# High-Throughput DNA Analysis Platform Based on an Optofluidic Ring Resonator Laser
## 2 Materials and Methods  
The microdroplets were generated with plastic tubing (Upchurch, P1476, inner diameter (ID) = 150 \(\upmu\)m) and T-junctions (Upchurch, P-890), as schematically shown in Figure 1a, in accordance with previous studies [23; 24; 25]. Silicon oil was introduced upstream as a hydrophobic carrier fluid, and an aqueous solution of DNA and intercalating dye (SYTO\({}^{\text{\textregistered}}\)-13 Green Fluorescent Nucleic Acid Stain, Life Technologies, Carlsbad, CA, USA) was injected into the T-junction. The flow rates of the carrier fluid and DNA samples were 10 \(\upmu\)L/min and 3 \(\upmu\)L/min, respectively. The immiscible phase forms individual microdroplets, of which approximately 125 nL can be derived from a generation rate of 0.4 Hz. Figure 1: (**a**) Schematic of microfluidic system for DNA/ dye microdroplet generation and optofluidic ring resonator (OFRR) laser experiments downstream. (**b**) When the microdroplets passing the glass capillary OFRR are optically pumped, the DNA/ dye gain interacts with light confined in the WGM in the evanescent field, providing enough feedback for laser emission. The optical signal, excluding undesired fluorescence background, is coupled into a fiber taper and collected using a spectrometer at distal end. The fused silica glass capillary was fabricated using the existing drawing method with CO\({}_{2}\) laser irradiation and an inner diameter and wall thickness of approximately 70 \(\upmu\)m and 7 \(\upmu\)m, respectively. The microdroplets of DNA/dye were infused downstream into the capillary OFRR. In a capillary with a smaller diameter, the DNA/dye microdroplets filled the entire cross-section, placing the DNA/dye samples in contact with the capillary wall. When the microdroplets passed a point of interest, the OFRR was optically pumped using an optical parametric oscillator (continuum, 488 nm wavelength, 5 ns pulse width, 20 Hz repetition rate). As the evanescent field of light confined in the OFRR whispering-gallery mode (WGM) interacted with the DNA/dye solution, a laser was emitted and coupled into the fiber taper in contact with the OFRR. Only the laser emission was carried into the spectrometer (iH+850, Horiba Jobin Yvon) connected to the distal end of the fiber taper, excluding the undesired fluorescence background. The microdroplets can also serve as high-Q OFRR cavities because they are natural spheres with smooth surfaces. However, the relatively low refractive index of the aqueous DNA/dye solution (\(\sim\)1.33) renders it impossible to confine light when the droplets are in carrier fluid with a higher index. Microdroplets in a carrier fluid are free from evaporation issues and are individual reaction chambers that can be easily manipulated. Thus, this platform is ideal for high-throughput biosensors and/or molecular detectors. However, a low water index significantly limits the choices of the continuous phase for light confinement in the WGM. This is one of the main reasons for utilizing a glass capillary in the laser cavity. Although most of the WGM-confined light resides inside the glass capillary, there is enough of an evanescent field inside, resulting in sufficient laser feedback from the laser gain, which is the DNA/dye mixture in this experiment. The arbitrary DNA sequences used in this study are listed in Table 1. The target DNA, which was perfectly matched double-stranded DNA, had 21 base pairs. Sequences 1 and 2 of single-stranded DNA (ssDNA) forming the target double-stranded DNA were complementary pairs. In control experiments, base mismatches in the middle of the sequences were intentionally introduced to verify that our DNA analysis platform was capable of distinguishing the non-target DNA with only a single base mismatch. In this study, we classified the base mismatched DNA described above using the suggested microdroplet OFRR laser system. Therefore, our DNA detection platform could distinguish any kind of non-target DNA with a thermodynamic difference larger than a single base mismatch.