Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

Abstract

An emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 10³ and 10⁶ CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 10⁶ CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

Figure 1

(A) Experimental methodology representation shows how potential contaminants are compartmentalized into their nanoliter-sized droplets. As a result, such contaminants cannot adversely affect the amplification (or lack thereof) of the target DNA, thus causing a decrease in droplet size for such amplified droplets. (B) A basic schematic illustrating the key materials necessary to conduct eLAMP experiments. It includes a hotplate, 3D-printed hotplate attachment, glass vial with micro stir bar, miniature spectrophotometer, fiber optic cables (to provide the light source and monitor light scatter), and the light source.

Figure 2

(A) Measured diameter from light microscope images of water–oil emulsions containing 10 µL of aqueous 1 and 0 µg DNA fragment mixture. (B) Emulsion light scatter intensity at 60° with respect to 650 nm incident light over time of 1, 0.5, and 0 µg DNA fragment mixture in 10 µL aqueous phase. (C) The 60° intensities at 30 s plotted against the DNA fragment amount.

Figure 3

Emulsion LAMP light scatter intensity via spectrophotometer over time at (A) 30° and (B) 60° angle with respect to 650 nm incident wavelength with varying initial bacteria concentration of 10⁶, 10³, 1, and 0 CFU/µL. (A and B) are the representatives chosen from 3 replicates for each concentration. Light scatter intensity at 3 min for (C) 30° and (D) 60° angle for bacteria concentrations of 10⁶, 10³, 1, and 0 CFU/µL.

Figure 4

Emulsion LAMP light scatter intensity with relation to time collected at (A) 30° and (B) 60° angles with respect to a 650 nm wavelength incident light with bacteria solution droplets (along with BSA as stabilizer), varying concentrations of 10⁶,10³, 1, and 0 CFU/µL. (A and B) are the representatives chosen from 3 replicates for each concentration. Light scatter intensity at 3 min collected from (C) 30° and (D) 60° angles at various bacterial concentrations.

Figure 5

Emulsion LAMP light scatter intensity via smartphone camera over time at (A) 30° and (B) 60° angle with respect to 650 nm incident wavelength with varying initial bacteria concentration of 10⁶, 10³, 1, and 0 CFU/µL. A and B are the representatives chosen from 3 replicates for each concentration. 30° light scatter red channel intensity at (C) 3 min and (D) 6 min. 60° light scatter red channel intensity at (E) 3 min and (F) 6 min with bacteria concentrations of 10⁶, 10³, 1, and 0 CFU/µL.

Figure 6

Sensitivity and specificity in comparison to conventional LAMP. (A) Average 30° light scatter intensity profiles for 10³ CFU/μL and NC (negative control) samples. (B) Comparison of light scatter intensity values at 5 min between initial target bacterial concentrations. (C) Gel image showing amplification of both positive (left lanes) and NC (right lanes) samples after conventional amplification for 30 min. (D) Table detailing sensitivity and specificity of both conventional amplifications combined with gel electrophoresis and emulsion LAMP.

Figure 7

Measured fluorescence of conventional LAMP amplification of positive control samples (red lines, n = 13) and negative control samples modeling contamination (black lines, n = 12).

Figure 8

Theoretical model equation and modeling results of LAMP amplicon creation and subsequent adsorption to the oil–water interface within the emulsified droplets.