M ICROARRAYS Microarrays contain minute amounts of materials (DNA, proteins, aptamers, etc.)

4.3.2 M ICROARRAYS Microarrays contain minute amounts of materials (DNA, proteins, aptamers, etc.)

that are placed onto a matrix in an array format. The matrix is a solid support onto which a biological or organic material is placed. The solid support material can be plastic, glass, complimentary metal oxide semiconductor (CMOS), gold, platinum, membranes, or other substance on which the reagents can be attached and still maintain function. The method of attachment can be covalent, hydrophobic, or through some tight-affinity reagent, such as a biotin=streptavidin couple [80].

A microarray can be defined in terms of the number of spots (or electrodes) per chip=slide. By this definition, a low-density array may contain as little as 16 spots or as many as 96 spots. High-density arrays may have >500,000 spots. Lower density arrays are considered to be sensors, as microsensor detection is typically at the lower end of array density. Based on these classifications, there are several companies that produce lower density microsensor arrays (Antara Biosciences, and Osmetech Inter- national, among others).

There are numerous methods used for array production. Arrays may result from ‘‘spotting’’ onto activated surfaces using robots to produce high-density arrays.

Proteins or DNA are spotted onto activated surfaces (aldehydes, amines, etc.) so that either a chemical bond is formed or proteins can adhere through hydrophobic interaction. Another means of producing arrays is by photolithography using masks or lasers. This method has been used to produce in situ DNA- or peptide-based

arrays. In this specific case, a photolabile group is used on the 5 0 -nucleotide end or photolabile groups are used as amino protection groups (peptides). The use of lasers

or masks removes the labile group from a specific electrode or spot, promoting peptide bond or oligonucleotide bond formation. Conversely, this can also be accomplished using acid that is generated at a specific electrode. DNA and peptides can also be synthesized in this manner. The protecting groups are removed only at specific electrodes that generate acid resulting in an elongated nucleotide or peptide. The oligomers or peptides can be used as aptamers to capture specific molecules, such as pesticides, heavy metals, or other environmental contaminants. The method can also be extended to any synthesis procedure, providing an acid- or base-labile group is present. Products from Antara Biosciences and Osmetech traditionally use cyclic voltammetry (CV). In this mode, a redox active species is used in conjunction with the assay. In arrays sold by CombiMatrix, the electrochemical amplification is enzyme-based and relies on a charge build up at a capacitor near that electrode. The capacitor is discharged and the quantity of charge is converted to nanoamps. As the current is determined by the charge buildup over time, this is an indirect measure- ment for the current developed.

In the early developmental stages of either a microarray or a large sensor technique, the starting point is typically one or two electrodes. Much of the recorded electrochemical sensor data are based on just a few electrodes, as a particular technique may or may not be converted to a microarray. The decision to convert to a high-density array is dependent on many parameters such as reading times and hardwire issues. Detection methods in microarrays employ various techniques including fluorescence, luminescence, visible, electrochemical, Raman scattering,

112 Analysis of Pesticides in Food and Environmental Samples SPR, and electrochemiluminescence, among others. The detection method used

depends on the matrix and if the chip is hardwired. Typically, the light-based method can accommodate almost any matrix and production method. However, a laser scanner or CCD camera is required, which tends to be very expensive increasing start-up costs (which may exceed $50K). Electrochemical methods require chip hardwire in tandem with various detection methods. Amperometric detection, cyclic voltammetry, and the evaluation of a charge build up on the electrode surface have all been employed.