Ultrasensitive detection of the H5N1 nucleic acid fragment by ICP-MS using DNA dendrimer-carried silver nanoparticle labeling.

The importance of avian influenza virus (AIV) detection in clinical diagnosis and prognosis has been deeply recognized. In this study, the ultrasensitive detection of AIV subtype H5N1 was achieved by ICP-MS combined with DNA dendrimer-carried silver nanoparticle (AgNP) labeling. First, a magnetic control system was constructed by anchoring double-strand DNAs (dsDNAs) which contained a complementary sequence of H5N1 and two locked triggers on the surface of magnetic beads (MBs). When H5N1 was present, the two triggers were released and initiated dendrimer hybridization chain reactions which led to the generation of DNA dendrimer-carried AgNPs on the surface of the MBs. Finally, the AgNPs were collected via magnetic separation, digested by nitric acid, and tested using ICP-MS. The signal intensities of 107Ag were positively correlated with the concentrations of H5N1. Notably, the DNA dendrimer assembly contributed to significant signal amplification and good sensitivity with the limit of detection as low as 2.0 × 10-11 mol L-1. Moreover, the method displayed favorable selectivity against mismatched H5N1 and good recoveries in human serum samples. It is a promising analytical tool for the H5N1 virus and other subtypes of AIV, and has potential value in clinical diagnosis and prognosis of infectious diseases.

Magnetic separation-assisted cluster-amplified versatile fluorescent aptasensors for the sensitive detection of target biomolecules.

A sensitive and versatile platform for detecting diverse target biomolecules was developed by combining a magnetic separation module and a fluorescence amplification module in a plug-and-play manner. The magnetic separation module was constructed using magnetic beads (MBs), whose surfaces were modified with aptamer-blocked captor DNAs. The fluorescence amplification module was constructed by loading the fluorescent dye rhodamine 6G (Rh6G) into the pores of mesoporous silica nanoparticles (MSNs). The MSN surfaces were modified with prey DNAs, of which the MSN-near ends hybridized with complementary DNAs (sealing DNAs) to form duplexes to seal the pores, and the free ends were designed to be single-stranded that were complementary to the captor DNAs. Upon binding of targets to their aptamers, the captor DNAs were unblocked and thus were able to hybridize with the prey DNAs, to capture Rh6G-laden MSNs, forming MB-MSN clusters. The clusters were isolated by magnetic separation and heated to dissociate the DNA duplexes, to unseal the MSN pores and release the inner Rh6G; thus a target was converted into a cluster of Rh6G dyes. By simply changing the target aptamers and related DNA connectors, this strategy detected ATP, thrombin, and platelet-derived growth factor BB with detection limits of 2.1 nM, 4.1 pM, and 2.4 pM, respectively. A wide range of targets, high amplification efficiency and universal functional modules endow the aptasensors with good potential as versatile platforms for detecting target molecules in vitro and in medical research.

A magnetic DNAzyme walker for both in-situ imaging and sensitive detection of MUC1 on living cells.

Mucin 1 (MUC1) is a transmembrane glycoprotein commonly expressed in epithelial cells with stable levels and polarized distribution. Their expression levels and spatial distribution abnormally altered during oncogenesis and play tumor-promoting roles synergistically. We herein propose a magnetic DNAzyme walker (MDW) for both in-situ imaging and sensitive detection of MUC1. This MDW was constructed by modifying specially designed track strands (TSs) and walking strands (WSs) on a streptavidin magnetic bead (SA-MB). The TSs contained cleavage sites for DNAzymes and were labeled with Cy3 at free ends. The WSs contained DNAzyme sequences and were firstly blocked by hybridizing with Cy5-labeled aptamers of MUC1. The DNAzymes were unlocked upon aptamers binding to MUC1 on cells. MDWs were then transferred to a buffer suitable for DNAzyme action, where the unlocked DNAzymes cleaved multiple TSs, releasing amplified Cy3-fragments, which were separated from the uncleaved ones by magnetic separation. In-situ imaging of MUC1 were achieved by the fluorescence of Cy5 on aptamers bound to MUC1. Sensitive detection of MUC1 were achieved by the amplified fluorescence of released Cy3. In-situ imaging and walker operation for detection were triggered by the same targets at the same time, ensuring the signals are real-time correlative. Moreover, MDWs' operation was separated from cells, reducing interference between imaging and detection. The proposed MDW offers a potential approach for comprehensive analysis of MUC1 in early diagnosis and progression assessment of tumor.

Primer-template conversion-based cascade signal amplification strategy for sensitive and accurate detection of polynucleotide kinase activity.

Here, a primer-template conversion-based cascade signal amplification strategy is described for the sensitive detection of polynucleotide kinase (PNK) activity. This strategy integrated rolling circle amplification (RCA) and multiple-repeated-strand displacement amplification (MRSDA) with G-quadruplex based fluorescence lighting-up assay. A delicate dumbbell-shaped DNA probe with 5'-hydroxyl terminus was designed, in which G-quadruplex and half recognition site of nicking enzyme Nb.BbvCI were encoded in two loops respectively. Under the action of PNK, the 5' terminus on dumbbell probe was firstly phosphorylated, and then the dumbbell was cyclized with the catalyzation of T4 ligase to become the RCA template. The RCA process produced multiple copies of the prolonged primer. After that, under the assistance of nicking enzyme Nb.BbvCI, a primer-template conversion occurred, which converted the primer and template of RCA into the template and primer of the subsequent MRSDA, respectively. The MRSDA generated multiple repeated ssDNA sequences which possessed G-quadruplexes for outputting signal by lighting-up fluorescence of thioflavin T (ThT). The cascade signal amplification of RCA and MRSDA provided high detection sensitivity, and the target-dependence of template in cascade signal amplification led to a low background. The method showed excellent detection limit of 0.2 × 10-6 U µL-1 in buffer and 5 cells in cell lysate sample. Moreover, this method displayed favorable selectivity when interfering proteins were present. The developed strategy has good practical potential for PNK activity detection in clinical diagnosis and medical research.

High-performance liquid chromatographic enantioseparation of 3,5-disubstituted hydantoins analogs and temperature-induced reversals of elution orders on a polysaccharide-based chiral stationary phase.

Enantioseparations were achieved for eleven 3,5-disubstituted hydantoins in HPLC under the normal phase mode using Chiralpak IA. The effects of polar alcoholic modifier and column temperature on retention and enantioseparation were determined. Importantly, we found two kinds of enantiomer elution order (EEO) reversals, which include solvent-induced EEO reversal for compound 9 and temperature-induced EEO reversals for compound 3 and compound 6. The phenomena of these EEO reversals were described for the first time in present work, which is helpful to elucidate the chiral separation mechanism of these hydantoins.