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Applications of conventional linear ligation-rolling circle amplification (RCA) are restricted by the sophisticated operation steps and unsatisfactory picomolar-level detection limits. We herein demonstrate an RCA-based cascade amplification reaction that converts a side-reaction to secondary amplification, which improves the detection limit and simplifies the operation compared to linear ligation-RCA assays. The proposed nicking-assisted enzymatic cascade amplification (NECA) comprises an on-loop amplification reaction using circular templates to generate intermediate amplicons, and an off-loop amplification reaction using intermediate amplicons as primers for end amplicons. The whole NECA reaction is homogeneous and isothermal. Amplicons anneal to detection probes that are grafted onto magnetic nanoparticles (MNPs), such that MNP clusters form and can be detected in real-time using optomagnetic measurements. The optomagnetic sensor detects the presence and size increase of MNP clusters by optical transmission measurements in an oscillating magnetic field. A detection limit of 2 fM was achieved with a total assay time of ca. 70 min. By combining optomagnetic readouts of signal phase lag and hydrodynamic size increase of MNPs, NECA-based target quantification provided a wide dynamic detection range of ca. 4.5 orders of magnitude. Moreover, the specificity and the serum detection capability of the proposed method were investigated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)