•Target-initiated modification of capture probes without prior need for a reporter probe or signal amplifier.•Duplex-specific nuclease and terminal deoxynucleotidyl transferase-based target recycling and labeling amplification.•Isothermal dual-amplification method for the detection of multiple miRNAs by a Luminex xMAP array.Herein we exploited a novel target-initiated labeling strategy for the multiplex detection of microRNAs (miRNAs) by coupling duplex-specific nuclease (DSN) with terminal deoxynucleotidyl transferase (TdT). In the presence of target miRNA, the immobilized and 3′-blocked capture probes hybridized with target and thus the formed DNA-RNA hybrid was recognized by DSN. DSN mediated the digestion of 3′-phosphated capture probes (CPs) in the hybrids and synchronously target was released and recycled for another round of hybridization and cleavage. The cleaved CP fragments with a free 3′-OH were then elongated and labeled with multiple biotin-dUTP nucleotides by TdT. Fluorescence reporter streptavidin-phycoerythin was finally added to react with the immobilized biotins and render fluorescence signals. This dual-amplification labeling strategy was successfully demonstrated to sensitively detect multiple miRNAs, taking advantage of DSN-mediated target recycling and TdT-catalyzed multiple signal modification with analysis by a commercial Luminex xMAP array platform. Our experimental results showed the simultaneous quantitative measurement of three sequence-specific miRNAs at concentrations from 1 pM to 2.5 nM. Attempts were also made to directly detect miRNAs in total RNA extracted from cancer cells. The dual-amplification labeling strategy reported here shows a great potential for the development of a method for the multiplexed, sensitive, selective, and simple analysis of multiple miRNAs in tissues or cells for biomedical research and clinical early diagnosis.Download high-res image (144KB)Download full-size image

Magnetic bead (MB)-based chemiluminescence (CL) ELISA can be a sample-thrifty, time-saving tool for evaluation of cigarette smoke-induced DNA single-strand breaks (SSBs) with high specificity. This article describes a novel approach using immobilized oligonucleotide on MBs to determine cigarette smoke-induced DNA SSBs and screen some protective natural compounds. Typically, fluorescein-labeled DNA (FAM-DNA) was immobilized on the MBs and then oxidized by the smoke in the absence or presence of natural compounds, and a part of FAM-DNA was fragmented due to cigarette smoke-induced DNA SSB and then detached from MBs whereas other non-broken FAM-DNA still remained on MBs. Then, any broken FAM-DNA fragments, complex tobacco smoke matrix, and other stuff related with natural compounds were conveniently washed away by a magnetic force, and thus possible interfering substances were completely removed. Finally, those remaining non-broken FAM-DNA on MBs were reacted with HRP-labeled anti-fluorescein antibody and then detected by CL ELISA. CL signal was converted to molar concentrations of the FAM-DNA by interpolation from a pre-determined standard linear calibration curve. The level of DNA SSBs induced by cigarette smoke was thus calculated using the method. A library of 30 natural products was subsequently screened, and two among them were found to protect DNA from oxidative damage and thus may be promising compounds for the development of new drugs. The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of DNA SSBs.

Apratoxin A, a cyclodepsipeptide isolated from a marine cyanobacterium, demonstrates potent cytotoxicity against cancer cell lines by a unique mechanism. A lactam analogue of apratoxin A, named as amidapratoxin A, was efficiently synthesized over 22 linear steps in 2.1% overall yield for the first time. The further conformation analysis was conducted by NMR techniques and computer-based molecular modeling. The results showed that the orientation of the benzene ring in tyrosine moiety, the iso-butyl in isoleucine moiety and hydroxyl group in Ahtmna moiety are quite different from that of apratoxin A, which might result in a significant decrease of the cytotoxicity. While further investigation is on the way, these results provide increased understanding of conformation-activity relationship for apratoxin family members which is an important complement to structure-activity relationship.Apratoxin A is a cyclodepsipeptide with potent cytotoxicity against several human cancer cell lines. To further explore the conformation-activity relationship of apratoxin family members, we designed a lactam analogue of apratoxin A named as amidapratoxin A and completed its first synthesis with a concise approach. Amidapratoxin A showed three orders of magnitude less inhibitory potency compared to apratoxin A. Based on the molecular modeling with constraints from the NMR data, we presented the structural model of amidapratoxin A in solvent and illustrated that the orientation variation of the benzene ring in tyrosine moiety, the iso-butyl in isoleucine moiety and hydroxyl group in Ahtmna moiety may play critical role in cytotoxicity decrease. While further investigation is underway, the results above provide an important reference for structural optimization and efficient synthesis of apratoxin A.

We have designed and evaluated novel stem-loop-structured probes for fluorescence detection of multiple microRNA (miRNA) targets. In the initial stage, the probes are in a closed stem conformation, shielding sterically a biotin label from being accessible to a fluorescence reporter. After hybridizing with target miRNAs, the probes undergo a conformational switch, restoring accessibility of the biotin to streptavidin–phycoerythin (SA–PE) for signal readout. Apparently, the bulky nature of the reporter SA–PE facilitates shielding of the biotin label in the absence of the target, thereby the stem–loop-structured probes allow sensitive detection of unlabeled miRNA targets, and xMAP array microspheres further realize simultaneous detection of multiple analytes using one fluorescence dye, SA–PE, for final readout. Here we demonstrated a successful multiplex assay for quantitative measurement of miRNA21, miRNA222, miRNA20a, and miRNA223, which are associated with nonsmall cell lung cancer. The approach can be extended to detecting an increasing number of targets for various indications. We believe such advancements represent a significant improvement for early disease diagnosis and prognosis.

In this article, we demonstrate the fabrication and simultaneous fluorescent detection of two biomarkers related to lung cancer. Polystyrene microspheres (PSM) were introduced as biomolecular immobilizing carriers and a 96-well filter plate was used as the separation platform. The whole experiment could be effectively carried out in a homogeneous system, as exemplified by the detection of carcinoembryonic antigen (CEA) and neuron specific enolase (NSE). First, two capture antibodies for CEA and NSE were immobilized on the PSM surface. Next, they reacted successively with two antigens and two modified detection antibodies. Finally, these two biomarkers could be recognized by streptavidin-conjugated quantum dots (QD) and goat-anti-FITC conjugated QD with a detection limit of 0.625 ng mL−1, which was lower than the clinical cut-off level. The protocol showed good precision within 6.36% and good recovery in the range of 90.86–105.02%. Compared with several other assay formats reported previously, our new technique is competitive or even better. Furthermore, the immunosensor was successfully illustrated in 20 serum samples. Overall, this new immunoassay offers a promising alternative for the detection of biomarkers related to cancer diseases, taking advantage of simplicity, specificity, sensitivity and cost-efficiency.Graphical abstractHighlights► A new cross-talk-free duplex fluoroimmunoassay for cancer related biomarkers was developed using multiple QD as detection elements with the LOD of 0.625 ng/mL. ► A fast homogeneous immunoreaction as well as a simple heterogeneous separation process was achieved by the coupling of the submicrometer-sized polystyrene microspheres as the carrier and the 96-well filter plate as the reaction and separation container. ► This new approach could also be extended to detect other biomarkers relating to other cancers, such as alpha fetoprotein and prostate specific antigen associated with liver cancer and prostrate cancer, etc.

A robust, selective and highly sensitive chemiluminescent (CL) platform for protein assay was presented in this paper. This novel CL approach utilized rolling circle amplification (RCA) as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier. Typically, the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer–primer sequence. This aptamer–primer sequence was then employed as the primer of RCA. Based on this design, a number of the biotinylated probes and streptavidin–horseradish peroxidase (SA–HRP) were captured on the plate, and the CL signal was amplified. In summary, our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized, and devoid of light source. Therefore, this new technique will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases, by taking advantages of high sensitivity and selectivity.