Droplet Microfluidic Device for Chemoenzymatic Sensing

Abstract

The rapid detection of pollutants in water can be performed with enzymatic probes, the catalytic light-emitting activity of which decreases in the presence of many types of pollutants. Herein, we present a microfluidic system for continuous chemoenzymatic biosensing that generates emulsion droplets containing two enzymes of the bacterial bioluminescent system (luciferase and NAD(P)H:FMN-oxidoreductase) with substrates required for the reaction. The developed chip generates "water-in-oil" emulsion droplets with a volume of 0.1 μL and a frequency of up to 12 drops per minute as well as provides the efficient mixing of reagents in droplets and their distancing. The bioluminescent signal from each individual droplet was measured by a photomultiplier tube with a signal-to-noise ratio of up to 3000/1. The intensity of the luminescence depended on the concentration of the copper sulfate with the limit of its detection of 5 μM. It was shown that bioluminescent enzymatic reactions could be carried out in droplet reactors in dispersed streams. The parameters and limitations required for the bioluminescent reaction to proceed were also studied. Hereby, chemoenzymatic sensing capabilities powered by a droplet microfluidics manipulation technique may serve as the basis for early-warning online water pollution systems.

Keywords: bioluminescence; chemoenzymatic system; droplet microfluidics; luciferase; sensing.

Figure 1

The schematic representation of a droplet reactor with the encapsulated chemoenzymatic system. The multienzyme part of the system consisted of luciferase and NAD(P)H:FMN–oxidoreductase, as well as substrates: flavin mononucleotide (FMN), reduced nicotinamide adenine dinucleotide (NADH) and oxygen.

Figure 2

Droplet formation from the two co-laminar flows of the basic and starting solutions. The results of the 2D numerical simulation of the droplet generation with a diameter of 0.8 mm in a channel 1 mm wide with a frequency of 12 drops per minute (a) showed that the mixing of laminar flows that are equal in volume was insignificant during the formation of droplets and their further flow along the straight channel. This result was experimentally confirmed for a droplet generator with a channel depth of 0.25 mm (b). For visualization, polymer microparticles with a nominal diameter of 1 $\mathsf{\mu }$m were added to one of the solutions.

Figure 3

Reinjection of the droplet reactors in an asymmetric T-junction: (a) numerical simulations of the droplet flow near the side channel; (b) time dependence of the mixing index Equation (2); and (c–e) experimental verification in the chip. It can be seen that the polymer microparticles with a nominal diameter of 1 $\mathsf{\mu }$m in a droplet before the side channel were only located in the zone of the upper Taylor vortex, and after the reinjection, they were distributed over the entire volume of the droplet (flow direction from left to right). For better visualization, the area where the distance between the particles in the figure was less than 100 $\mathsf{\mu }$m was tinted with green using the ImageJ software.

Figure 4

The principle of operation of the microfluidic device for the regular immobilization of Red&Luc with substrates, products and other chemical compounds in droplet reactors (above) and the design of the PDMS microfluidic chip (below). The channels of the chip presented in the figure were filled with Coomassie Brilliant Blue G-250 for better visualization.

Figure 5

Assembled, tuned and functioning microfluidic device for the regular immobilization of the multienzyme system in droplet reactors (also see video S1 in supplementary). The developed microfluidic chip (a) consisting of Y- (behind the frame of the picture on the left), X- and T-crosshairs, in which the basic and starting solutions are coupled, droplets are formed, mixed and distanced. One of the components of the dispersed phase was colored with Coomassie Brilliant Blue G-250 for better visualization. The fluid supply pressures were selected to ensure the generation of droplets in a volume of 0.13 $\mathsf{\mu }$L with a frequency of 12 drops per minute (b), and allowing the droplets to move away from each other at a distance of 2–2.5 cm (c).

Figure 6

(a) Dependence of luminescence intensity on the presence of the tetrodecanal (+/−) in the carrier (O) and dispersed (W) phases. In the presence of tetradecanal (+), its concentration in the carrier and dispersed phase was 10 mM and 0.33 mM, respectively. (b) The intensity of luminescence in time was recorded by the photomultiplier tube (PMT) 20 seconds after the start of the enzymatic cascade. Flares with a glow intensity in the region of 6–8 kRLU were recorded at the moments when the droplet was opposite the PMT aperture. When the droplet was carried away with the flow and only the carrier phase appeared at the aperture, the glow intensity corresponding to the level of the PMT dark noise was recorded (approximately 20 RLU).

Figure 7

Luminescence intensity inhibition of the multienzyme bioluminescent system in droplets with the addition of model pollutants: (a) copper sulfate; (b) 1,4-benzoquinone; and (c) 1,3-dihydroxybenzene.