Microsoft Paint (Microsoft Inc. Redmond, WA) and imported using the Dimatix Materials Printer with Dimatix Printer Controller software..