Target-triggered cyclic assembly of DNA-protein hybrid nanowires for dual-amplified fluorescence anisotropy assay of small molecules.

Aptamer-based fluorescence anisotropy (FA) assays have attracted great interest in recent years. However, a key factor that determines FA value is molar mass, thus limiting the utility of this assay for the detection of small molecules. To solve this problem, streptavidin, as a molar mass amplifier, was used in a hybridization chain reaction (HCR) to construct a target-triggered cyclic assembly of DNA-protein hybrid nanowires for highly sensitive detection of small molecules by fluorescence anisotropy. In this assay, one blocking DNA strand is released by target-aptamer recognition. The DNA then serves as an initiator to trigger enzyme-free autonomous cross-opening of hairpin probes via HCR to form a DNA nanowire for further assembly of streptavidin. Using adenosine triphosphate (ATP) as the small molecule target, this novel dual-amplified, aptamer-based FA assay affords high sensitivity with a detection limit of 100 nM. This limit of detection (LOD) is much lower than that of the disassembly approach without HCR amplification or the assembly strategy without streptavidin. In contrast to the previous turn-off disassembly approaches based on nonspecific interactions between the aptamer probe and amplification moieties, the proposed aptamer-based FA assay method exhibits a turn-on response to ATP, which can increase sensing reliability and reduce the risk of false hits. Moreover, because of its resistance to environmental interferences, this FA assay has been successfully applied for direct detection of 0.5 µM ATP in complex biological samples, including cell media, human urine, and human serum, demonstrating its practicality in real complex biological systems.

High-sensitivity naphthalene-based two-photon fluorescent probe suitable for direct bioimaging of H2S in living cells.

H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S in living systems are desired. Although quite a few one photon fluorescence probes have been reported for H2S, two-photon (TP) probes are more favorable for intracellular imaging. In this work, by employing a donor-π-acceptor-structured naphthalene derivative as the two-photon fluorophore and an azide group as the recognition unit, we reported a new two-photon bioimaging probe 6-(benzo[d]thiazol-2'-yl)-2-azidonaphthalene (NHS1) for H2S with improved sensitivity. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for both one photon and TP excited fluorescence were observed, resulting in a high sensitivity to H2S in aqueous solutions with a detection limit of 20 nM observed, much lower than the previously reported TP probe. The probe also exhibits a wide linear response concentration range (0-5 µM) to H2S with high selectivity. All these features are favorable for direct monitoring of H2S in complex biological samples. It was then applied for direct TP imaging of H2S in living cells with satisfactory sensitivity, demonstrating its value of practical application in biological systems.

Asymmetric Construction of Multifunctional γ-Lactams from 1,3-Dienes and α-Ketoamides via Pd(0)-π-Lewis Base Catalysis.

A straightforward approach for the asymmetric synthesis of multifunctionalized γ-lactams, including those bearing two tetrasubstituted stereogenic centers, has been developed through a palladium-catalyzed vinylogous addition/allylic amination process between 1,3-dienes and α-ketoamides. This protocol features advantages of ready substrate availability, broad applicability, high efficiency, and excellent stereoselectivity, making it an attractive complementary tool to the previous strategies.

Through bond energy transfer: a convenient and universal strategy toward efficient ratiometric fluorescent probe for bioimaging applications.

Fluorescence resonance energy transfer (FRET) strategy has been widely applied in designing ratiometric probes for bioimaging applications. Unfortunately, for FRET systems, sufficiently large spectral overlap is necessary between the donor emission and the acceptor absorption, which would limit the resolution of double-channel images. The through-bond energy transfer (TBET) system does not need spectral overlap between donor and acceptor and could afford large wavelength difference between the two emissions with improved imaging resolution and higher energy transfer efficiency than that of the classical FRET system. It seems to be more favorable for designing ratiometric probes for bioimaging applications. In this paper, we have designed and synthesized a coumarin-rhodamine (CR) TBET system and demonstrated that TBET is a convenient strategy to design an efficient ratiometric fluorescent bioimaging probe for metal ions. Such TBET strategy is also universal, since no spectral overlap between the donor and the acceptor is necessary, and many more dye pairs than that of FRET could be chosen for probe design. As a proof-of-concept, Hg(2+) was chosen as a model metal ion. By combining TBET strategy with dual-switch design, the proposed sensing platform shows two well-separated emission peaks with a wavelength difference of 110 nm, high energy transfer efficiency, and a large signal-to-background ratio, which affords a high sensitivity for the probe with a detection limit of 7 nM for Hg(2+). Moreover, by employing an Hg(2+)-promoted desulfurization reaction as recognition unit, the probe also shows a high selectivity to Hg(2+). All these unique features make it particularly favorable for ratiometric Hg(2+) sensing and bioimaging applications. It has been preliminarily used for a ratiometric image of Hg(2+) in living cells and practical detection of Hg(2+) in river water samples with satisfying results.

Efficient fluorescence turn-on probe for zirconium via a target-triggered DNA molecular beacon strategy.

It is well-known that Zr(4+) could selectively bind with two phosphate-functionalized molecules through a coordinate covalent interaction to form a sandwich-structured complex (-PO(3)(2-)-Zr(4+)-PO(3)(2-)-). In this paper, we for the first time converted such interaction into fluorescence sensing systems for Zr(4+) via a target-triggered DNA molecular beacon strategy. In the new designed sensing system, two phosphorylated and pyrene-labeled oligonucleotides were chosen as both recognition and reporter units, which will be linked by target Zr(4+) to form a hairpin structure and bring the two labeled pyrene molecules into close proximity, resulting in a "turn-on" excimer fluorescence signal. Moreover, γ-cyclodextrin was introduced to afford an amplified fluorescence signal and, therefore, provided an improved sensitivity for the target Zr(4+). This allows detection of Zr(4+) with high sensitivity (limit of detection, LOD = 200 nM) and excellent selectivity. The proposed sensing system has also been used for detection of Zr(4+) in river water samples with satisfactory result.

An efficient rhodamine thiospirolactam-based fluorescent probe for detection of Hg2+ in aqueous samples.

This paper described the optimized design, synthesis and application of a novel rhodamine thiospirolactam derivative as an 'off-on' fluorescent probe for the detection of Hg(2+) in aqueous samples. The 'off-on' fluorescence and color signal change of the probe is based on an Hg(2+)-triggered domino reaction which brings on the opened-ring form of the rhodamine spirolactam to regain the conjugated system of the rhodamine skeleton. In the well designed probe, the thiospirolactam serves as both Hg(2+) binding unit and electron-defect carbon centre, a phenolic hydroxyl with very strong nucleophilicity after deprotonation is chosen as the attacking unit, and a benzene ring is introduced on the linker to afford steric effects, which benefits an efficient nucleophilic reaction, with a high sensitivity towards Hg(2+). It exhibits a stable response for Hg(2+) from 1.0 × 10(-8) to 1.0 × 10(-6) M, with a detection limit of 3.0 × 10(-9) M. The response of the probe to Hg(2+) is highly selective and pH-insensitive, with a fast response time. All these unique features make it particularly favorable for cellular Hg(2+) imaging applications. It has been preliminarily used for highly sensitive monitoring of Hg(2+) levels in living cells with satisfying resolution.

Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule.

In this contribution, an electrochemical aptameric sensing scheme for the sensitive detection of small molecules is proposed using adenosine as a target model. A ferrocene (Fc)-functionalized thiolated aptamer probe is adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only suppresses the peak current corresponding to blank sample but also provides a signal-on response mechanism. In the absence of adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc is capable of being removed from electrode surface by treatment with endonuclease, and almost no peak current is observed. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissociating the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, adenosine even at a low concentration can give a detectable current signal. Thus, a detection limit of 10(-10) M and a linear response range from 3.74×10(-9) to 3.74×10(-5) M are achieved. The proposed proof-of-principle of a novel electrochemical sensing is expected to extend to establish various aptameric platforms for the analysis of a broad range of target molecules of interest.

Highly sensitive and selective bifunctional oligonucleotide probe for homogeneous parallel fluorescence detection of protein and nucleotide sequence.

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 × 10(-10) M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-of-concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.

Molecular beacon-based junction probes for efficient detection of nucleic acids via a true target-triggered enzymatic recycling amplification.

This work reports the development of a new molecular beacon-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNA-cleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system.

Unimolecular catalytic DNA biosensor for amplified detection of L-histidine via an enzymatic recycling cleavage strategy.

Fluorescence catalytic beacons have emerged as a general platform for sensing applications. However, almost all such sensing systems need covalent modification of the DNAzymes with fluorophore-quencher pairs, which may require elaborate design of the synthetic routes and many heavy and complicated synthetic steps and result in increased cost and lower synthesis yield. Here we report the construction of fluorescent cascadic catalytic beacons. With separation of the molecular recognition module from the signal reporter, this new design both avoids DNAzyme modifications and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification. This allows detection of L-histidine with high sensitivity (LOD = 200 nM) and excellent specificity. The proposed sensing system has also been used for detection of L-histidine in cellular homogenate with satisfactory results.

Graphene-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb2+ with a high selectivity.

On the basis of the remarkable difference in affinity of graphene (GO) with ssDNA containing a different number of bases in length, we for the first time report a GO-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb(2+). A FAM-labeled DNAzyme-substrate hybrid acted as both a molecular recognition module and signal reporter and GO as a superquencher. By taking advantage of the super fluorescence quenching efficiency of GO, our proposed biosensor exhibits a high sensitivity toward the target with a detection limit of 300 pM for Pb(2+), which is lower than previously reported for catalytic beacons. Moreover, with the choice of a classic Pb(2+)-dependent GR-5 DNAzyme instead of 8-17 DNAzyme as the catalytic unit, the newly designed sensing system also shows an obviously improved selectivity than previously reported methods. Moreover, the sensing system was used for the determination of Pb(2+) in river water samples with satisfying results.

DNA-encoded signal conversion for sensitive microgravimetric detection of small molecule-protein interaction.

Identification and quantification of small organic molecules capable of binding to a protein of interest with reasonable affinity and specificity is a central problem. Via developing DNA-encoded recognizing probe, we validate a proof-of-principle for constructing of small target-to-DNA conversion that screens the small molecule-protein interaction. Successful identification of ß-indole acetic acid, abscisic acid, or 2,4-dichlorophenoxyacetic acid/corresponding antibody binding implies its fascinating potential for interrogating small molecule/protein interaction.

Universal aptameric system for highly sensitive detection of protein based on structure-switching-triggered rolling circle amplification.

A universal approach is proposed in this study for the development of an aptameric assay system for proteins based on aptamer structure-switching-triggered ligation-rolling circle amplification (L-RCA) upon target binding. The strategy chiefly depends on the competition for binding the aptamer probe between target protein and a complementary single-stranded DNA (CDNA) that can induce the circularization of the padlock probe. Introduction of target protein into the assay system inhibits the hybridization of the CDNA with the aptamer probe because of the formation of the target/aptamer duplex. The free CDNA can only hybridize with the padlock probe. With the assistance of DNA ligase, the padlock probe is circularized, and the subsequent RCA process can be accomplished by Phi 29 DNA polymerase. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In contrast, in the absence of target protein, no obvious change in the fluorescence intensity of the detection probe is observed. This signaling mode for target recognition and transduction events is based on the combination of aptamer recognition elements and L-RCA technology with high specificity and sensitivity. The proposed assay system not only exhibits excellent analytical characteristics (e.g., the detection limit on attomolar scale and a linear dynamic range of more than 3 orders of magnitude) but also possesses significant advantages over existing aptameric assays. The proposed strategy is universal since the sequences of aptamer probe, CDNA, and padlock probe could be easily designed to be compatible with the L-RCA based detection of other proteins without other conditions.

Fluorescence aptameric sensor for strand displacement amplification detection of cocaine.

A new fluorescence method based on aptamer-target interactions has been developed for cocaine detection with target-induced strand displacement. Here we describe new probes, the hairpin-probe and the single strand-probe (ss-probe), that possess two recognition sequences of cocaine aptamer. In the presence of cocaine, both probes would associate with the target to form a tripartite complex. The conformational change in the hairpin-probe causes the opening of a hairpin structure and the hybridization to primer. With polymerase and the dNTPs, the replication of the single-stranded domain of hairpin-probe triggers the process of primer extension. When the hairpin-probe is converted into a fully double-stranded form, the ss-probe and cocaine are displaced to bind another hairpin-probe and initiate new amplification cycles. Fluorescence signal generation would be observed upon SYBR Green I intercalating into the new DNA double helix. The new protocol design permits detection of as low as 2 nM cocaine in a closed tube, offering a convenient approach for a homogeneous assay. Compared with previously reported cocaine aptameric sensors, our new method is highly sensitive, selective, and economical.

Clicking fluoroionophores onto mesoporous silicas: a universal strategy toward efficient fluorescent surface sensors for metal ions.

Mesoporous SBA-15 silica is an excellent support for constructing fluorescent surface sensors. In this letter, we reported a two-step surface reaction involved strategy to construct efficient fluorescent surface sensors for metal ions by clicking fluoroionophores onto azide-functionalized SBA-15. Our experimental results indicate that such a strategy exhibits an obviously higher loading efficiency within commercial SBA-15 than a previously reported strategy. As a proof-of-concept, a newly designed alkyne-functionalized Hg(2+) fluoroionophore was grafted onto SBA-15 to form a fluorescent Hg(2+) surface sensor. It shows improved sensitivity and selectivity than the fluoroionophore itself working in the solution phase with a detection limit of 2.0 x 10(-8) M for Hg(2+).

Efficient fluorescence resonance energy transfer-based ratiometric fluorescent cellular imaging probe for Zn(2+) using a rhodamine spirolactam as a trigger.

This letter described the design and synthesis of a novel fluorescein-appended rhodamine spirolactam derivative and its preliminary application as a ratiometric fluorescent cellular imaging probe for Zn(2+). The ratiometric fluorescent signal change of the probe is based on an intramolecular fluorescence resonance energy transfer (FRET) mechanism modulated by a specific metal ion induced ring-opening process of the rhodamine spirolactam (acting as a trigger). In the new developed sensing system, the emission peaks of the two fluorophores are well-resolved, which can avoid the emission spectra overlap problem generally met by spectra-shift type probes and benefits for observation of fluorescence signal change at two different emission wavelengths with high resolution. It also benefits for a large range of emission ratios, thereby a high sensitivity for Zn(2+)detection. Under optimized experimental conditions, the probe exhibits a stable response for Zn(2+) over a concentration range from 2.0 x 10(-7) to 2.0 x 10(-5) M, with a detection limit of 4.0 x 10(-8) M. Most importantly, the novel probe has well solved the problem of serious interferences from other transition metal ions generally met by previously reported typical fluorescent probes for Zn(2+) with the di(2-picolyl)amine moiety as the receptor (in this case, the fluorescence response induced by Cd(2+)is even comparable to that of Zn(2+)) and shows a reversible and fast response toward Zn(2+). All these unique features make it particularly favorable for ratiometric cellular imaging investigations. It has been preliminarily used for ratiometric imaging of Zn(2+) in living cells with satisfying resolution.

Surface-enhanced Raman scattering based detection of bacterial biomarker and potential surface reaction species.

Gold nanoparticles immobilized on gold surfaces (AuNPs/Au) function as an excellent SERS substrate for the detection of bacteria biomarkers. The possibility of the reactivity of bacteria biomarkers on such a nanoparticle-based substrate poses complications for the spectroscopic identification and quantification. This report describes new findings of an investigation of the SERS characteristics for the competitive adsorption of dipicolinic acid (DPA), which is an important biomarker from bacterial spores, and pyridine (Py), which is a possible decarboxylation product of DPA. The comparison focuses on the diagnostic region of 900-1100 cm(-1) associated with the ring-breathing modes of the two molecules. While the SERS spectra in this region appeared to display some similarities between DPA and Py, distinctive differences in the detailed band characteristics were revealed for both individual and competitive adsorptions on the AuNPs/Au substrates. The fact that the equilibrium constant for the adsorption of Py on the substrate (~8 × 10(5) M(-1)) is larger than that for DPA (~2 × 10(5) M(-1)) in the measured concentration region is attributed to a stronger binding of Py to Au surface than that for DPA. The analysis of the differences has provided not only accurate speciation of the biomarker molecules on the gold nanoparticle based substrates under the SERS measurement conditions, but also has implications for expanding the application of the nanoparticle substrates for highly sensitive and selective detection of bacterial biomarkers under various reactive or non-reactive conditions.

Sub-attomolar HIV-1 DNA detection using surface-enhanced Raman spectroscopy.

A subattomolar HIV-1 DNA detection assay based on multilayer metal-molecule-metal nanojunctions (NJs) has been developed using surface-enhanced Raman spectroscopy (SERS). There is a two-step mechanism involved. First, label free target DNA facilitated the precipitation of Detection Unit I on the substrate through forming a sandwiched structure based on the capture probe, resulting in the first level amplification of target. Following that, the binding site on Detection probe I was further recognized by Detection Unit II. These two complementary probes acted as bricks to build up the multi-metal-molecule-metal NJs between Au nanoparticles (NPs) that not only created SERS "hot spots" by the conjugated Au NPs, but also obviously decreased the distance between Au NPs and Raman labels. Therefore, the Raman signal of the tag molecules on these detection probes was significantly enhanced due to the distance dependent electromagnetic enhancement (EM) of SERS. With regards to a HIV-1 DNA sequence, the platform could detect a concentration as low as 10(-19) M (approximately 10(-23) mol) with the ability of single base mismatch discrimination.

Biocatalytic growth of gold agglomerates on an electrode for aptamer-based electrochemical detection.

Here we describe the biocatalytic growth of high-density gold agglomerates on a gold electrode surface to form a carrier for aptamer probe immobilization. The present approach provides a simple strategy to promote the seed-mediated deposition of Au from AuCl(4)(-) onto surface-attached 12 nm diameter Au nanoparticles (AuNPs) in the presence of reductive coenzyme and surfactant. The growth process was studied by electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). This nanostructured platform is effective and prospective toward the aptamer probe immobilization. For the nice performance of enhanced substrate, the aptamer-sensing interface showed excellent applicability under the investigations such as alternating current voltammetry (ACV) and surface-enhanced Resonance Raman scattering (SERRS) spectra.

Terminal protection of small-molecule-linked DNA for sensitive electrochemical detection of protein binding via selective carbon nanotube assembly.

Small-molecule-linked DNA has emerged as a versatile tool for the interaction assay between small organic molecules and their protein receptors. We report herein the proof-of-principle of a terminal protection assay of small-molecule-linked DNA. This assay is based on our new finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule is protected from the degradation by exonuclease I (Exo I) when the small molecule moiety is bound to its protein target. This finding translates the binding of small molecules to proteins into the presence of a specific DNA sequence, which enables us to probe the interaction between small organic molecules and their protein targets using various DNA sequence amplification and detection technologies. On the basis of selective assembly of single-walled carbon nanotubes (SWNTs) with surface-tethered small-molecule-linked ssDNA not protected by protein binding, a novel electrochemical strategy for terminal protection assay has been developed. Through detecting the redox signal mediated by SWNT assembly on a 16-mercaptohexadecanoic acid-blocked electrode, this strategy is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor (FR), and a detection limit of 3 pM FR is readily achieved with desirable specificity and sensitivity, indicating that the terminal protection assay can offer a promising platform for small molecule-protein interaction studies.