•AuNPs/G/MWCNTs nanostructure was prepared.•Glucose oxidase was immobilized onto the structure via electrostatic attraction.•The direct electron transfer was achieved between the enzyme and the electrode.•The glucose biosensor fabricated exhibited satisfactory analytical performance.In this work, poly (diallyldimethylammonium chloride) (PDDA)-capped gold nanoparticles (AuNPs) functionalized graphene (G)/multi-walled carbon nanotubes (MWCNTs) nanocomposites were fabricated. Based on the electrostatic attraction, the G/MWCNTs hybrid material can be decorated with AuNPs uniformly and densely. The new hierarchical nanostructure can provide a larger surface area and a more favorable microenvironment for electron transfer. The AuNPs/G/MWCNTs nanocomposite was used as a novel immobilization platform for glucose oxidase (GOD). Direct electron transfer (DET) was achieved between GOD and the electrode. Field emission scanning electron microscopy (FESEM), UV–vis spectroscopy and cyclic voltammetry (CV) were used to characterize the electrochemical biosensor. The glucose biosensor fabricated based on GOD electrode modified with AuNPs/G/MWCNTs demonstrated satisfactory analytical performance with high sensitivity (29.72 mA M−1 cm−2) and low limit of detection (4.8 µM). The heterogeneous electron transfer rate constant (ΚS) and the apparent Michaelis–Menten constant (Km) of GOD were calculated to be 11.18 s−1 and 2.09 mM, respectively. With satisfactory selectivity, reproducibility, and stability, the nanostructure we proposed offered an alternative for electrode fabricating and glucose biosensing.

•Differential pulsed amperometry on microchip is reported for the first time.•The nature of the novel pulse electrochemical technique is discussed.•Comparable detection sensitivity to triple pulsed amperometry is demonstrated.•The present detection strategy broadens electroanalytical community.•This electrochemical protocol has potential for other fluid analysis methods.In this work, we describe a novel electrochemical detection method, differential pulsed amperometry (DPA) on microchip capillary electrophoresis (MCE). In a pulse period, a sequential two-step sampling is executed at two different potentials (E1 and E2). Differential current signal of the duplex sampling events is recorded that functions as time domain. The performance of this detection scheme was evaluated by separating and detecting three model analytes including tyramine (Tym), tryptophan (Trp), and p-aminobenzoic acid (PABA). Multiple parameters that would affect electrochemical response and peak shape, such as sampling potential, sampling time, and electrode cleaning time, were investigated. This pulse technique exhibits better sensitivity over constant potential amperometry (CPA), nearly equal to triple pulsed amperometry (TPA). More importantly, DPA can generate more stable baseline than TPA, primarily due to the background subtraction through the two-step sampling, which is beneficial to further improve analytical sensitivity. In the optimal condition, the limits of detection for Tym, Trp and PABA, were down to 0.27 μM, 0.32 μM and 1.1 μM, respectively. DPA detection opens up a new avenue for microchip electrochemistry, and can be virtually extended to other fluid analysis techniques.