Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging.

Accurate identification of cancer cells is an essential prerequisite for cancer diagnosis and subsequent effective curative interventions. The logic-gate-assisted cancer imaging system that allows a comparison of expression levels between biomarkers, rather than just reading biomarkers as inputs, returns a more comprehensive logical output, improving its accuracy for cell identification. To fulfill this key criterion, we develop a compute-and-release logic-gated double-amplified DNA cascade circuit. This novel system, CAR-CHA-HCR, consists of a compute-and-release (CAR) logic gate, a double-amplified DNA cascade circuit (termed CHA-HCR), and a MnO2 nanocarrier. CAR-CHA-HCR, a novel adaptive logic system, is designed to logically output the fluorescence signals after computing the expression levels of intracellular miR-21 and miR-892b. Only when miR-21 is present and its expression level is above the threshold CmiR-21 > CmiR-892b, the CAR-CHA-HCR circuit performs a compute-and-release operation on free miR-21, thereby outputting enhanced fluorescence signals to accurately image positive cells. It is capable of comparing the relative concentrations of two biomarkers while sensing them, thus allowing accurate identification of positive cancer cells, even in mixed cell populations. Such an intelligent system provides an avenue for highly accurate cancer imaging and is potentially envisioned to perform more complex tasks in biomedical studies.

A protein enzyme-free strategy for fluorescence detection of single nucleotide polymorphisms using asymmetric MNAzymes.

To establish protein enzyme-free and simple approach for sensitive detection of single nucleotide polymorphisms (SNPs), the nucleic acid amplification reactions were developed to reduce the dependence on protein enzymes (polymerase, endonuclease, ligase). These methods, while enabling highly amplified analysis for the short sequences, cannot be generalized to long genomic sequences. Herein, we develop a protein enzyme-free and general SNPs assay based on asymmetric MNAzyme probes. The multi-arm probe (MNAzyme-9M-13) with two asymmetric recognition arms, containing a short (9 nt) and a long (13 nt) arm, is designed to detect EGFR T790 M mutation (MT). Owing to the excellent selectivity of short recognition arm, MNAzyme-9M-13 probe can efficiently avoid interferences from wild-type target (WT) and various single-base mutations. Through a one-pot mixing, MNAzyme-9M-13 probe enables the sensitive detection of MT, without protein enzyme or multi-step operation. The calculated detection limit for MT is 0.59 nM and 0.83%. Moreover, this asymmetric MNAzyme strategy can be applied for SNPs detection in long genomic sequences as well as short microRNAs (miRNAs) only by changing the low-cost unlabeled recognition arms. Therefore, along with simple operation, low-cost, protein enzyme-free and strong versatility, our asymmetric MNAzyme strategy provides a novel solution for SNPs detection and genes analysis.

A DNAzyme-based normalized fluorescence strategy for direct quantification of endogenous zinc in living cells.

Taking the maximum fluorescence of an identical fluorophore as a reference, a DNAzyme-based normalized strategy is developed to unify the output signals under external interferences. This makes it possible to directly quantify endogenous zinc in living cells by in situ fluorescence imaging, implying promising potential in fundamental study and early disease diagnosis.

Covalent Amide-Bonded Nanoflares for High-Fidelity Intracellular Sensing and Targeted Therapy: A Superstable Nanosystem Free of Nonspecific Interferences.

A nanoflare, a conjugate of Au nanoparticles (NPs) and fluorescent nucleic acids, is believed to be a powerful nanoplatform for diagnosis and therapy. However, it highly suffers from the nonspecific detachment of nucleic acids from the AuNP surface because of the poor stability of Au-S linkages, thereby leading to the false-positive signal and serious side effects. To address these challenges, we report the use of covalent amide linkage and functional Au@graphene (AuG) NP to fabricate a covalent conjugate system of DNA and AuG NP, label-rcDNA-AuG. Covalent coating of abundant amino groups (-NH2) onto the graphitic shell of AuG NP efficiently facilitates the coupling with carboxyl-labeled capture DNA sequences through simple, but strong, amide bonds. Importantly, such an amide-bonded nanoflare possesses excellent stability and anti-interference capability against the biological agents (nuclease, DNA, glutathione (GSH), etc.). By accurately monitoring the intracellular miR-21 levels, this covalent nanoflare is able to identify the positive cancer cells even in a mix of cancer and normal cells. Moreover, it allows for efficient photodynamic therapy of the targeted cancer cells with minimized side effects on normal cells. This work provides a facile approach to develop a superstable nanosystem showing promising potential in clinical diagnostics and therapy.