Rapid and ultrasensitive electrochemical detection of circulating tumor DNA by hybridization on the network of gold-coated magnetic nanoparticles

An accurate and robust method for quantifying the levels of circulating tumor DNA (ctDNA) is vital if this potential biomarker is to be used for the early diagnosis of cancer. The analysis of ctDNA presents unique challenges because of its short half-life and ultralow abundance in early stage cancers. Here we develop an ultrasensitive electrochemical biosensor for rapid detection of ctDNA in whole blood. The sensing of ctDNA is based on hybridization on a network of probe DNA modified gold-coated magnetic nanoparticles (DNA-Au@MNPs). This DNA-Au@MNPs biosensor can selectively detect short- and long-strand DNA targets. It has a broad dynamic range (2 aM to 20 nM) for 22 nucleotide DNA target with an ultralow detection limit of 3.3 aM. For 101 nucleotide ctDNA target, a dynamic range from 200 aM to 20 nM was achieved with a detection limit of 5 fM. This DNA-Au@MNPs based sensor provides a promising method to achieve 20 min response time and minimally invasive cancer early diagnosis.

This study introduces a new electrochemical sensing strategy for the rapid detection of circulating tumor DNA (ctDNA) from whole blood in combination with a network of DNA-Au@MNPs with high sensitivity and excellent selectivity.     

Ultrasensitive detection of β-lactamase-associated drug-resistant bacteria using a novel mass-tagged probe-mediated cascaded signal amplification strategy

The emergence and spread of drug-resistant bacteria (DRB) is a global health threat. Early and accurate detection of DRB is a critical step in the treatment of DRB infection. However, traditional assays for DRB detection are time-consuming and have inferior analytical sensitivity and quantification capability. Herein, a mass-tagged probe (MP-CMSA)-mediated enzyme- and light-assisted cascaded signal amplification strategy was developed for the ultrasensitive detection of β-lactamase (BLA), an enzyme closely associated with most DRB. Each MP-CMSA probe contained multiple poly(amidoamine) (PAMAM) dendrimer molecules immobilized on a streptavidin agarose bead via a BLA-cleavable linker, and each dendrimer was modified with multiple mass tags via a photo-cleavable linker. In BLA detection, BLA could cleave the BLA-cleavable linker, leading to dendrimers shedding from the MP-CMSA probe to achieve enzyme-assisted signal amplification. Then, each dendrimer can further release mass tags under UV light to achieve light-assisted signal amplification. After this cascaded signal amplification, the released mass tags were ultimately quantified by mass spectrometry. Consequently, the sensitivity of BLA detection can be significantly enhanced by four orders of magnitude with a detection limit of 50.0 fM. Finally, this approach was applied to the blood samples from patients with DRB. This platform provides a potential strategy for the sensitive, rapid and quantitative detection of DRB infection.

Development of a mass-tagged probe-mediated enzyme- and light-assisted cascaded signal amplification strategy for the ultrasensitive detection of β-lactamase.     

Programmable mismatch-fueled high-efficiency DNA signal amplifier

Herein, by introducing mismatches, a high-efficiency mismatch-fueled catalytic multiple-arm DNA junction assembly (M-CMDJA) with high-reactivity and a high-threshold is developed as a programmable DNA signal amplifier for rapid detection and ultrasensitive intracellular imaging of miRNA. Compared with traditional nucleic acid signal amplification (NASA) with a perfect complement, the M-CMDJA possesses larger kinetic and thermodynamic favorability owing to the more negative reaction standard free energy (ΔG) as driving force, resulting in much higher efficiency and rates. Once traces of the input initiator react with the mismatched substrate DNA, it could be converted into amounts of output multiple-arm DNA junctions via the M-CMDJA as the functional DNA conversion nanodevice. Impressively, the mismatch-fueled catalytic four-arm DNA junction assembly (M-CFDJA) exhibits high conversion efficiency up to 1.05 × 10⁸ in 30 min, which is almost ten times more than those of conventional methods. Therefore, the M-CMDJA could easily address the challenges of traditional methods: slow rates and low efficiency. In application, the M-CFDJA as a DNA signal amplifier was successfully used to develop a biosensing platform for rapid miRNA detection with a LOD of 6.11 aM and the ultrasensitive intracellular imaging of miRNA, providing a basis for the next-generation of versatile DNA signal amplification methods for ultimate applications in DNA nanobiotechnology, biosensing assay, and clinical diagnoses.

We proposed an ingenious mismatch-enhanced catalytic multiple-arm DNA junction assembly (M-CMDJA) which possesses more negative reaction standard free energy (ΔG) as the driving force, resulting in quite high conversion efficiency and much faster reaction speed.     

Bioorthogonal regulation of DNA circuits for smart intracellular microRNA imaging

Catalytic DNA circuits represent a versatile toolbox for tracking intracellular biomarkers yet are constrained with low anti-interference capacity originating from their severe off-site activation. Herein, by introducing an unprecedented endogenous DNA repairing enzyme-powered pre-selection strategy, we develop a sequential and specific on-site activated catalytic DNA circuit for achieving the cancer cell-selective imaging of microRNA with high anti-interference capacity. Initially, the circuitry reactant is firmly caged by an elongated stabilizing duplex segment with a recognition/cleavage site of a cell-specific DNA repairing enzyme, which can prevent undesired signal leakage prior to its exposure to target cells. Then, the intrinsic DNA repairing enzyme of target cells can liberate the DNA probe for efficient intracellular microRNA imaging via the multiply guaranteed molecular recognition/activation procedures. This bioorthogonal regulated DNA circuit presents a modular and programmable amplification strategy for highly reliable assays of intracellular biomarkers, and provides a pivotal molecular toolbox for living systems.

An on-site bioorthogonal regulated DNA circuit was developed by introducing an endogenous DNA repairing enzyme-mediated sequential activation strategy to achieve cancer cell-selective microRNA imaging with high anti-interference ability.     

Selectively detecting attomolar concentrations of proteins using gold lined nanopores in a nanopore blockade sensor

Disease diagnosis at earlier stages requires the development of ultrasensitive biosensors for detecting low-abundance biomarkers in complex biological fluids within a reasonable time frame. Here, we demonstrate the development of an ultrasensitive nanopore blockade biosensor that can rapidly diagnose a model protein biomarker, prostate-specific antigen (PSA) with high selectivity. The solid-state nanopores have gold located only along the length of the nanopore whilst the rest of the membrane is silicon nitride. The orthogonal use of materials allows nanopore arrays with a different surface chemistry inside the nanopore relative to the rest of the membrane to be fabricated. The importance of this differential surface chemistry is it can improve the detection limit of nanopore blockade sensors in quantitative analysis. Based on such functionalized nanopore devices, nanopore blockade sensors lower the limit of detection by an order of magnitude and enable ultrasensitive detection of PSA as low as 80 aM. The findings from this study open new opportunities for nanopore sensors in further developments including optical detection and ultralow detection limit biosensing at complex biological fluids.

Selective detection of attomolar proteins was achieved using gold lined nanopores in a nanopore blockade sensor.     

Standard-free single magnetic bead evaluation: a stable nanoplatform for prostate disease differentiation

Explicit interpretation of heterogeneity between prostate-specific antigen (PSA) subtypes is essential for prostate cancer differentiation during different disease courses, whereas a universal protocol with uniform criteria is still lacking across the globe. In this work, a standard-free single magnetic bead (SMB) nanoplatform utilizing metal nanoparticles with optimal diameters was proposed for prostate disease differentiation in a 134-donor model. The inaccuracy of detection in absolute quantification was diminished via evaluations of metal intensities on the single magnetic bead. The intrinsic proportion of fPSA in tPSA was successfully evaluated by direct use of the Pt to Au intensity ratio (Pt/Au ratio), exhibiting better differentiation between healthy and unhealthy, benign prostatic hyperplasia (BPH) and cancer individuals compared with solo fPSA or tPSA. We generated thresholds respectively for prostate disease differentiation, envisioning that this standard-free SMB nanoplatform would establish a standardized methodology with uniform criteria worldwide in cancer diagnosis, staging, and postoperative assessments.

A standard-free stable single magnetic bead nanoplatform was proposed in this work. The use of metal signal ratio was directly applied for intrinsic biological fPSA to tPSA ratio evaluations for prostate disease differentiation.     

Nickel-catalyzed cross-electrophile allylation of vinyl bromides and the modification of anti-tumour natural medicine β-elemene

Herein, we present a facile and efficient allylation method via Ni-catalyzed cross-electrophile coupling of readily available allylic acetates with a variety of substituted alkenyl bromides using zinc as the terminal reductant. This Ni-catalyzed modular approach displays excellent functional group tolerance and a broad substrate scope, which the creation of a series of 1,4-dienes including several structurally complex natural products and pharmaceutical motifs. Moreover, the coupling strategy has the potential to realize enantiomeric control. The practicality of this transformation is demonstrated through the potent modification of the naturally antitumor active molecule β-elemene.

Herein, we present a facile and efficient allylation method via Ni-catalyzed cross-electrophile coupling of readily available allylic acetates with a variety of substituted alkenyl bromides using zinc as the terminal reductant.     

Molecularly pure miktoarm spherical nucleic acids: preparation and usage as a scaffold for abiotic intracellular catalysis

We present a fullerene-based strategy that allows the synthesis of molecularly pure miktoarm spherical nucleic acids (SNAs) with diverse structures, which, with post-functionalization, could serve as efficient scaffolds for intracellular catalysis. The SNA structure promotes cell permeability, nucleic acid stability, and catalytic efficiency, making the platform ideal for in cellulo reactions. Consequently, the tris(triazole)-bearing miktoarm SNA was able to effectively mediate intracellular copper-catalyzed alkyne–azide cycloaddition at nanomolar level of copper, and facilitate the same reaction in live zebrafish.

Biocompatible nano-constructs, with definite molecular structures and programmable subunits, can potentially be used as biocatalysts with modular functional moieties.     

Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes

Cell-surface proteins, working as key agents in various diseases, are the targets for around 66% of approved human drugs. A general strategy to selectively detect these proteins in a real-time manner is expected to facilitate the development of new drugs and medical diagnoses. Although brilliant successes were attained using small-molecule probes, they could cover a narrow range of targets due to the lack of suitable ligands and some of them suffer from selectivity issues. We report herein an antibody-based fluorogenic probe prepared via a two-step chemical modification under physiological conditions, to fulfill the selective recognition and wash-free imaging of membrane proteins, establishing a modular strategy with broad implications for biochemical research and for therapeutics.

A modular strategy to convert commercially available antibodies into fluorogenic probes has been developed, enabling selective recognition and wash-free imaging of endogenous membrane proteins.      

Silacyclization through palladium-catalyzed intermolecular silicon-based C(sp2)–C(sp3) cross-coupling

Silicon-based cross-coupling has been recognized as one of the most reliable alternatives for constructing carbon–carbon bonds. However, the employment of such reaction as an efficient ring expansion strategy for silacycle synthesis is comparatively little known. Herein, we develop the first intermolecular silacyclization strategy involving Pd-catalyzed silicon-based C(sp²)–C(sp³) cross-coupling. This method allows the modular assembly of a vast array of structurally novel and interesting sila-benzo[b]oxepines with good functional group tolerance. The key to success for this reaction is that silicon atoms have a stronger affinity for oxygen nucleophiles than carbon nucleophiles, and silacyclobutanes (SCBs) have inherent ring-strain-release Lewis acidity.

Herein, we develop the first silacyclization between 2-halophenols and SCBs, which allows the modular assembly of sila-benzo[b]oxepines with good functional group tolerance and can be applied for the late-stage modification of biologically active molecules.     

Multicomponent formation route to a new class of oxygen-based 1,3-dipoles and the modular synthesis of furans

A new class of phosphorus-containing 1,3-dipoles can be generated by the multicomponent reaction of aldehydes, acid chlorides and the phosphonite PhP(catechyl). These 1,3-dipoles are formally cyclic tautomers of simple Wittig-type ylides, where the angle strain and moderate nucleophilicity in the catechyl-phosphonite favor their cyclization and also direct 1,3-dipolar cycloaddition to afford single regioisomers of substituted products. Coupling the generation of the dipoles with 1,3-dipolar cycloaddition offers a unique, modular route to furans from combinations of available aldehydes, acid chlorides and alkynes with independent control of all four substituents.

A new class of phosphorus-containing 1,3-dipoles has been developed, which, when coupled with cycloaddition, offers modular synthesis of furans with independent control of all four substituents.     

A redox-enabled strategy for intramolecular hydroamination

Metal- or acid-catalyzed intramolecular hydroamination and Cope-type intramolecular hydroamination, a distinct concerted approach using hydroxylamines, typically suffer from significant synthetic limitations. Herein we report a process for intramolecular hydroamination that uses a redox-enabled strategy relying on efficient in situ generation of hydroxylamines by oxidation, followed by Cope-type hydroamination, then reduction of the resulting pyrrolidine N-oxide. The steps are performed sequentially in a single pot, no catalyst is required, the conditions are mild, the process is highly functional group tolerant, and no chromatography is generally required for isolation. A robustness screen and a gram-scale example further support the practicality of this approach.

A redox strategy enables hydroaminations: mild conditions allows efficient hydroxylamine formation & cyclization, then B₂(OH)₄ as reductant also facilitates isolation!     

Decarboxylative 1,4-carbocyanation of 1,3-enynes to access tetra-substituted allenes via copper/photoredox dual catalysis

We disclose herein the first example of merging photoredox catalysis and copper catalysis for radical 1,4-carbocyanations of 1,3-enynes. Alkyl N-hydroxyphthalimide esters are utilized as radical precursors, and the reported mild and redox-neutral protocol has broad substrate scope and remarkable functional group tolerance. This strategy allows for the synthesis of diverse multi-substituted allenes with high chemo- and regio-selectivities, also permitting late stage allenylation of natural products and drug molecules.

An efficient synthesis of multi-substituted allenes by metallaphotoredox-catalyzed decarboxylative 1,4-carbocyanation of 1,3-enynes is described.     

In situ lattice tuning of quasi-single-crystal surfaces for continuous electrochemical modulation

The ability to control the atomic-level structure of a solid represents a straightforward strategy for fabricating high-performance catalysts and semiconductor materials. Herein we explore the capability of the mechanically controllable surface strain method in adjusting the surface structure of a gold film. Underpotential deposition measurements provide a quantitative and ultrasensitive approach for monitoring the evolution of surface structures. The electrochemical activities of the quasi-single-crystalline gold films are enhanced productively by controlling the surface tension, resulting in a more positive potential for copper deposition. Our method provides an effective way to tune the atom arrangement of solid surfaces with sub-angstrom precision and to achieve a reduction in power consumption, which has vast applications in electrocatalysis, molecular electronics, and materials science.

We reported a new method capable of adjusting the lattice structure of solid surfaces with sub-angstrom precision and achieved in situ and continuous control over electrochemical activity.     

Atroposelective synthesis of N-aryl peptoid atropisomers via a palladium(ii)-catalyzed asymmetric C–H alkynylation strategy

The introduction of chirality into peptoids is an important strategy to determine a discrete and robust secondary structure. However, the lack of an efficient strategy for the synthesis of structurally diverse chiral peptoids has hampered the studies. Herein, we report the efficient synthesis of a wide variety of N-aryl peptoid atropisomers in good yields with excellent enantioselectivities (up to 99% yield and 99% ee) by palladium-catalyzed asymmetric C–H alkynylation. The inexpensive and commercially available l-pyroglutamic acid was used as an efficient chiral ligand. The exceptional compatibility of the C–H alkynylation with various peptoid oligomers renders this procedure valuable for peptoid modifications. Computational studies suggested that the amino acid ligand distortion controls the enantioselectivity in the Pd/l-pGlu-catalyzed C–H bond activation step.

The introduction of chirality into peptoids is an important strategy to determine a discrete and robust secondary structure.     

An expeditious FeCl3-catalyzed cascade 1,4-conjugate addition/annulation/1,5-H shift sequence for modular access of all-pyrano-moiety-substituted chromenes

ortho-Alkynyl quinone methides are well-known four-atom synthons for direct [4 + n] cycloaddition in constructing useful oxa-heterocyclic compounds owing to their high reactivity as well as the thermodynamically favored aromatization nature of this process. Herein we report an operationally simple and eco-friendly protocol for the modular and regioselective access of (E)-4-(vinyl or aryl or alkynyl)iminochromenes from propargylamines and S-methylated β-ketothioamides in the presence of FeCl₃, and particularly under undried acetonitrile and air atmosphere conditions. This method exhibits a broad substrate scope and displays nice functional group compatibility, thus providing an efficient access of 3,4-disubstituted iminochromenes.

An operationally simple protocol is described for the facile, modular and regioselective access of all-pyrano-moiety-substituted iminochromenes, particularly under undried acetonitrile and air atmosphere.     

A core–brush 3D DNA nanostructure: the next generation of DNA nanomachine for ultrasensitive sensing and imaging of intracellular microRNA with rapid kinetics

A highly loaded and integrated core–brush three-dimensional (3D) DNA nanostructure is constructed by programmatically assembling a locked DNA walking arm (DA) and hairpin substrate (HS) into a repetitive array along a well-designed DNA track generated by rolling circle amplification (RCA) and is applied as a 3D DNA nanomachine for rapid and sensitive intracellular microRNA (miRNA) imaging and sensing. Impressively, the homogeneous distribution of the DA and HS at a ratio of 1 : 3 on the DNA track provides a specific walking range for the DA to avoid invalid and random self-walking and notably improve the executive ability of the core–brush 3D DNA nanomachine, which easily solves the major technical challenges of traditional Au-based 3D DNA nanomachines: low loading capacity and low executive efficiency. As a proof of concept, the interaction of miRNA with the 3D DNA nanomachine could initiate the autonomous and progressive operation of the DA to cleave the HS for ultrasensitive ECL detection of target miRNA-21 with a detection limit as low as 3.57 aM and rapid imaging in living cells within 15 min. Therefore, the proposed core–brush 3D DNA nanomachine could not only provide convincing evidence for sensitive detection and rapid visual imaging of biomarkers with tiny change, but also assist researchers in investigating the formation mechanism of tumors, improving their recovery rates and reducing correlative complications. This strategy might enrich the method to design a new generation of 3D DNA nanomachine and promote the development of clinical diagnosis, targeted therapy and prognosis monitoring.

This study designed a highly loaded and integrated core–brush 3D DNA nanomachine for miRNA imaging and sensing, which easily solves the major technical challenges of traditional Au-based 3D nanomachines: low loading capacity and low executive efficiency.     

Construction of sterically congested oxindole derivatives via visible-light-induced radical-coupling

The oxindole scaffold represents an important structural feature in many natural products and pharmaceutically relevant molecules. Herein, we report a visible-light-induced modular methodology for the synthesis of complex 3,3′-disubstituted oxindole derivatives. A library of valuable fluoroalkyl-containing highly sterically congested oxindole derivatives can be synthesized by a catalytic three-component radical coupling reaction under mild conditions (metal & photocatalyst free, >80 examples). This strategy shows high functional group tolerance and broad substrate compatibility (including a wide variety of terminal or non-terminal alkenes, conjugated dienes and enynes, and a broad array of polyfluoroalkyl iodide and oxindoles), which enables modular modification of complex drug-like compounds in one chemical step. The success of solar-driven transformation, large-scale synthesis, and the late-stage functionalization of bioactive molecules, as well as promising tumor-suppressing biological activities, highlights the potential for practical applications of this strategy. Mechanistic investigations, including a series of control experiments, UV-vis spectroscopy and DFT calculations, suggest that the reaction underwent a sequential two-step radical-coupling process and the photosensitive perfluoroalkyl benzyl iodides are key intermediates in the transformation.

Simple, modular assembly of complex fluoroalkyl-containing oxindole derivatives with a broad scope and excellent functional group tolerance under mild conditions (metal- and photocatalyst-free). Benzyl iodides were identified as key intermediates.     

Three-component carboacylation of alkenes via cooperative nickelaphotoredox catalysis

Various commercially available acyl chlorides, aldehydes, and alkanes were exploited for versatile three-component 1,2-carboacylations of alkenes to forge two vicinal C–C bonds through the cooperative action of nickel and sodium decatungstate catalysis. A wealth of ketones with high levels of structural complexity was rapidly obtained via direct functionalization of C(sp²)/C(sp³)–H bonds in a modular manner. Furthermore, a regioselective late-stage modification of natural products showcased the practical utility of the strategy, generally featuring high resource economy and ample substrate scope.

Various commercially available acyl chlorides, aldehydes, and alkanes were exploited for versatile three-component 1,2-carboacylations of alkenes to forge two vicinal C–C bonds through the cooperative action of nickel and sodium decatungstate catalysis.     

O-GlcNAcylation mapping of single living cells by in situ quantitative SERS imaging

O-GlcNAcylation is involved in many biological processes including cancerization. Nevertheless, its in situ quantification in single living cells is still a bottleneck. Here we develop a quantitative SERS imaging strategy for mapping the O-GlcNAcylation distribution of single living cells. O-GlcNAcylated compounds (OGCs) can be quantified through their in situ azide labeling and then a click reaction competing with azide and Raman reporter labeled 15 nm-gold nanoparticles (AuNPs) for linking to dibenzocyclooctyne labeled 40 nm-AuNPs to produce OGC-negatively correlated SERS signals. The calibration curve obtained in vitro can be conveniently used for detecting OGCs in different areas of single living cells due to the negligible effect of cell medium on the click linkage and Raman signal. This method has been successfully applied in mapping O-GlcNAcylation distribution in different cell lines and monitoring O-GlcNAcylation variation during cell cycling, which demonstrate its great practicability and expansibility in glycosylation related analysis.

A quantitative SERS imaging strategy is developed for O-GlcNAcylation mapping of single living cells through a competitive click reaction.