Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics

Abstract

Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications.

Keywords: Isothermal nucleic acid amplification; Paper-based diagnostic device; Point-of-care diagnostics; Polyethersulfone.

Fig. 1

Scanning electron micrograph of CHR (a, f), PES (b, g), PC (c, h), GF (d, i), and NC (e, j) at 1000× (a–e) and 5000× (f–j) magnification. Scale bars represent 10 µm (a–e) and 2 µm (f–j)

Fig. 2

Fluidic absorbency of 1 cm² materials. Average absorbency +/− standard deviations are shown (n = 5 for each sample type). Materials with similar symbols (* or +) are not statistically significantly different than one another using a one-way ANOVA followed by Tukey’s HSD test

Fig. 3

Results of LAMP performed on B. pertussis (Left) and Influenza A (H1N1) (Right). Detection by agarose gel electrophoresis (a) and LFD strips (b). Intensity of the LFD test line as a percentage of the control line in the strips is shown in (c). Box plots indicate the minimum, maximum, and median intensities for each sample. (*p < 0.05, **p < 0.005, ***p < 0.001 using Dunnett’s test versus the negative control)

Fig. 4

Results of tHDA performed on C. trachomatis (Left) and N. gonorrhoeae (Right). Detection by agarose gel electrophoresis (a) and LFD strips (b). Intensity of the test line as a percentage of the control line in the strips is shown in (c). Box plots indicate the minimum, maximum, and median intensities for each sample. (*p < 0.05 using Dunnett’s test versus the negative control)