Nano-structured arrays for multiplex analyses and Lab-on-a-Chip applications Chandra K. Dixit a,b , Ajeet Kaushik c,⇑ a National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland b Schools of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland c Bio-MEMS and Microsystem Lab, Department of Electrical Engineering, University of South Florida, 4202 E. Fowler Avenue, ENB 118, Tampa, FL 33620, United States article info Article history: Received 25 January 2012 Available online 11 February 2012 Keywords: Nanosphere lithography Microfluidics Lab-on-a-Chip Antibody ELISA Immunofluorescence abstract Nanospheres lithographic (NSL) method has been used to fabricate nano-structured arrays (NAs) of hex- agonally close-packed gold (Au) using polystyrene beads [PS, diameter 300 nm] as mask. The developed NA was incorporated with a customized and cheap microfluidics system to demonstrate its applicability as an alternative easy and efficient platform for multiplex analysis and Lab-on-a-Chip applications. The chip functionality was demonstrated with horseradish peroxidase (HRP) and anti-HRP antibody as model for recognition system. The enzyme-linked immunosorbent assay (ELISA) performed on fabricated pro- tein biochip had a detection limit 100 pg/mL for HRP. The antibody chip was also checked for the shelf-life and it was found that these chips could be stored for 50 days when stored at 4 °C without any significant loss of activity. Therefore, NAs based protein biochip with the correct microfluidics could find huge potential application in diagnostics and biosensing technology. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction An orderly arrangement of nanomaterials on the surface and their size controls the performance of the system depending on the application [1,2]. In this context, the fabrication of periodically arranged nano-structured arrays (NAs) of different materials is cru- cial and ability of generating desired functionality has been of con- siderable interest [1–4]. Nanosphere lithography (NSL), among the various techniques such as nano-imprinting lithography [5], hole mask colloidal lithography [6] and electron beam and focused- ion beam lithography [7], has been used to fabricate patterns with excellent size, shape, and spacing control. In addition, NSL is a ver- satile, high-throughput, and cost-effective technique. NAs of desired material could be fabricated with NSL technique, wherein primary step is an appropriate choice of deposition mask that governs the shape and order of the developed NAs. Polysty- rene (PS) bead-based deposition masks are commonly used for NAs development in NSL [1–3]. Therefore, a deposition mask can be developed by employing a coating of PS beads of appropriate size onto the desired solid support viz. glass and plastics. Further, spatially arranged patterns of the deposited material can be ob- tained by removing the polystyrene mask [1]. Depending on the nature of the deposited material these NAs may be employed for optical, electrical, magnetic and molecular properties, have got applications in sensors [8–10], optical and opto-electronic [3,11], photovoltaic devices [12] along with magnetic data storage mem- ory component designing [13,14]. In spite of mentioned significant applications, the application of NSL based NAs as effective electro- active surfaces for biosensor development is not yet well explored. The current generation of biosensor, there is considerable scope for fabricate nano-structured supported platform to achieve high sen- sitivity, ultra low detection limit, multiplexing capability, low cost, portability and easy operation [15,16]. Among these properties, multiplex application of protein detection is urgently required to detect ultra low concentration of proteins with high sensitivity in clinical diagnosis for human healthcare [17]. Some of these chal- lenges could be achieved using NSL based efficient highly elec- tro-active and sensitive platforms that can either be used as fabricated or in conjunction with microfluidics channel systems for the development of biosensors. In addition, due to the two dimensional electro-actively ordered array formation and ability to oriented immobilization of biomolecules (enzyme, DNA, anti- body, microbial cells), these may also be employed for the develop- ment of cheap alternative microarray-based immunosensor to perform multiplexed diagnostics along with high sensitivity. There are several diagnostics approaches such as, microtiter plate-based assays, and microarrays. These approaches have cer- tain associated drawbacks such as cumbersome antibody immobi- lization procedures and antibody functionality related issues. In this manuscript we are addressing the problems associated with the pre-existing approaches by employing NAs with a simple model of microfluidics system to demonstrate the use of NAs as an 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.018 ⇑ Corresponding author. E-mail addresses: Chandra.dixit2@mail.dcu.ie (C.K. Dixit), ajeet.npl@gmail.com (A. Kaushik). Biochemical and Biophysical Research Communications 419 (2012) 316–320 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc