Origami-based "Book" shaped three-dimensional electrochemical paper microdevice for sample-to-answer detection of pathogens

Abstract

Herein, an ease-of-use and highly sensitive origami-based "book" shaped three-dimensional electrochemical paper microdevice based on nucleic acid testing (NAT) methodology was developed for sample-to-answer detection of pathogens from whole blood and food samples. The whole steps of NAT, including sample preparation, amplification and detection, were performed by alternately folding the panels of the microdevice, just like flipping a book. The screen-printing electrodes were combined with wax-printing technology to construct a paper-based electrochemical unit to monitor Loop-mediated isothermal amplification (LAMP) reaction with an electrochemical strategy. After nucleic acid extraction and purification with the glass fiber, the LAMP reaction was performed for 45 min to amplify the extracted nucleic acid sequence, followed by the execution of the electrochemical interrogation reaction based on methylene blue (MB) and double-stranded LAMP amplicons. Starting with whole blood and food samples spiked with Salmonella typhimurium, this microdevice was successfully applied to identify pathogens from biological samples with satisfactory sensitivity and specificity. Therefore, the proposed origami-based "book" shaped three-dimensional paper microdevice has great potential for disease diagnosis, food safety analysis applications in the future.

Fig. 1. Schematic of the “book” shaped three-dimensional electrochemical paper microdevice. (a) Foldable paper fluidic device: dark and gray areas are printed with hydrophobic wax. The device consists of five panels (1–5) and a plastic plate (b) for LAMP processing to avoid evaporation. (c) An image of the prototype device. (d) Schematic illustration of the electrochemical interrogation based on a redox active molecule methylene blue (MB) and double-stranded LAMP reaction products.

Fig. 2. General operation of the electrochemical paper microdevice: (a) flipping the panel 3 & 4 and (b) continuously adding sample and buffer; (c) flipping the panel 3 onto panel 4 and (d) adding elution buffer; (e) flipping the panel 3 onto panel 2 and (f) sealing the panel 4 and 5 with an acetate film to prevent evaporation during amplification; (f) measuring current three times after LAMP reaction.

Fig. 3. The DNA recovery rates of paper extraction and traditional centrifugation extraction. Error bars: standard deviation, n = 3. Percentage values indicate the DNA recovery of paper extraction compared to traditional centrifuge control.

Fig. 4. Optimization of experimental parameters. (a) Effect of the volumes of washing buffer and elution buffer (p < 0.05). (b) Effect of number of washes (p < 0.05). (c) Effect of the LAMP amplification time (p < 0.05). (d) Effect of the MB concentration.

Fig. 5. The analytical sensitivity of the “book” shaped three-dimensional electrochemical paper microdevice for the detection of pathogens was tested by using purified genomic DNA of S. typhimurium. (a) DPVs of detection purified genomic DNA (GDNA) at different concentrations: 0–10⁵ CFU mL⁻¹ in whole blood. (b) The calibration plot of changes of current intensity vs. log  G_DNA.

Fig. 6. Assay integrated with electrochemical paper microdevice using spiked-human whole blood sample. (a) DPVs of detection S. typhimurium at different concentrations: 0–10⁵ CFU mL⁻¹ in whole blood. (b) The calibration plot of changes of current intensity vs. log C_S.