Heteromultivalent DNA Enhances the Assembly Yield of Hybrid Nanoparticles and Facilitates Dynamic Disassembly for Bioanalysis Using ICP-MS.

To obtain enhanced physical and biological properties, various nanoparticles are typically assembled into hybrid nanoparticles through the binding of multiple homologous DNA strands to their complementary counterparts, commonly referred to as homomultivalent assembly. However, the poor binding affinity and limited controllability of homomultivalent disassembly restrict the assembly yield and dynamic functionality of the hybrid nanoparticles. To achieve a higher binding affinity and flexible assembly choice, we utilized the paired heteromultivalency DNA to construct hybrid nanoparticles and demonstrate their excellent assembly characteristics and dynamic applications. Specifically, through heteromultivalency, DNA-functionalized magnetic beads (MBs) and gold nanoparticles (AuNPs) were efficiently assembled. By utilizing ICP-MS, the assembly efficiency of AuNPs on MBs was directly monitored, enabling quantitative analysis and optimization of heteromultivalent binding events. As a result, the enhanced assembly yield is primarily attributed to the fact that heteromultivalency allows for the maximization of effective DNA probes on the surface of nanoparticles, eliminating steric hindrance interference. Subsequently, with external oligonucleotides as triggers, it was revealed that the disassembly mechanism of hybrid nanoparticles was initiated, which was based on an increased local concentration rather than toehold-mediated displacement of paired heteromultivalency DNA probes. Capitalizing on these features, an output platform was then established based on ICP-MS signals that several Boolean operations and analytical applications can be achieved by simply modifying the design sequences. The findings provide new insights into DNA biointerface interaction, with potential applications to complex logic operations and the construction of large DNA nanostructures.

Protein-Recognition-Initiated Exponential Amplification Reaction (PRIEAR) and Its Application in Clinical Diagnosis.

Isothermal exponential amplification technology has rarely been fabricated as a universal sensing platform for the detection of proteins. To broaden their application, we have developed a strategy, named protein-recognition-initiated exponential amplification reaction (PRIEAR) using protein recognition to induce DNA assembly, which converts protein recognition events into ssDNA amplicons and combines two-stage amplification to achieve exponential amplification. Taking advantage of this principle, diverse biomarkers can be quantified at sub-picomolar concentrations in a homogenous manner, making PRIEAR suitable for clinical settings. Therefore, this strategy can expand the powerful isothermal exponential amplification technology to protein targets and thus provide a new toolbox in clinical and biomedical applications.

Activation of catalytic DNAzyme by binding-induced DNA displacement for homogeneous assay.

The sensitive assays for protein, especially for the DNA-based assay, are often dependent on the amplification procedure with assistance of enzyme. Compared with a protein enzyme, deoxyribozyme (DNAzyme) exhibits similar catalytic activity and specificity and better flexibility. In this work, we streamlined the binding induced DNA displacement (BINDD) strategy for the activation of DNAzyme cleavage. Since the intrinsic element of DNAzyme is the nucleic acids, it is easy to join the BINDD by hybridization with an affinity probe. The activity of DNAzyme was initiated by the BINDD reaction mediated by the recognition affinity probe with target proteins. Upon DNAzyme release, it was able to catalyze and cleave the predesigned substrates, generating the enhanced fluorescence signal indicating the protein concentration. Such a constructed homogeneous assay is available and effective in human serum when it was used for detection of platelet derived growth factor-BB (PDGF-BB) and prostate specific antigen (PSA), with detection limits of 100 pM and 200 pM, respectively.

Spatially Constrained DNA Nanomachines To Accelerate Kinetics in Response to External Input: Design and Bioanalysis.

Cells take advantage of the spatial organization to accelerate the reaction kinetics of diverse components within a crowded intracellular environment. Inspired by this, we hereby designed a principle of spatial constraint to overcome limitations of response kinetics in DNAzyme-powered DNA nanomachines. First, we proposed the type-1 of spatially constrained DNA nanomachines (scDN-1) by co-localizing the aptamer probe and power unit (DNAzyme), allowing the DNA nanomachines to accomplish faster cyclic cleavage of DNAzyme as intramolecular reactions. To expand the scDN into the clinical practice, Type 2 spatially constrained DNA nanomachines (scDN-2) with constrained antibody probes were then constructed through Holliday junction assembly, which increased the effective local concentration to obtain the improved kinetics. With an accelerated response kinetics, this design principle allows DNA nanomachines to accomplish the response to tumor markers in real patients' samples within 30 min, significantly broadening the bioanalytical applications of DNA nanomachines to clinical practice.

Development of a highly sensitive and specific monoclonal antibody-based ELISA coupled with immuno-affinity extraction for the detection of anticancer drug 5-fluorouracil in blood samples.

5-Fluorouracil (5-FU) is an effective anticancer drug widely used in cancer treatment. In this study, two 5-FU derivatives containing a spacer arm with the carboxylic group at the end were synthesized, which were linked to the carrier proteins to form 5-FU-protein conjugates used as the immunogens for the production of monoclonal antibody (mAb). Based on the produced mAb, the highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for 5-FU detection was established. The IC50 and LOD values of the assay were found to be 19.5 ng mL-1 and 0.5 ng mL-1, respectively. There was no cross-reactivity (CR) of the ELISA with cytosine, thymine and uracil, which avoided the interference from inherent pyrimidines. The CR values of the assay with three substitutes of 5-FU (tegafur, 5-fluoro-2'-deoxyuridine, carmofur) were within 9.7%-17.6%. The produced mAb was also applied in sample extraction. The immuno-affinity column capable specific capturing 5-FU was prepared by immobilizing the mAb on Sepharose-4B gel and filling into a SPE column. The recoveries of 5-FU in spiked samples measured by ELISA were 72.4%-90.7% with RSD of 3.6%-8.3%. Five blood samples collected from patients were extracted by immuno-affinity column, then measured by ELISA and confirmed by HPLC-MS/MS. There was a good correlation between HPLC-MS/MS and ELISA. It is demonstrated that the developed ELISA combined with immuno-affinity extraction can be a powerful alternative method for the detection of 5-FU in blood samples.

Monitoring nucleic acid amplification process using a UiO-66-NH2-based fluorescent sensor.

Herein, we developed a nucleic acid amplification process monitoring scheme by the use of UiO-66-NH2, in which pyrophosphate ion (PPi) released from the amplification can competitively coordinate with Zr to weaken the ligand-to-metal charge transfer and cause fluorescence recovery, thus reflecting the amplification process. This is the first attempt at using a MOF for monitoring the nucleic acid amplification process.

Biomolecule-guided co-localization of intermolecular G-rich strands for the construction of a tetramolecular G-quadruplex sensing strategy.

We herein introduce the principle of proximity assay into tetramolecular G-quadruplexes guided by various biomolecules for the construction of a sensing strategy. Our strategy is based on the co-localization of intermolecular G-rich strands guided by a recognition event of a specific biomolecule to its corresponding affinity ligand. In such case, the local concentration among intermolecular strands is significantly increased to trigger the following self-assembly that served as the peroxidase-mimicking activity. This strategy is versatile, homogenous and adaptable to different types of biomolecules.