An integrated E-Tube cap for sample preparation, isothermal amplification and label-free electrochemical detection of DNA

Abstract

A simple, disposable, and integrated electronic-tube cap (E-tube cap) for DNA detection at the point-of-care was designed, fabricated, and tested. The E-tube cap contains a 3D printed electrode substrate for DNA extraction and label-free pH sensing detection. One Flinders Technology Associates (Whatman FTA) membrane was incorporated into the 3D printed electrode substrate for the isolation, concentration, and purification of DNA. The E-tube cap with captured DNA by the membrane was inserted directly into a reaction tube for loop-mediated isothermal amplification (LAMP). The isothermal amplification process was monitored in real-time by a 3D printed electrochemical electrode coated with pH-sensitive material (carbon/iridium oxide layer). The pH sensing electrode showed an excellent linear response within the pH range of 6-9 with a slope of -31.32 ± 0.5 mV/pH at room temperature. The utility of the integrated E-tube cap was demonstrated by detecting the presence of lambda DNA spiked in saliva samples with a sensitivity of 100 copies per mL sample within 30 min. Such a simple, rapid, and affordable diagnostic device is particularly suitable for point-of-care molecular diagnostics of infectious diseases.

Keywords: 3D-printing; Electrochemical detection; Isothermal amplification (LAMP); Loop-mediated; Point-of-care molecular diagnostics; pH-sensitive electrode.

Figure 1.

Schematic illustration of the electronic-tube cap (E-tube cap) system consisting of: i) E-tube cap with pH sensing electrode, FTA membrane and tube cap, and ii) 3D printed absorbent case with cotton absorbent. The left bottom insert is a photo of the assembled E-tube cap.

Figure 2.

(A) Energy dispersive spectroscopy (EDS) image /element map (i), electrochemical impedance spectroscopy (EIS) curves (ii), and EIS simulation (iii) of the carbon paste (CP) electrode. (B) Energy dispersive spectroscopy (EDS) image /element map (i), EIS (ii), and EIS simulation (iii) of the IrO_x/CP electrode.

Figure 3.

Real-time relative potential of the different concentration samples (0 (negative control), 10², 10³, and 10⁴ copies/test) during LAMP reaction. The electrochemical LAMP reaction system contains 7.5 μL non-buffered LAMP reaction solution, 3.6 μL ddH₂O, 0.8 μL LAMP primers mix, and 1.2 μL 100 mM MgSO₄ with/without 1 μL target DNA. The incubation temperation of LAMP assay is 63 °C.

Figure 4.

(A) Real-time relative potential monitoring of LAMP amplification of saliva samples containing 0 (negative control), 10², 10³ and 10⁴ copies DNA/mL saliva sample in our integrated E-tube cap system. The electrochemical LAMP reaction system contains 7.5 μL non-buffered LAMP reaction solution, 3.6 μL ddH₂O, 0.8 μL LAMP primers mix, and 1.2 μL 100 mM MgSO₄. The incubation temperation of LAMP assay is 63 °C. (B) Relative potential change after 30-minute incubation as function of log of initial concentration of DNA in saliva samples (0, 10², 10³ and 10⁴ copies/mL) (n=3).