Fabrication of immunosensor microwell arrays from gold compact discs for detection of cancer biomarker proteins

Abstract

A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm(2) with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL(-1) (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL(-1). These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.

Fig. 1

Fitting the 8-electrode immunoarray into the microfluidic device. The array is sandwiched between two layers of PMMA and one layer of PDMS acting as microfluidic channel above the sensor electrodes. The red arrows indicate the flow of buffer.

Fig. 2

(a) Computer generated design of the gold CD-R array showing microwells around electrodes. (b) Completed array of 8 electrodes with individual microwells containing 1 μL aqueous droplets.

Fig. 3

Tapping mode AFM images of (a) exposed bare gold CD-R surface, (b) anti-IL-6 capture antibody attached to the gold CD-R surface. (c) Same as (b) at 10-fold higher resolution showing globular structures characteristic of attached antibodies.

Fig. 4

Array reproducibility: (a) CVs at 50 mV s ⁻¹ on 8-electrodes of a gold CD-R array in 5 mM ([Ru(NH₃)₆]-Cl₃) in aqueous 0.1 M sodium trifluoroacetate (NaTFA). (b) Amperometric response of the 8-electrode array in the microfluidic device to injection of 100 μL of 100 μM ([Ru (NH₃)₆]-Cl₃) at 100 μL min ⁻¹.

Fig. 5

Amperometric data for immunoarray chips after exposure to diluted serum containing IL-6 and HRP-labeled secondary antibody: (a) responses for IL-6 in the microfluidic device at 0.3 V developed by injecting 100 μL 1 mM hydroquinone +100 μM H₂O₂. (b) Calibration plot for IL-6 standards in diluted serum after the subtraction of control (n = 6).

Scheme 1

Steps in the fabrication of gold arrays from commercial gold CD-Rs. (a) Remove the protective layer of the CD-R to expose the gold film; (b) print the array pattern on wax paper using a laserjet printer and transfer onto the gold surface by applying heat and pressure, contacts are the squares at the bottom of each chip, electrodes are the circles in the middle of chip; (c) use a Sharpie ® pen to cover contact pads and electrodes; (d) remove unprotected gold in etchant solution; (e) wash with ethanol/water to expose contact pads and electrodes; (f) transfer second layer of toner by heat to form wells.

Scheme 2

Schematic of an amplification strategy using streptavidin poly-HRP. The streptavidin poly-HRP is attached to the biotinylated anti-human IL-6 detection antibody.