Sensitive and specific detection of tumor exosomes is of great significance for early cancer diagnosis. In this paper, we report an aptamer strategy for exosome detection based on aptamer recognition-induced multi-DNA release and cyclic enzymatic amplification. First, we use aptamer-magnetic bead bioconjugates to capture tumor exosomes derived from LNCaP cells, leading to the release of three kinds of messenger DNAs (mDNAs). After magnetic separation, the released mDNAs hybridized with the probe DNAs immobilized on a gold electrode. Electroactive Ru(NH) was used as the signal reporter because of its electrostatic attraction to DNA. Subsequent Exo III cyclic digestion caused the electrochemical signal to "turn off". Because the electrochemical signal reflects the concentration of Ru(NH) and the concentration of Ru(NH) is correlated with the mDNA concentration, which is correlated with the exosome concentration, the tumor exosomes can be detected by examining the decrease in the peak current of Ru(NH). In this paper, the signal was amplified by the numerous mDNAs released from the magnetic bead and the Exo III-assisted mDNA recycling. Under the optimal conditions, a detection limit down to 70 particles/μL was achieved, which is lower than the LODs of most currently available methods. Furthermore, this assay can be used to detect tumor exosomes in complex biological samples, demonstrating potential application in real sample diagnosis.

Effective detection of DNA methyltransferase (DNMT) activity is significant for cancer research. Herein, we developed a sensitive electroanalytical method to detect human DNA (cytosine-5)-methyltransferase 1 (DNMT1) from crude lysates of cancer cells. In this assay, capture DNA having a preferred DNMT1 methylation site was immobilized on a gold electrode and then hybridized with gold nanoparticle (Au NP)-DNA complexes. The modified electrodes were equilibrated with the lysate and then incubated with methylation-sensitive restriction enzyme. If the lysate was negative for DNMT1 activity, the Au NP-DNA complexes would be cut by the restriction enzyme and released from the electrode. Conversely, restriction enzyme cleavage would be blocked by the fully methylated duplexes, and the Au NP-DNA complexes would remain on the electrode. Electroactive Ru(NH) was used as the signal reporter, because of its electrostatic attraction to DNA, resulting in an electrochemical signal. Since the electrochemical signal reflects the amount of Ru(III) redox and the amount of Ru(III) redox is correlated with the activity of DNMT1, the activity of DNMT1 is proportional to the electrochemical signal. The signal could be amplified by the numerous DNAs on the Au NPs and further amplified by Ru(III) redox recycling. With this method, a detection limit down to 0.3 U/mL for pure DNMT1 and 8 MCF-7 cells was achieved. DNMT1 activities of different cell lines were also successfully evaluated.

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Existing disulfide-rich peptides, both naturally occurring and de novo designed, only represent a tiny amount of the possible sequence space because natural evolution and de novo design only keep sequences that are structurally approachable by correct disulfide pairings. To bypass this limitation for designing new peptide scaffolds beyond the natural sequence space, we dedicate to developing novel disulfide-rich peptides with predefined disulfide pairing patterns irrelevant to primary sequences. However, most of these designed peptides still suffer from disulfide rearrangements to at least one to three possible isomers. Here, we report a general and reliable strategy for the design and synthesis of a range of structurally diverse cross-link-dense peptide (CDP) scaffolds with two orthogonal disulfide bonds and a bisthioether bridge that are not subject to disulfide isomerizations. Altering the pattern of cysteine and penicillamine generates hundreds of different CDP scaffolds tolerant to extensive sequence manipulations. This work thus provides many useful scaffolds for the design of functional molecules such as protein binders with improved proteolytic stability (e.g., designed by epitope grafting).