1. Introduction

Droplet-based microreactors have attracted great research interest in physical, chemical and biological fields due to their advantages such as fast thermal- and mass-transfer rates, low sample and reagent consumption, and cost-effective fabrication [1,2]. Their potential application areas rang from materials synthesis, bioanalysis, to high throughput screening [3–5]. A key requirement for many biological and chemical applications is efficient heating of the samples or reactants. For example, the ability to perform polymerase chain reaction (PCR) with high efficiency in microfluidic devices is critically dependent on rapid and precise heat transfer [6].

To date, on-chip heating mainly relies on electrical [7–9] and optical means [10–12]. The use of embedded electrodes not only limits the spatial resolution of localized heating, but also increases the complexity of microchip fabrication. In contrast, the optical means do not need electric connections, and the heating locations are more controllable due to the small light spot. However, high-power light source and materials with strong photothermal effect are often needed to enhance the heating efficiency. The use of high-power light source introduces additional safety problems and the intensive incident light may also interfere with on-chip optical detection. Therefore, safe and effective means of photothermal conversion on microfluidic chips without interference with detection are highly desired.

Gold nanoparticles (GNPs) have large absorption cross sections (more than two orders of magnitude higher than that of organic fluorophores) at their localized surface plasmon resonance (LSPR) wavelengths [13]. Under the resonant excitation, a small number of GNPs can absorb a large amount of light and convert it into heat with very high efficiency [14]. The fundamental aspects of the photothermal conversion arising from the LSPR of metal nanoparticles have been carefully studied [15–18]. In spite of the success of heating large volume solution [17] and bulk polymer [19] by employing GNPs, well-controlled heating of nanoliter level droplet is a great challenge.

Here we demonstrate an optical droplet heating method by employing gold nanorods (GNRs) as photothermal conversion materials. Localized heating of individual and stream droplets containing GNRs was achieved with fast response, large temperature range, and precise temperature control. By varying the incident laser power, we further demonstrated the possibility of ultra-fast PCR in individual droplets.

2. Experimental section

In this work, a polydimethylsiloxane (PDMS) microfluidic chip, as schematically illustrated in Fig. 1(a) , was used for generating droplets and localized heating. It was fabricated using the standard soft lithography described elsewhere [20]. The depth of the microchannel is about 130 μm, and the thickness of the chip is about 800 μm. Two multi-mode optical fibers (62.5/125 μm) were inserted into the fiber channels for heating and fluorescence excitation, respectively. To generate aqueous droplets, paraffin oil was employed as carrier phase. Two syringe pumps (Pico Plus, Harvard Apparatus, USA) were used for driving the paraffin oil and GNR solution, respectively. By tuning the flow rates of paraffin oil and GNR solution, the volume and interval of the droplets can be well controlled. Figure 1(b) and 1(c) show microscope images of the droplet generation zone and laser heating zone, respectively. The uniform droplets indicate a high reproducibility of the composition in the droplets, which is a key factor affecting the heating performance.

 

Fig. 1 (a) Schematic illustration of the microfluidic chip. (b and c) Optical microscope images of (b) droplets generation and (c) droplets flowing through the heating zone.

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GNRs with an aspect ratios of 3.4 ± 0.6 (length 70.8 ± 8.9 nm, width 20.7 ± 2.8 nm) and longitudinal surface plasmon resonance wavelength of 781 nm was used through this work due to its high photothermal conversion efficiency (~70%) [17] and well-established synthesis method. The preparation of the GNRs followed the same procedure described in our previous work [21] except that the volume of the seed solution was increased to 0.070 mL. Figure 2 shows the TEM image and extinction spectrum of as-prepared GNRs, respectively.

 

Fig. 2 (a) TEM image of the GNRs. (b) Extinction spectrum of the GNRs dispersed in water.

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Rhodamine B was used to determine the temperature of droplets, because its quantum yield is strongly temperature dependent in the range between 0 and 100 °C [22]. To avoid the adsorption of rhodamine B on the PDMS channel wall, sodium dodecyl sulfate (SDS) was employed as a dynamic coating reagent [23]. To calibrate the dependence of fluorescence intensity on temperature, 1.5 mL of an aqueous rhodamine B (0.1 mM) and SDS (50 mM) solution (95 °C) was injected into a quartz cuvette mounted with a K-type thermocouple. The temperature and fluorescence spectra, excited by a 532-nm-wavelength laser, were dynamically recorded by a digital thermometer and a spectrometer (QE65000, Ocean optics, USA), respectively. For the calibration curve, peak fluorescence intensity at different temperature ranging from 90 to 22 °C (environment temperature) were normalized by the intensity at 22 °C. The temperature is a function of normalized intensity as shown in Eq. (1):

3. Results and discussion

3.1 Individual droplets heating

For characterization of this localized heating method, we firstly investigated the heating performance for individual stable droplets by employing stop flow technique. When a 2.5 nL droplet containing GNRs (9 nM), rhodamine B (0.1 mM), and SDS (50 mM) was located at the laser heating zone, as shown in Fig. 1(a), an intensity-tunable 808-nm-wavelength laser was used to illuminate the droplet from the left, and a 532-nm-wavelength laser was used to excite rhodamine B from the right. The fluorescence of rhodamine B was collected via an optical microscope (Eclipse 90i, Nikon, Japan) with a 10 × objective and then directed to the spectrometer. 532 nm and 808 nm notch filters were used to eliminate the scattered light. Figure 3(a) shows the fluorescence spectra obtained at increasing laser powers from 0 to 13.6 mW. The fluorescent intensity decreased monotonically with the increasing laser power, indicating the gradual increase of the droplet temperature. The insets of Fig. 3(a) show two typical images of a droplet without (top) and with (bottom) laser heating. As shown in Fig. 3(b), the droplet temperature increases from 22 to 95 °C, and is linearly proportional to the laser power. Thus, by adjusting the power of the heating laser, the temperature of droplets can be precisely controlled. Also, no gas bulbs were observed during the heating process because the confinement effect assisted the prevention of water evaporation [24]. For a droplet without GNRs, no obvious fluorescence intensity change was observed before and after the illumination of 808-nm-wavelength laser, which confirms that the heating of droplets was due to the photothermal effect of GNRs.

 

Fig. 3 (a) Fluorescence spectra of rhodamine B in the droplet obtained at increasing laser powers. The insets are optical microscope images of a droplet containing GNRs taken without (top) and with (bottom) a 13.6 mW 808-nm-wavelength laser heating. The scale bars: 100 μm. (b) Temperature of the droplet as a function of laser power.

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To investigate the reproducibility and response time of this method, the time-dependent fluorescence intensity at peak wavelength of 588 nm was traced. Figure 4(a) shows reversible response of the droplet temperature when an 11 mW 808-nm-wavelength laser was switched on and off alternatively, indicating a good reproducibility of droplet temperature. As shown in Fig. 4(b), the heating and cooling times are 200 and 800 ms, respectively, which are 1 or 2 orders of magnitude faster than those of previously reported methods [19, 25]. It is worth mentioning that the toxicity of CTAB may have negative effects on some biological samples, but this can be solved by coating GNRs with biocompatible polymers, such as polyethylene glycol.

 

Fig. 4 (a) Reversible response of the droplet temperature when the laser was switched on (11 mW) and off alternatively. (b) Typical time-dependent temperature of the droplet reveals the heating and cooling times of about 200 and 800 ms, respectively.

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3.2 Stream droplets heating

Furthermore, we employed the aforementioned configuration for stream droplets heating by setting the flow rates of paraffin oil and GNRs solutions at 0.12 μL/min and 0.08 μL/min, respectively. In this case, the time-interval between two adjacent droplets was 1.2 s. There was only one droplet flowing through the 500-μm-length heating zone once a time, and thus, the interference between adjacent droplets was eliminated. Figure 5 shows the fluorescence intensity of 45 consecutive droplets, and each peak indicates a droplet. When a 20 mW 808-nm-wavelength laser was illuminated on the heating zone, the fluorescence intensity of droplets decreased to 18% of the original intensity. According to Eq. (1), the droplets were heated to 87 °C when they flowed through the laser heating zone. The good reversibility of fluorescence intensity not only indicates a good reproducibility of droplet temperature, but also demonstrates the uniformity of the droplets.

 

Fig. 5 Reversible response of fluorescence intensity of stream droplets by alternately switching on and off a 20 mW 808-nm-wavelength laser.

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3.3 Temperature cycle for rapid PCR amplification

The precise temperature control, rapid response, and small heating volume make this localized heating method attractive for many applications such as rapid PCR amplification [26]. To demonstrate the temperature cycles of ultra-fast PCR amplification, we calculated the laser powers for DNA denaturation (94~98 °C), primer annealing (50~65 °C), and DNA extension (75~80 °C), and controlled the illumination time for each step. Figure 6 shows two typical thermal cycling profiles for PCR amplification, the droplet temperature is very stable during the three steps. Owing to the fast thermal-transfer rate of droplets and high photothermal conversion efficiency, the transition from one step to another was achieved within 1 s, and the time for one cycle is only 75 s, which means 48 cycles can be accomplished in one hour. This represents a considerable time saving on previously reported microwave PCR systems where 33 cycles took 127 minutes [27].

 

Fig. 6 Typical thermal cycling profiles of the laser heating system for rapid PCR amplification. The DNA denaturation, primer annealing, and DNA extension temperatures were of 96 °C, 60 °C and 78 °C, respectively.

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4. Conclusion

In conclusion, we have demonstrated an optical localized heating method for microfluidic applications. Individual and stream nanoliter level droplets containing GNRs were heated by a low power 808-nm-wavelength laser. The droplet temperature was linearly proportional to the heating laser power. A temperature of 95 °C was achieved by employing a 13.6 mW 808-nm-wavelength laser to an individual droplet. Owing to the fast thermal-transfer rates of droplets, the heating and cooling times were less than 1 s. Moreover, temperature cycles of PCR were demonstrated by adjusting the heating laser power and controlling the heating time. We envision this method to be quite suitable for a wide range of applications in microfluidics requiring rapid and localized heating, including cell treatment, enzyme activity research, chemical and biology assays.