A near-infrared light-controlled, ultrasensitive one-step photoelectrochemical detection of dual cell apoptosis indicators in living cancer cells.

Here, a near-infrared (NIR) light-controlled, ultrasensitive one-step photoelectrochemical (PEC) strategy was constructed to simultaneously detect cell apoptosis indicators, phosphatidylserine (Pho) and sodium-potassium adenosine triphosphatase (Sat), on living cancer cells. Using NIR light as excitation, the signal probe methylene blue (Tagkinetic) could be released, leading to a gradually decreased photocurrent signal Ikinetic; meanwhile, the photocurrent Istable of the signal probe carbon quantum dots (Tagstable) remained stable. The simultaneous detection of Pho and Sat could be achieved based on rapid one-step PEC detection under single NIR light with the assistance of a smart signal decryption strategy with Ikinetic and Istable.

Highly stable Ru-complex-grafted 2D metal-organic layer with superior electrochemiluminescent efficiency as a sensing platform for simple and ultrasensitive detection of mucin 1.

This work utilized ultrathin metal-organic layer (MOL) to immobilize luminophores for effectively shortening the ion/electron-transport distance and relieving the diffusional constraints of ion/electron, which greatly enhanced the ECL efficiency and intensity. Moreover, the MOL's immobilization amount of luminophores should be higher than these of bulk MOFs because MOLs possess more accessible postmodification sites for the luminophores with minimal diffusion barriers. As expected, our proof-of-concept experiment indicated that the Hf-MOL's loading number of Ru(bpy)(mcpbpy) was about 1.

An ATP-fueled nucleic acid signal amplification strategy for highly sensitive microRNA detection.

Herein, an adenosine triphosphate (ATP)-fueled nucleic acid signal amplification strategy based on toehold-mediated strand displacement (TMSD) and fluorescence resonance energy transfer (FRET) was proposed for highly sensitive detection of microRNA-21. More importantly, the target microRNA-21 could be regenerated with ATP as the fuel rather than a nucleotide segment in conventional approaches, which made the proposed strategy simple and efficient due to the high affinity and strength of the aptamer-target interaction.

Ultrasensitive Fluorescent Assay Based on a Rolling-Circle-Amplification-Assisted Multisite-Strand-Displacement-Reaction Signal-Amplification Strategy.

Heavy metal ions are persistent environmental contaminants and pose a great threat to human health, which has prompted demand for new methods to selectively identify and detect these metal ions. Herein, a novel fluorescent assay based on a rolling-circle-amplification (RCA)-assisted multisite-strand-displacement-reaction (SDR) signal-amplification strategy was proposed for the ultrasensitive detection of heavy metal ions with lead ions (Pb) as a model. The proposed strategy not only achieved the target recycling but also introduced RCA induced by released DNAzyme.

Highly Stable Mesoporous Luminescence-Functionalized MOF with Excellent Electrochemiluminescence Property for Ultrasensitive Immunosensor Construction.

In this work, a novel mesoporous luminescence-functionalized metal-organic framework (Ru-PCN-777) with high stability and excellent electrochemiluminescence (ECL) performance was synthesized by immobilizing Ru(bpy)(mcpbpy) on the Zr cluster of PCN-777 via a strong coordination bond between Zr and -COO. Consequently, the Ru(bpy)(mcpbpy) could not only cover the surface of PCN-777 but also graft into the interior of PCN-777, which greatly increased the loading amount of Ru(bpy)(mcpbpy) and effectively prevented the leaching of the Ru(bpy)(mcpbpy) resulting in a stable and high ECL response. Considering the above merits, we utilized the mesoporous Ru-PCN-777 to construct an ECL immunosensor to detect mucin 1 (MUC1) based on proximity-induced intramolecular DNA strand displacement (PiDSD).

Electrochemiluminescent Pb-Driven Circular Etching Sensor Coupled to a DNA Micronet-Carrier.

Herein, an ultrasensitive electrochemiluminescent (ECL) strategy was designed based on the fabrication of a multi-interface DNA micronet-carrier via layer by layer hybridization of double-stranded DNAzyme-substrate to immobilize large amounts of ECL indicator, [Ru(dcbpy)dppz], in double-strand DNA on the electrode surface, generating enhanced ECL signals. When the double-stranded structures were cleaved circularly via Pb in the detection sample, the ECL indicator was released, which resulted in a decreased ECL signal associated with the concentration of Pb, that had higher sensitivity and wider linear range. As a result, the developed ECL strategy exhibited a linear range from 50 pM to 500 μM with a detection limit of 4.

Intramolecular Self-Enhanced Nanochains Functionalized by an Electrochemiluminescence Reagent and Its Immunosensing Application for the Detection of Urinary β2-Microglobulin.

In this study, polyethylenimine (PEI) is discovered to possess a noticeable amplification effect for the electrochemiluminescence (ECL) of N-(aminobutyl)-N-(ethylisoluminol) (ABEI); thus, a novel self-enhanced ECL reagent (ABEI-PEI) is prepared by covalent cross-linking. Because of the shortened electron-transfer path and reduced energy loss, the intramolecular ECL reaction between ABEI and PEI exhibited enhanced luminous efficiency compared with the traditional intermolecular ECL reaction. Owing to the amine-rich property of PEI, abundant ABEI could be immobilized on the molecular chains of PEI to strengthen the luminous intensity of ABEI-PEI.

Universal Ratiometric Photoelectrochemical Bioassay with Target-Nucleotide Transduction-Amplification and Electron-Transfer Tunneling Distance Regulation Strategies for Ultrasensitive Determination of microRNA in Cells.

A universal ratiometric photoelectrochemical (PEC) bioassay, which could be readily expanded for ultrasensitive determination of various targets in complex biological matrixes, was established by coupling a target-nucleotide transduction-amplification with DNA nanomachine mediated electron-transfer tunneling distance regulation strategies. With the help of target-nucleotide transduction-amplification strategy, the one input target signal could be transducted to corresponding multiple output DNA signals by nucleotide specific recognition technology, simultaneously leading to an efficient signal amplification for target. Then the output DNA could initiate the formation of four-way junction DNA nanomachine through binding-induced combination, by which the electron-transfer tunneling distance between photoactive materials and sensing interface could be regulated, simultaneously resulting an enhanced photocurrent signal from SiO@methylene blue (SiO@MB) as wavelength-selective photoactive material in close proximity to sensing interface and a reduced photocurrent signal from another wavelength-selective photoactive material CdS quantum dots (CdS QDs) away from sensing interface for photocurrent signal ratio calculation.

An efficient target-intermediate recycling amplification strategy for ultrasensitive fluorescence assay of intracellular lead ions.

An ultrasensitive fluorescence assay for intracellular Pb determination was proposed through target-intermediate recycling amplification based on metal-assisted DNAzyme catalysis and strand displacement reactions. Compared with only target recycling-based fluorescence assay with an M amplification ratio, the proposed assay could achieve an M × N amplification ratio to obtain an improved sensitivity by more than 10 times, in which M and N are the amplification ratios of target recycling and intermediate recycling, respectively. Remarkably, this proposed ultrasensitive fluorescence assay could be applied to the determination of various analytes with the well-designed detection probe, especially in intracellular assay, providing a promising tool for clinical diagnosis and biomedical detection.

Competitive method-based electrochemiluminescent assay with protein-nucleotide conversion for ratio detection to efficiently monitor the drug resistance of cancer cells.

A simple and highly-efficient approach to monitor the expression of P-glycoprotein (P-gp) in cells was urgently needed to demonstrate the drug resistance of cancer cells. Herein, a competitive method-based electrochemiluminescent (ECL) assay with a single ECL indicator was proposed for the first time to efficiently estimate the concentration ratio of two proteins. By converting the different proteins to partially coincident nucleotide sequences a sandwich type immunoassay on magnetic beads, the concentration ratio related ECL signals could be obtained competitive nucleotide hybridization on an electrode surface.

Self-Enhanced Ultrasensitive Photoelectrochemical Biosensor Based on Nanocapsule Packaging Both Donor-Acceptor-Type Photoactive Material and Its Sensitizer.

In this work, a self-enhanced ultrasensitive photoelectrochemical (PEC) biosensor was established based on a functionalized nanocapsule packaging both donor-acceptor-type photoactive material and its sensitizer. The functionalized nanocapsule with self-enhanced PEC responses was achieved first by packaging both the donor-acceptor-type photoactive material (poly{4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl-alt-3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophene-4,6-diyl}, PTB7-Th) and its sensitizer (nano-C60, fullerene) in poly(ethylene glycol) (PEG) to form a nanocapsule, which significantly enhanced PEC signal and stability of the PEC biosensor. Moreover, a quadratic enzymes-assisted target recycling amplification strategy was introduced to the system for ultrasensitive determination.

Luminescence-Functionalized Metal-Organic Frameworks Based on a Ruthenium(II) Complex: A Signal Amplification Strategy for Electrogenerated Chemiluminescence Immunosensors.

Novel luminescence-functionalized metal-organic frameworks (MOFs) with superior electrogenerated chemiluminescence (ECL) properties were synthesized based on zinc ions as the central ions and tris(4,4'-dicarboxylicacid-2,2'-bipyridyl)ruthenium(II) dichloride ([Ru(dcbpy)3](2+)) as the ligands. For potential applications, the synthesized MOFs were used to fabricate a "signal-on" ECL immunosensor for the detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP). As expected, enhanced ECL signals were obtained through a simple fabrication strategy because luminescence-functionalized MOFs not only effectively increased the loading of [Ru(dcbpy)3](2+), but also served as a loading platform in the ECL immunosensor.

Development of multivalent radioimmunonanoparticles for cancer imaging and therapy.

Background: Noninvasive, focused hyperthermia can be achieved by using an externally applied alternating magnetic field (AMF) if effective concentrations of nanoparticles can be delivered to the target cancer cells. Targeting agents, for example, monoclonal antibodies or peptides, linked to magnetic iron oxide nanoparticles (NP), represent a promising strategy to target cancer cells and hyperthermia.

Methods: We have developed a new radioconjugate NP ((111)In-DOTA-di-scFv-NP), using recombinantly generated antibody fragments, di-scFv-c, for the imaging and therapy of anti-MUC-1-expressing cancers, because aberrant MUC-1 is abundantly expressed on the majority of human epithelial cancers.

Construction of di-scFv through a trivalent alkyne-azide 1,3-dipolar cycloaddition.

Heterofunctional azide and alkyne PEG-linkers have been synthesized and site specifically conjugated to scFv via a reactive thiol functionality; two scFv were coupled by copper catalyzed 1,3-dipolar cycloaddition to make divalent scFv (di-scFv) with an inter-scFv distance defined to provide divalent binding; antigen binding was maintained for the di-scFv construct and increased several times compared to that of the parent scFv; the cycloaddition reaction reported herein represents an important ligation strategy to covalently link macromolecular proteins and retain sensitive structural conformations.

Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding.

MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation.

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals.

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity.

Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand.

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain.