Single-particle electrochemical collision has gained great achievements in fundamental research, but it is challenging to use in practice on account of its low collision frequency and the interference of the complex matrix in actual samples. Here, magnetic separation and DNA walker amplification were integrated to build a robust and sensitive single-particle electrochemical biosensor. Magnetic nanobeads (MBs) can specifically capture and separate targets from complex samples, which not only ensures the anti-interference capability of this method but also avoids the aggregation of platinum nanoparticles (Pt NPs) caused by numerous coexisting substances. A low amount of targets can lead to the release of more Pt NPs and the generation of more collision current transients, realizing cyclic amplification. Compared with simple hybridization, a DNA walker can improve the collision frequency by about 3-fold, greatly enhancing detection sensitivity, and a relationship between collision frequency and target concentration is used to realize quantification. The biosensor realized an ultrasensitive detection of 4.86 fM human immunodeficiency virus DNA (HIV-DNA), which is 1–4 orders of magnitude lower than that of traditional methods. The successful HIV-DNA detection in complex systems (serum and urine) demonstrated a great promising application in real samples and in the development of new single-entity biosensors.

Sensitive detection of human immunodeficiency virus DNA (HIV-DNA) is essential for timely diagnosis and cure of the illness. Herein, a novel “enrichment–stowage–cycle” strategy was proposed to fabricate a multiple amplified electrochemiluminecence (ECL) biosensor for HIV-DNA detection. On the basis of the enrichment role of magnetic nanobeads, assembly role of copolymer nanospheres and strand displacement amplification (SDA), the processes were named as “enrichment”, “stowage”, and “cycle”, respectively. The method employed electrochemiluminescent nanospheres (ENs) as signal labels by assembling three layers of CdSe/ZnS quantum dots (QDs) onto the surface of copolymer nanospheres. Compared to QDs, the same concentration of ENs can the enhance the ECL intensity by about 11.3-fold. SDA could further amplify the signals by about 3.77-fold, possessing high sensitivity for low-abundant biomarkers detection. The integration of magnetic separation improved detection specificity and stability, making the method possible for practical application. On the basis of magnetic separation, ENs and SDA, the ECL biosensor realized ultrasensitive detection of 39.81 fM HIV-DNA, which was more sensitive than other HIV-DNA analytical methods, with a wide dynamic range of 0.05 pM to 50 nM. The successful detection of HIV-DNA in complex samples with good sensitivity and accuracy indicated its potential utilization in early judgment of diseases and fabrication of signal amplification platforms.

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