Nanosphere lithography-based platform for developing rapid and high sensitivity microarray systems Chandra K. Dixit a,b,⇑ , Ashish Kumar c , Ajeet Kaushik d a School of Biotechnology, National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland b Schools of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland c Molecular Diagnostics Division, Vishwakarma Biotech Pvt. Ltd., Haridwar, India d Bio-MEMS and Microsystem Lab, Department of Electrical and Computer Engineering, Florida International University, Miami, FL 33174, United States article info Article history: Received 23 May 2012 Available online 5 June 2012 Keywords: Nanosphere lithography NSL Microarray Human fetuin A High sensitivity One-step immobilization abstract A novel gold nanoarray (NA)-based platform was developed for microarray applications. This novel approach is based upon the principle of nanosphere lithography and can be used for one-step antibody immobilization. The developed platform was checked by functionalizing with cysteine followed by cap- turing biotinylated antibody and detecting it with dye-conjugated steptravidin. An immunoassay was performed with spiked samples containing human fetuin A antigen. The minimum limits of detection (LOD) of human fetuin A for NA-based and conventional microarray platforms were 50 pg/mL and 50 ng/mL, respectively. The developed approach was highly reproducible and unlike conventional micro- array approaches the use of a spotting system was omitted because immobilization was controlled and directed on the predefined arrays. This approach could be an ideal alternative for developing microarrays. And, the ease of the strategy also allows the high throughput production of the microarrays. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Microarray, with the advent of high density printing, has be- come a high-throughput and cost-effective bioanalytical technique allowing the fabrication and exploitation of DNA and protein microarrays [1–4]. Various protein microarray platforms have been developed for interactome analysis that includes screening of anti- bodies and antigens, novel proteins, protein-nucleic acid and pro- tein–protein interactions [5–10]. Immuno-proteomic analyses, that assess antibody-antigen interactions, are among the most established microarray applications [11–14]. These array-based as- says have the potential to impact the field of infectious disease diagnostics and greatly facilitate the design of subunit vaccines [11,13]. Additionally, the development of microarray applications for the functional characterization of proteins of unknown or un- proven function continues to be a major challenge [14–17]. Whereas, the array capacity for protein screening is limited to 1000–10,000, which is relatively low compared to DNA arrays [18–20]. Requirement of precision spotting and tendency of pro- teins to leach out from the polyion-grafted surface are two major drawbacks associated with the current microarray techniques. The generation and immobilization of pure protein reagents are critical to the quality of protein microarrays. However, the protein attachment to the surface, that may entail covalent immobilization or adsorption, involves surface preparation which universally is performed by generating an adsorbed polylysine matrix on the support of choice [21–23]. Protein leaching and non-specificity [24] are few of the major addressable technical difficulties associ- ated with planar microarray platforms. Despite the development of many strategies for protein capture there is an immense need to devise an efficient generic surface and a relatively easy modifica- tion procedure that can increase the reproducibility of the array performance and reduce the requirement of cumbersome and sophisticated instrumentation. In addition, such platform should also support the development of highly sensitive antibody-bound microarray platforms with a high specificity, irrespective of the nature of solid-support matrix employed. Microarrays were reportedly developed either by spin-coating [25] or drop-coating [26,27] for grafting polylysine/silane on to the solid supports. Additionally, many reports pertaining to the development of microarrays claim to use nitrocellulose mem- branes [28]. We report a method to develop a generic microarray platform by first generating a highly ordered network of gold is- lands with nanosphere lithography (NSL) [29] followed by func- tionalizing these gold nanoarrays (NA/s) using either cysteine or cysteine-conjugated streptavidin, which binds to the gold surface via thiol bonds such that either, amino and carboxyl groups remain free for further chemistries or streptavidin is available for biotin- 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.05.144 ⇑ Corresponding author at: School of Biotechnology, National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland. E-mail addresses: Chandra.dixit2@mail.dcu.ie (C.K. Dixit), genebio7@gmail.com (A. Kumar), ajeet.npl@gmail.com (A. Kaushik). Biochemical and Biophysical Research Communications 423 (2012) 473–477 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc