Label-free electrical monitoring of nucleic acid amplification with integrated hydrogel ionic diodes

Abstract

We demonstrate here for the first time the utility of a monolithically integrated hydrogel ionic diode for label-free quantitative DNA detection and real-time monitoring of nucleic acid amplification. The hydrogel ionic diode presented herein, unlike nanomaterial-based field-effect biosensors, features high cost-effectiveness and convenient fabrication. This is realized by patterning a micrometer-sized heterojunction consisting of adjacent segments of polycationic and polyanionic hydrogels on a microfluidic chip through simple photocuring steps. The integrated diode rectifies ionic currents being sensitive to the charge of DNA adsorbed onto the polycationic chains through electrostatic associations. Based on the mechanism, we show that the ionic biosensor can electrically quantify DNA in a dynamic range relevant to typical nucleic acid amplification assays. Utilizing the device, we demonstrate the evaluation of a PCR assay amplifying a 500-bp DNA fragment of E. coli, an infection-causing pathogen, and real-time in situ monitoring of an isothermal assay amplifying E. coli whole genome. We anticipate that the device could potentially pave the way for miniaturized optics-free platforms for quantifying nucleic acid amplification at point-of-care.

Keywords: Electrical biosensor; Hydrogel; Ionic diode; Nucleic acid; Point-of-care.

Graphical abstract

Fig. 1

Schematic overview of the hydrogel ionic diode biosensor principle. Electrical detection of DNA based on its intrinsic negative charge is realized through monitoring the modulated ionic rectification behavior induced by electrostatic adsorption of DNA molecules to oppositely charged polycationic hydrogel of the ionic diode.

Fig. 2

(a) Schematic presentation of the monolithically integrated ionic diode microchip and the relevant measurement setup. The superimposed fluorescent micrograph displays the hydrogel heterojunction patterned between the sample and recording channels. The polyelectrolyte hydrogel segments are labeled with respective charged dyes for illustration (green: fluorescein, red: rhodamine B). Scale bar: 100 ​μm. (b) I–V curves depicting the initial state of a representative ionic diode device and the states upon sequential exposure to 80 ​ng/μL negatively charged DNA (500 bp, 10-min incubation) and 1 mg/μL positively charged pLL solutions (5-min incubation). (c) Rectification ratios of a representative device subject to four repeated rounds of DNA and pLL treatments.

Fig. 3

Dose-sensitive detection of DNA with the ionic diode. (a) A real-time trajectory showing the shift in rectification ratio of a representative ionic diode upon exposure to various mass concentrations of 500-bp DNA fragments. (b) Biosensor response to DNA fragments of varying lengths at constant mass (left) and mole (right) concentrations, respectively. (c) Dose-response plot of the presented ionic diodes based on the shift in rectification ratio as a function of DNA mass concentration (incubation time: 10 ​min). The symbols and bars in (b) and (c) represent the mean and standard deviation derived from parallel tests using separate devices (n ​= ​3 for b and n ​= ​5 for c).

Fig. 4

Monitoring PCR progress with the ionic diode. (a) Changes in rectification ratio induced by individual components in a typical PCR: primers (0.4 ​μM each), dNTP (50 ​μM each), polymerase (0.05 units/μL), and purified 500-bp PCR products (80 ​ng/μL). (b) The rectification ratio of the same device subjected to unpurified PCR products terminated at various cycles. After each test, the ionic rectification behavior of the diode was recovered by treating with pLL. (c) PCR amplification curves obtained electrically from the presented ionic diode (red) and optically from real-time quantitative PCR (blue). The symbols and bars represent the mean and standard deviation derived from three separate I–V measurements.

Fig. 5

In situ real-time monitoring of an isothermal MDA reaction amplifying E. coli whole genome. (a) A modified microchip integrating a 2-μL reaction chamber to accommodate the MDA reaction and an adjacent hydrogel ionic diode for in situ monitoring. (b) Fluorescent micrographs (top) and intensity (bottom) of the N-type gel, P-type gel, and reaction chamber, respectively, during the DNA amplification process. The symbols and bars represent the mean and standard deviation derived from fluorescence measured at different positions (n ​= ​3). (c) The electrical and optical readouts recording the progress of a typical MDA reaction. The electrical readout refers to the shift in the rectification ratio of the ionic diode. The optical readout is measured from a parallel tube-based reaction using a commercial optical instrument, Qubit.