Method for fabrication and verification of conjugated nanoparticle-antibody tuning elements for multiplexed electrochemical biosensors Jeffrey T. La Belle a,b,⇑ , Aaron Fairchild a,b , Ugur K. Demirok b , Aman Verma a,b a Harrington Program of Biomedical Engineering, in the School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85287, USA b Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA article info Article history: Available online 26 April 2013 Keywords: Biosensor Health management Disease monitoring Electrochemical impedance spectroscopy abstract There is a critical need for more accurate, highly sensitive and specific assay for disease diagnosis and management. A novel, multiplexed, single sensor using rapid and label free electrochemical impedance spectroscopy tuning method has been developed. The key challenges while monitoring multiple targets is frequency overlap. Here we describe the methods to circumvent the overlap, tune by use of nanopar- ticle (NP) and discuss the various fabrication and characterization methods to develop this technique. First sensors were fabricated using printed circuit board (PCB) technology and nickel and gold layers were electrodeposited onto the PCB sensors. An off-chip conjugation of gold NP’s to molecular recognition ele- ments (with verification technique) is described as well. A standard covalent immobilization of the molecular recognition elements is also discussed with quality control techniques. Finally use and verifi- cation of sensitivity and specificity is also presented. By use of gold NP’s of various sizes, we have dem- onstrated the possibility and shown little loss of sensitivity and specificity in the molecular recognition of inflammatory markers as ‘‘model’’ targets for our tuning system. By selection of other sized NP’s or NP’s of various materials, the tuning effect can be further exploited. The novel platform technology developed could be utilized in critical care, clinical management and at home health and disease management. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction With the advent and interest in genomics [44], proteomics [48], metabolomics [1], vaccine development [4], and personalized med- icine [49], high throughput systems are of great desire [41]. How- ever, many of these technologies are time consuming, labor intensive, have issues with lower limits of detection or multiplex factor, or require the use of special reagents and sample prepara- tion or labels on the target(s) of interest. Typical modes of detec- tion are optical based [6] or electrochemical in nature [33]. Likewise due to the variety of techniques, platforms, and sen- sors there are many means to assemble these systems to do the high throughput multiplexed assays. One common method, uti- lized in many industries for mass fabrication is printed circuit board design [35]. Here, typically a fiberglass substrate is copper- clad, then coated with photoresist which protects the copper in subsequent steps. The pattern can be negatively or positively developed in the photoresist. The excess copper is removed leaving behind circuitry, leads and/or sensors in copper. Ideal substrate layers, such as gold or other metals can then be deposited on via an electrodeless process [28] or electrodeposited [29] onto the cop- per or other interface layers. The deposition of gold allows for immobilization of molecular recognition elements such as antibod- ies using covalent attachment methods [23]. Basically, a long car- bon chain alkanethiol can self-assemble via thiol linkage to the hydroxyl group on the gold surface. Through the use of zero- crosslength linkers such as sulfo-N-Hydroxysuccinimid (sulfo- NHS) and ethyl(dimethylaminopropyl) carbodiimide (EDC). Then the molecular recognition elements are added and the sensors are ready for testing. A common method of electrochemical testing begins with a cyc- lic voltammogram (CV) in order to determine the formal potential of the electrochemical cell or sensor. This determines the DC offset for the AC sweep used in electrochemical impedance spectroscopy (EIS) based techniques. Next, a common means to validate sensor performance is to run a concentration gradient or scale against the sensors to determine many typical parameters such as: lower limits of detection (LLD), upper limits of detection (ULD), from these one obtains a dynamic range. Other parameters include responsivity (slope of the gradient), reproducibility (standard devi- ation divided by the average in percent), tightness of fit (R-square of trend line applied and behavior (liner, non-linear, etc.). One un- ique parameter in EIS is optimal binding frequency, which can be determined across the frequency spectra of the gradient by looking 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.04.015 ⇑ Corresponding author. Address: 550 East Orange St., P.O. Box 85287-9709, Tempe, AZ 9709, USA. E-mail address: jeffrey.labelle@asu.edu (J.T. La Belle). Methods 61 (2013) 39–51 Contents lists available at SciVerse ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth