Laser-induced heating integrated with a microfluidic platform for real-time DNA replication and detection

Abstract

This study developed a microfluidic platform for replicating and detecting DNA in real time by integrating a laser and a microfluidic device composed of polydimethylsiloxane. The design of the microchannels consisted of a laser-heating area and a detection area. An infrared laser was used as the heating source for DNA replication, and the laser power was adjusted to heat the solutions directly. In addition, strong biotin–avidin binding was used to capture and detect the replicated products. The biotin on one end was bound to avidin and anchored to the surface of the microchannels, whereas the biotin on the other end was bound to the quantum dots (Qdots). The results showed that the fluorescent intensity of the Qdots bound to the replicated products in the detection area increased with the number of thermal cycles created by the laser. When the number of thermal cycles was ≥10, the fluorescent intensity of the Qdots was directly detectable on the surface of the microchannels. The proposed method is more sensitive than detection methods entailing gel electrophoresis.

Laser‐induced heating for in situ DNA replication and detection in microchannels

“…4 b ). This finding corresponds to those of our previous studies, in which BSA blocked adsorption of Qdots onto the glass surface [10, 22]. Fig.…”

“…The relationship between the laser power and temperature was calibrated using a fluorescent dye, namely rhodamine B; the fluorescence intensity varied according to temperature changes [31]. Very recently, we have demonstrated the temperature calibration of IR laser spot based on the fluorescence intensity of the rhodamine B [22]. The laser power was measured under the objective lens with a power meter, whereas the temperature in the microchannels was measured using a thermocouple.…”

“…In a previous study, we proposed and demonstrated the use of a microfluidic platform integrated with laser‐induced heating and fluorescence intensity of quantum dots (Qdots) for replicating and detecting DNA in real time [22]. In the present study, rather than the rapid replication and detection of DNA in extracted bio‐samples, the goal was to propose a method for accurately replicating and detecting the targeted location of a DNA strand from individual cells at the single‐cell level.…”

“…In the present study, rather than the rapid replication and detection of DNA in extracted bio‐samples, the goal was to propose a method for accurately replicating and detecting the targeted location of a DNA strand from individual cells at the single‐cell level. The strength of our previous study [22] is that it illustrated the use of a single platform to both replicate and detect DNA. The laser‐induced heating and fluorescent detection methods were also shown to be readily integrated with microelectrode arrays for accurate DNA detection through single measurement.…”

Magnetic Bead-Quantum Dot (MB-Qdot) Clustered Regularly Interspaced Short Palindromic Repeat Assay for Simple Viral DNA Detection

“…After magnetic isolation, a high fluorescence intensity (∼14 counts) was detected, indicating that the MB-Qdot conjugate is mainly achieved by DNA complementary hybridization rather than non-specific absorption (control, blue). In the future, streptavidin-coated magnetic beads can be treated with blocking reagents such as bovine serum albumin 32 to reduce the non-specific binding. To further confirm the MB-Qdot conjugation, the beads were washed 5 times followed by DNase I (EN0521, ThermoFisher Scientific) treatment to degrade the hybridized DNA strands.…”