Norland optical adhesive 81 noa 81

Preparation of the striated substrate is based on soft lithography and micromoulding in capillaries [22 ]. Briefly, a PDMS (polydimethylsiloxane) stamp was prepared by pouring dimethylsiloxane plus catalyst (Sylgard, Dow Corning Corp, Midland, MI) onto an SU-8-silicon master grid (gift from Prof. Superfine, University of North Carolina, Chapel Hill) in a Petri dish. The polymer was cured at 70°C for 1 h. The PDMS stamp was removed from the master and pressed into a 10 µl drop of Norland Optical Adhesive-81 (NOA-81, Norland Products, Cranbury, NJ) on top of a 60 mm × 24 mm, #1.5 microscope cover slide (Thomas Scientific, Swedesboro, NJ). The NOA-81 was cured for 70 s with UV light (365 nm setting, UVP 3UV transilluminator, Upland, CA) and the stamp was removed. The substrate pattern had 6.5 µm wide ridges separated by 13.5 µm wide and 6 µm deep channels.

Silicon wafers (4 in.) were purchased from Nova Electronic Materials (Richardson, TX, USA). Photolithography masks were printed on mylar films at 10,160 dpi by Fine Line Imaging (Colorado Springs, Colorado, USA). SU8-3035 photoresist was obtained from MicroChem (Newton, MA, USA). Sylgard 184 PDMS kits were manufactured by Dow Corning (Midland, MI, USA) and 10:1 weight ratio (pre-polymer to curing agent) was used in this work. Norland optical adhesive 81 (NOA81) was ordered from Norland Products (Cranbury, NJ, USA). 3-Aminopropyltriethoxysilane (APTES, 97%) and 2-hydroxyethyl methacrylate (HEMA) were received from Sigma–Aldrich.
Agarose beads were prepared by emulsification of 2% type I-B agarose purchased from Sigma. Briefly, 1 g of agarose was stirred and dissolved in 50 mL of nano-pure water at 60°C. A suspending solution of 10 mL of Span85 (Sigma) and 90 mL hexanes was heated to 60° C and stirred at 900 rpm. The agarose solution was poured into the suspending solution followed by stirring at 900 rpm at 59° C for 1 min. The stirring was then adjusted to 600 rpm with heat off to allow the agarose to gel to 25°C. Beads were collected, washed with 50/50 mixture of ethanol/water and sorted using a sieve that screens out beads of 250–280 μm in diameter, which were then glyoxylated to transform the hydroxyl groups to aldehyde groups.
Rabbit anti-human CRP antibody used both as capture and detection antibody, was purchased from Accurate Chemical Corp (Westbury, NY, USA). AlexaFluor^®488 protein labeling kit from Invitrogen was conjugated to the detection antibody following instructions from the manufacturer. CRP antigen was from Fitzgerald (Acton, MA, USA), and the Helicobacter pylori antibody for the control beads, was purchased from Meridian Life Science (Memphis, TN, USA). Prior to the assay, CRP antigen was diluted with phosphate buffered saline (PBS) blocking buffer containing 1% bovine serum albumin (BSA). Secondary detection antibody was diluted with PBS in 0.4% (v/v). A 500 μL volume of homemade glyoxylated 2% agarose beads were coupled to 9 mg/mL polyclonal rabbit anti human CRP antibody in a 1.5 mL solution overnight and blocked with tris solution for 1 h prior to final wash. Negative control beads were prepared similarly, by incubating 2% agarose beads with a polyclonal antibody irrelevant to the CRP target and specific to H. pylori.