Microarray technologies have become a tool of choice for multiparametric diagnostic assays and for gene expression profiling (7
). The ability to perform sensitive and reproducible microarray hybridizations depends to a great extent on the quality of the microarray substrate. Microbiological diagnostic assays on glass slide microarrays for highly sensitive and specific detection of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 in human blood samples have been demonstrated recently (8
). Nonetheless, plastic materials are likely more promising than glass supports for the future of microarrays dedicated to microbiological diagnostics, because the former have excellent microfabrication properties and are more easily amenable to integration into low-cost, portable microanalysis systems. The development of microanalysis systems for the molecular detection of infectious disease agents requires the development of assays that are both rapid and highly sensitive. However, the high autofluorescence of standard polymeric materials is not compatible with a highly sensitive detection method that employs fluorescent targets. Plastic microarray hybridizations compatible with fluorescent dyes have been developed previously (5
). However, these investigators reported only hybridizations with labeled oligonucleotides, and the specific hybridization signal-to-background ratios were relatively low. Others have avoided this issue by using nonfluorescent detection technologies such as radiolabeling (13
) or enzymatic reactions (17
), but none of these detection technologies fulfill the diagnostic requirement in terms of both rapidity and safety.
In this work, we report ultrasensitive microarray hybridization of fluorescently labeled PCR-amplified target DNA using two high-quality plastic substrates for molecular diagnostic purposes. Both plastics (PMMA-VSUVT and Zeonor 1060R) were previously selected for their premium optical properties and low autofluorescence background (2
). They have been specifically functionalized in order to reduce background hybridization and increase specific hybridization signals. We observed that the hybridization background signals were as low as, or even lower than, those measured with commercially available high-quality functionalized microarray glass slides. The amplicons generated via multiplex RT-PCR from as few as 25 to 100 viral particles of IAV, EV, RSV, or MPV could be detected and discriminated, respectively, on plastic microarrays using either 1-h passive hybridization or 3-min microfluidic hybridization without any amplicon purification step. Specific fluorescence signals were nonambiguous and were as high as (Zeonor 1060R), or higher than (PMMA-VSUVT), those obtained with a glass substrate. Similar results were obtained using the microfluidic platform (20
). To our knowledge, this is by far (at least 100-fold) the lowest detection limit achieved with DNA microarray hybridizations on plastic supports. Indeed, most published studies of microarray hybridizations on plastic supports did not report any analytical sensitivity data (2
). Lenigk et al. (11
) have described microfluidic hybridization of PCR-amplified, Cy- labeled staphylococcal DNA onto a polycarbonate microarray, detecting a minimum of 10,000 genome copies. This analytical sensitivity may be insufficient for reliable detection of microbial pathogens, which can be found at lower clinically relevant loads. For example, infections associated with viral loads as low as 100 viruses per ml of respiratory tract sample may be encountered (1
). A highly sensitive hybridization on a plastic microarray using DNA capture probes spotted onto microfabricated micropillars has been described recently (19
). In this system, the background fluorescence from the area surrounding the pillars is rejected through confocal detection. An excellent signal-to-background ratio was obtained for hybridizations using labeled and reverse-transcribed total human RNA, but the fabrication and functionalization of those micropillars are far more complex than those of flat plastic substrates such as those described in the present study.
High background fluorescence on plastic supports is often due to nonspecific adsorption of the labeled target onto the substrate (4
). In the present study, we report high signal-to-background ratios. On average, these ratios reached 250 for hybridization using 5 nM labeled oligonucleotide and 137 for hybridization of nonpurified labeled amplicons generated from 100 viral genome copies. This means that the background fluorescence represents as little as 0.4% or 0.7% of the signal, respectively. By comparison, Fixe et al. (5
), using 40 times more labeled oligonucleotides (0.2 μM) on PMMA slides, reported a signal-to-background ratio of 90.
We previously described a centrifuge-based microfluidic system for rapid and efficient microarray hybridization on standard glass slides (20
). In the present study, we obtained high fluorescence signal intensities and low background with both plastic and glass slides using this 3-min microfluidic microarray hybridization system, in which hybridizations are performed at room temperature (23°C) using nonpurified PCR products. The only other reported study of room temperature hybridization on plastic supports was performed using a 16-h protocol, and the hybridization signal-to-background ratios obtained were low (approximately 10) (4
). Others have performed hybridization experiments on plastic supports at 30 to 65°C, requiring special heating devices to carry out the hybridizations (5
). In addition, stability tests demonstrated that when stored in a vacuum desiccator, the chemically activated plastic slides were stable for at least 10 weeks.
In conclusion, we have developed two functionalized plastic supports (PMMA-VSUVT and Zeonor 1060R) that allow efficient DNA microarray hybridizations at room temperature, with signal-to-background ratios and analytical sensitivities comparable to those obtained with high-quality standard glass slides. These plastic supports, which were shown to be highly stable, have been validated for microbiological diagnostic applications with both passive hybridization and active hybridization in a microfluidic system. Considering that both plastics are suitable for low-cost fabrication, this plastic microarray technology is promising for the development of fully integrated medical diagnostic devices, such as portable μ-TASs.