Cascaded and nonlinear DNA assembly amplification for sensitive and aptamer-based detection of kanamycin.

Simple, selective and sensitive monitoring of antibiotic residues in food is essential for food safety and human health because of its side effects upon inappropriate usage. Here, with a new label- and enzyme-free significant signal enhancement approach by the coupling of catalytic hairpin assembly (CHA) with nonlinear hybridization chain reaction (nHCR), we developed a fluorescence aptamer sensor for the detection of trace kanamycin in milk samples with high selectivity and sensitivity. The binding of the target kanamycin to the aptamer probe could initiate the CHA between two hairpins for the formation of partial DNA duplexes, which further triggered the nHCR of other three hairpins to yield branched DNA complexes with a multitude of active G-quadruplex structures. The subsequent intercalation of the organic dye, thioflavin T, into G-quadruplex structures resulted in significantly enhanced fluorescence responses for realizing sensitive sensing of kanamycin in the dynamic range of 0.1-300 nM with a detection limit of 46.1 pM. Besides, this strategy could also achieve the monitoring of kanamycin selectively in spiked milk samples. With features of high sensitivity and simplicity in a non-enzyme/label fashion, our signal amplification strategy has high potentials for establishing sensitive and convenient means to monitor various antibiotics.

Nonlinear hybridization chain reaction-based functional DNA nanostructure assembly for biosensing, bioimaging applications.

Hybridization chain reaction (HCR) can be divided into two categories: linear HCR and nonlinear HCR. In traditional linear HCR, the relatively slow kinetics and less sufficient sensitivity largely limit its scope of application. In the nonlinear HCR system, under the trigger of the initiator, the judicious designed substrate sequences (hairpin or hairpin-free) will self-assembly to dendritic or branched DNA nanostructures with exponential growth kinetics. Given the advantages of its enzyme-free, high-order growth kinetic, high sensitivity, and simple operation, nonlinear HCR is regarded as a powerful signal amplifier for the detection of biomarkers by integrating with versatile sensing platforms in the past few decades. In this review, we describe the basic features of nonlinear HCR mechanism and classify the nonlinear HCR into several categories based on their self-assembly mechanisms: the branched HCR, dendritic HCR, hydrogel-based clamped HCR, and other types of HCR. Then, we summarize the recent development of nonlinear HCR in biosensing, such as nucleic acid, protein, enzyme activities, and cancer cell detection, etc., and we also review the current applications of nonlinear HCR in bioimaging (mRNA in situ imaging). We choose several representative works to further illustrate the analysis mechanisms via various detection platforms, such as fluorescence, electrochemical, colorimetric, etc. At last, we also review the challenges and further perspectives of nonlinear HCR in the use of bioanalysis.

Combining competitive sequestration with nonlinear hybridization chain reaction amplification: an ultra-specific and highly sensitive sensing strategy for single-nucleotide variants.

Highly specific and sensitive detection of single-nucleotide variants (SNVs) is of central importance in disease diagnosis and pharmacogenomics. However, it remains a great challenge to successfully detect very low amounts of mutant SNV sequences in real samples in which a SNV sequence may be surrounded by high levels of closely related wild-type sequences. Herein, we propose an ultra-specific and highly sensitive SNV sensing strategy by combining the competitive sequestration with the nonlinear hybridization chain reaction (HCR) amplification. The rationally designed sequestration hairpin can effectively sequester the large amount of wild-type sequence and thus dramatically improve the hybridization specificity in recognizing SNVs. To improve the detection sensitivity, a new fluorescent signal probe is fabricated by intercalating SYBR Green I dye into the nonlinear HCR based DNA dendrimer to further bind with SNVs for signal amplification. The hyperbranched DNA dendrimer possesses large numbers of DNA duplexes for dye intercalation, thus the signal probe shows strong fluorescence intensity, leading to large fluorescence signal amplification. Taking advantage of the improved hybridization specificity of the competitive sequestration and the enhanced fluorescence response of the nonlinear HCR amplification, the developed sensing strategy enables ultra-specific and highly sensitive detection of SNVs. Taking human pancreatic cancers and colorectal carcinomas related KRAS gene mutations as models, the developed strategy shows remarkably high specificity against 17 SNVs (discrimination factors ranged from 126 to 1001 with a median of 310), and achieves high sensitivity for 6 KRAS mutations (the best resultant detection limit reached 15 pM for KRAS G13D (c.38G > A)). Notably, combined with PCR amplification, our SNV sensing strategy could detect KRAS G12D (c.35G > A) from extracted human genomic DNA samples at abundance as low as 0.05%. This work expands the rule set of designing specific and sensitive SNV sensing strategies and shows promising potential application in clinical diagnosis.

An enzyme-free surface plasmon resonance biosensing strategy for detection of DNA and small molecule based on nonlinear hybridization chain reaction.

A label-free and enzyme-free surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive and specific detection of target DNA by employing the nonlinear hybridization chain reaction (HCR) amplification. Nonlinear HCR is a hairpin-free system in which double-stranded DNA monomers could dendritically assemble into highly branched nanostructure upon introducing a trigger sequence. The target DNA partly hybridizes with capture probe on the gold sensing chip and the unpaired fragment of target DNA works as a trigger to initiate the nonlinear HCR, forming a chain-branching growth of DNA dendrimer by self-assembly. Real-time amplified SPR response is observed upon the introduction of nonlinear HCR system. The method is capable of detecting target DNA at the concentration down to 0.85 pM in 60min with a dynamic range from 1 pM to 1000 pM, and could discriminate target DNA from mismatched sequences. This biosensing strategy exhibits good reproducibility and precision, and has been successfully applied for detection of target DNA in complex sample matrices. In addition, the nonlinear HCR based SPR biosensing methodology is extended to the detection of adenosine triphosphate (ATP) by aptamer recognition. Thus, the versatile method might become a potential alternative tool for biomolecule detection in medical research and early clinical diagnosis.

Highly sensitive electrochemical biosensor based on nonlinear hybridization chain reaction for DNA detection.

In the present work we demonstrated an ultrasensitive detection platform for specific DNA based on nonlinear hybridization chain reaction (HCR) by triggering chain-branching growth of DNA dendrimers. HCR was initiated by target DNA (tDNA) and finally formed dendritic structure by self-assembly. The electrochemical signal was drastically enhanced by capturing multiple catalytic peroxidase with high-ordered growth. Electrochemical signals were obtained by measuring the reduction current of oxidized 3, 3', 5, 5'-tetramethylbenzidine sulfate (TMB), which was generated by HRP in the presence of H2O2. This method exhibited ultrahigh sensitivity to tDNA with detection limit of 0.4 fM. Furthermore, the biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences.