Label-free detection of biomolecules on microarrays using surface–colloid interaction

Abstract

IntroductionMicroarrays are rapidly advancing the technology for analysis of expression of tens of thousands of genes in a single experiment by employing hybridization of target genes to probe molecules immobilized on solid surface [1][2][3]. DNA microarrays usually are small glass, plastic, silicon substrate, or nylon membrane onto which the probe molecules are tethered in the arrays of spots. Each array's spot carries probe molecules specific to one particular gene. The target molecules contained in a sample solution are captured on the array by hybridization to corresponding homologous probe molecules on the array surface. To quantify the number of captured targets on each array spot, the target molecules are usually modified (labeled) by chemical attachment of a fluorescent group [4,5]. The intensity of fluorescence detected from the array spot is the measure of gene expression, i.e., the measure of the abundance of the corresponding mRNA specie in sample solution [6]. Currently, the need for chemical modification of target molecules is a significant limitation of the microarray technology: (1) t chemical modification of targets introduces inaccuracy of quantification due to labeling biases, (2) the chemical labeling requires expensive labeling reagents, and finally (3) the labeling process is time consuming and is often accompanied by a significant loss of sample materials during the post-labeling purification steps.Recently we have reported development of the microarray system for gene expression analysis using cationic gold nanoparticles [7,8]. This approach is significantly different from the previous methods of using gold nanoparticles with oligonucleotides attached to the gold particles for sequence-specific recognition of targets [9,10]. In our approach there is no need to attach a sequence-specific target-recognition agent to the particle surface. The nanoparticles precipitate, under selected conditions, on the hybridized array spots due to electrostatic (e.g., ionic) attraction of the cationic particles and the anionic phosphate groups in the target DNA backbone. The most important aspect of this detection approach is that no covalent chemical modification of target molecules is required for detection. Furthermore, the number of hybridized molecules can be quantified by detecting absorption or light scattering by nanoparticles using a flatbed scanner instead of expensive confocal laser scanners required by conventional fluorescent microarray technologies. The ionic labeling by nanoparticles greatly simplifies procedures and reduces the cost of microarray analysis. In this report we follow up our previous demonstration of the method for label-free detection of DNA hybridization [8], and further investigate the physical chemistry of interaction of colloidal particles and the * Corresponding author. Fax: +1 865 671 2167 E-mail address: golovlev@scien-tec.com (V. Golovlev).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our cus...

Rapid and quantitative quality control of microarrays using cationic nanoparticles

“…1B and C). Two computational models were developed to study the ionic interaction of gold particles and DNA molecules on a microarray surface (Sun et al, 2007). The theoretical models illustrated the effects of the chemical composition and density of chemical groups on the gold particles, the size of gold particles as well as the size of DNA molecules on the binding of gold particles and DNA.…”

“…The labeling procedure takes about 10 min and is able to detect less than 0.3 pg of probes on the array spot. Gold nanoparticles with different diameters were investigated for the DNA binding capability and discussed in a previous paper (Sun et al, 2007). Briefly, the gold particles with a size of less than 50 nm absorb light and are better detected by the scanner in the bright-field detection mode.…”

“…The method is based on an electrostatic attraction of positively charged 250 nm gold nanoparticles with the immobilized DNA probes that bear negative charges in the phosphate backbone (Sun et al, 2005(Sun et al, , 2007. The entire staining process takes about 10 min and no hybridization of labeled oligonucleotides is required for the detection, which is simpler and faster.…”

Carbon Nanoparticles as Detection Label for Diagnostic Antibody Microarrays

“…Lönnberg and Carlsson were the first to combine carbon nanoparticles with flatbed scanning (Lonnberg & Carlsson 2001). Some other authors also mentioned the use of flatbed scanning using gold nanoparticles (Han et al 2003;Sun et al 2007;Taton et al 2000) or enzyme (Petersen et al 2007). Inexpensive flatbed scanners and CCD-based detectors open new opportunities to develop microarray applications, even for low-facility laboratories and under field conditions (Rasooly & Herold 2008).…”

Food Microbial Pathogen Detection and Analysis Using DNA Microarray Technologies

“…A flatbed scanner application was reported for analysis of cDNA labeled with gold nanoparticles with sensitivity of < 2 pg of DNA molecules captured on the array surface (Sun et al, 2007). Lai et al (2005) used a flatbed scanner for microarray-based quantitative gene expression analysis.…”