Sensitive detection of low-abundance driver mutations may provide valuable information for precise clinical treatment. Compared to next-generation sequencing and droplet digital PCR methods, fluorescent probes show great flexibility in rapid detection of specific mutations with high sensitivity and easily accessible instruments. However, existing approaches with fluorescent probes need an additional step to convert duplex DNA to single-stranded DNA (ssDNA) before the detection step, which increases the time, cost, and risk of loss of low-input target strands. In this work, we attempt to integrate the ssDNA-generation step with the subsequent detection into a programable one-pot reaction by employing lambda exonuclease (λ exo), a versatile nanopore nuclease which exercises different functions on different substrates. The capability of λ exo in discrimination of mismatched bases in 5′- FAM-ended 2 nt-unpaired DNA duplexes was first demonstrated. Specific fluorescent probes were developed for EGFR exon 19 E746-A750del and PIK3CA E545K mutations with discrimination factors as high as 8470 and 884, respectively. By mixing the probes and λ exo with the PCR products of cell-free circulating DNA extracted from plasma samples, the reaction was immediately initiated, which allowed sensitive detection of the two types of mutations at an abundance as low as 0.01% within less than 2 h. Compared to existing approaches, the new method has distinct advantages in simplicity, low cost, and rapidity. It provides a convenient tool for companion diagnostic tests and other routine analysis targeting genetic mutations in clinical samples.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and “interactomes” of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio‐nanocomposite with antibody‐like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin‐modified surface of silica‐coated magnetic nanoparticles, we first added 3‐aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2‐acrylamido‐2‐methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG‐NH2). The resulting molecularly imprinted polymer‐based bio‐nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio‐nanocomposite with high activity. The bio‐nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.

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