Nanostructured biochip for label-free and real-time optical detection of polymerase chain reaction

Real-time monitoring of polymerase chain reaction (PCR) has been established as the main technique for the specific nucleic acid identification of biological samples [1–3]. Real-time PCR technology, which generally uses fluorescence from released dye molecules, enables the measurement of amplified DNA products after each cycle with a small value of standard deviation [4,5].Nevertheless, the requirements for complex optical components and the inhibitory effects from the fluorescent reagents make this approach difficult for point-of-care testing (POCT) applications in a miniaturized portable instrument.Toward the forthcoming goal of performing an overall PCR anal-ysis on chip without labeling the DNA samples, many alternative detection schemes with the robust quantifications of direct PCR products have been proposed based on DNA intrinsic properties[6–12]. One of the promising candidates relies on the detection of the PCR products using relative reflected intensity (RRI) of the nanostructured substrates in connection with collinear optical systems [13–15]. Moreover, the direct relationship between DNA concentration and the refractive index has been exploited for label-free optical biosensors [16,17]. The interaction of an oligonucleotide with its complementary DNA probe that was immobilized on a gold surface, was detected by monitoring the changes in the intensity and peak wavelength shift of the RRI before and after hybridization [13,18]. To the best of our knowledge, a complete PCR including simultaneous amplification and label-free RRI-based quantification on a single chip has not been reported yet. For this purpose, the strong attachment of oligonucleotide primers to the Au surface specifically via the 5 -end is needed to ensure the availability of the free 3 -ends for the polymerase activity and the annealing of base pairs. Recent studies on oligonucleotides and their interactions with Au nanoparticles provide the fundamental knowledge for developing a novel label-free PCR system on our Aucoated nanostructured substrate. First, several chemical strategies were tested for the direct attachment of 5 -end oligonucleotides primers on the solid surface with the previously reported conditions for the surface density of probe oligonucleotides [19–21].Then, the linkage between the thiolated primers and Au surface was evaluated against the repeated heating and cooling cycles of PCR [22–24]. In order to determine whether the oligonucleotides were desorbed from the surface of the Au nanoparticles after the repeated temperature cycles, Kim et al. prepared oligonucleotidemodified Au nanoparticles in buffer used for the PCR assay with and without dNTPs and primers were subjected to the repetitive cycles of temperature changes used for PCR [22]. After 40 cycles,the color of the solution containing the oligonucleotide-modified Au nanoparticles with and without dNTPs and primers remained red, indicating that the oligonucleotides were not desorbed from the nanoparticle surface [22]. In this work, the RRI properties of Au-coated nanostructured substrates were thoroughly examined throughout the visible region in various refractive index solutions and their surface modifications with different lengths of alkanethiol molecules were investigated using UV–vis spectroscopy. As a result, the RRI of our nanostructured surface was closely related to the change in the RI of surrounding medium and the formation of self-assembled monolayer (SAM) of alkanethiols on the deposited gold layer.Subsequently, Au-coated nanostructured substrates were used to immobilize thiolated oligonucleotide primers on their surfaces and for the first time, we performed label-free and real-time optical detection of PCR.

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