Switching of the enzymatic activity synchronized with signal recognition by an artificial DNA receptor on a liposomal membrane

We constructed a supramolecular system on a liposomal membrane that is capable of activating an enzyme via DNA hybridization. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, providing a platform for the system, was prepared by the self-assembly of an oligonucleotide lipid, a phospholipid and a cationic synthetic lipid. The enzyme was immobilized on the liposomal surface through electrostatic interactions. Selective recognition of DNA signals was achieved by hybridizing the DNA signals with the oligonucleotide lipid embedded in the liposome. The hybridized DNA signal was sent to the enzyme by a copper ion acting as a mediator species. The enzyme then amplified the event by the catalytic reaction to generate the output signal. In addition, our system demonstrated potential for the discrimination of single nucleotide polymorphisms.     

人工细胞膜上天然酶与人工受体的分子间通讯

在集成的分子系统中,分子间的通讯联系是设计分子器件和分子机械的重要方式[1].在水相介质中自组装形成的脂质体可以作为分子间通讯的平台,各种分子可以依靠弱作用力,按照设计思路有组织、有计划地排布其上,构成一个功能化的超分子体,即纳米器件(nanodevice).在脂质体上模拟生物膜上发生的细胞信号转导,成为在分子和超分子水平上开发具有仿生特征的新型纳米器件的重要方法和手段,近年来成为关注的热点[2-6].     

Lateral spread of an amplification signal using an enzymatic system on a conductive surface

In this letter, we report a novel signal amplification phenomenon that rapidly and dramatically increases both the magnitude and the lateral extent of the original signal. This phenomenon utilizes an enzyme immobilized on a conductive surface to generate amplified signals at locations remote from the original site of enzyme activity. The result is demonstrated on a microfluidic platform using the established precipitating enzyme-substrate system of horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) on a surface plasmon resonance (SPR) imaging system.     

Modeling cell membrane transport: interaction of guanidinylated poly(propylene imine) dendrimers with a liposomal membrane consisting of phosphate-based lipids

Mixed anionic liposomes consisting of dihexadecyl phosphate, phosphatidylcholine, and cholesterol were employed as model systems for assessing the ability of a series of functionalized dendrimers, bearing a varying number of guanidinium groups at their surface, to translocate across the liposomal bilayers. At low guanidinium/phosphate molar ratios or when weakly guanidinylated dendrimeric derivatives were employed, the dendrimeric derivative acted as a kind of "molecular glue" leading to a simple adhesion of the liposomes. Liposomal fusion occurred to a certain extent at high guanidinium/phosphate molar ratios or when highly guanidinylated dendrimeric derivatives were employed. Furthermore, translocation of these dendrimeric derivatives to the liposomal core was observed for low to medium guanidinylation and at low guanidinium/phosphate molar ratios which was, however, enhanced when the lipid bilayer was in its fluid liquid-crystalline phase. Thus, an optimum balance is required between the binding strength of guanidinium with the phosphate groups and the degree of hydrophilicity of the guanidinylated dendrimers for the transport of the latter to the liposomal core to occur.     

Glutamate receptor incorporated in a mixed hybrid bilayer lipid membrane array, as a sensing element of a biosensor working under flowing conditions

The realization of a reliable receptor biosensor requires stable, long-lasting, reconstituted biomembranes able to supply a suitable biomimetic environment where the receptor can properly work after incorporation. To this end, we developed a new method for preparing stable biological membranes that couple the biomimetic properties of BLMs (bilayer lipid membranes) with the high stability of HBMs (hybrid bilayer membranes); this gives rise to an innovative assembly, named MHBLM (mixed hybrid bilayer lipid membrane). The present work deals with the characterization of biosensors achieved by embedding an ionotropic glutamate receptor (GluR) on MHBLM. Thanks to signal (transmembrane current) amplification, which is typical of natural receptors, the biosensor here produced detects glutamate at a level of nmol L(-1). The transmembrane current changes linearly vs glutamate up to 100 nmol L(-1), while the limit of detection is 1 nmol L(-1). In addition, the biosensor response can be modulated both by receptor agonists (glycine) and antagonists (Mg(2+)) as well, and by exploiting the biosensor response, the distribution of different kinds of ionotropic GluR present in the purified sample, and embedded in MHBLM, was also evaluated. Finally, one of the most important aspects of this investigation is represented by the high stability of the biomimetic system, which allows the use of biosensor under flowing conditions, where the solutions flow on both biomembrane faces.     

Chemical conjugation of an mRNA cap analogue with a cell-penetrating peptide as a potential membrane permeable translation inhibitor

We present a versatile method for chemical conjugation of a dinucleotide cap analogue with a cell-penetrating peptide. The final coupling reaction is between an azide-modified peptide (MPS-N₃)—a fragment that is responsible for transport of the conjugate through the cell membrane, with a biologically active compound—and an alkynylated cap structure, using the Cu(I)-catalyzed click reaction.     

Diarylurea-linked zinc porphyrin dimer as a dual-mode artificial receptor: supramolecular control of complexation-facilitated photoinduced electron transfer

A novel porphyrinic receptor 1 in which two zinc porphyrins are bridged by two diarylurea linkers was developed for recognition of a viologen derivative (hexyl viologen, HV). The electronic absorption spectra as well as the 1H NMR experiments revealed that the HV molecule was bound to the cleft in 1 mainly through carbonyl dipole-charge interactions to afford a 1:1 complex. From the steady-state fluorescence spectroscopic study, the photoinduced electron transfer (PET) from 1 to HV was extremely facilitated by the receptor-substrate complexation. The receptor 1 also formed a 1:1 complex with 1,4-diazabicyclo[2.2.2]octane (DABCO) through two Zn-N coordination interactions, and, using DABCO as an inhibitor, we suppressed the PET reaction via the substrate exchange.     

MALDI-TOF mass spectrometry of a combinatorial peptide library: effect of matrix composition on signal suppression

The effect of matrix composition on signal suppression caused by a dominant compound under MALDI ionization was studied using the combinatorial TQTXT pentapeptide library as a model system. The peptide library is composed of 19 components with all proteinogenic amino acids except cysteine in position X. From these compounds, only the Arg peptide (TQTRT) was detected with sufficient intensity in the MALDI-TOF mass spectrum under typical MALDI conditions (CCA matrix). The analysis of a set of compounds utilized as different matrix components, additives and a cationizing agent revealed that the composition of the matrix is a critical point in signal suppression. Highly improved ion yields were achieved by using a CCA/DHB mixture as a matrix. The addition of K(+) as a cationizing agent to the CCA matrix resulted in MALDI-TOF mass spectra with relative ion intensities very similar to those obtained by electrospray ionization.     

Intermolecular Communication on a Liposomal Membrane: Enzymatic Amplification of a Photonic Signal with a Gemini Peptide Lipid as a Membrane‐Bound Artificial Receptor

A supramolecular system that can activate an enzyme through photo‐isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self‐assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH‐dependent L ‐lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane‐bound receptor induced photo‐isomerization, which significantly altered the receptor’s metal‐binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu²⁺ ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.     

Effect of peptide lipidation on membrane perturbing activity: a comparative study on two trichogin analogues

The effect of lipidation on the membrane perturbing activity of peptaibol antibiotics was investigated by performing a comparative study on two synthetic analogues of the natural peptide trichogin GA IV. Both analogues were labeled with a hydrophobic fluorescent probe, but one of them lacked the N-terminal n-octanoyl chain, present in the natural peptide. Spectroscopic studies show that the fatty acyl chain produces two opposite effects: it increases the affinity of the monomeric peptide for the membrane phase, but, at the same time, it favors peptide aggregation in water, thus inhibiting membrane binding by reducing the effective monomer concentration. In the membrane phase the two analogues exhibit the same aggregation and orientation behavior, indicating that the n-octanoyl chain plays no specific role in determining their orientation or membrane perturbing activity. Indeed, the dependence of peptide-induced membrane leakage on total peptide concentration is basically the same for the two analogues, because the aforementioned opposite effects, caused by peptide lipidation, tend to balance. These findings make questionable the use of lipidation as a general method for increasing the peptide membrane-perturbing activity, as its validity seems to be restricted to parent compounds of limited overall hydrophobicity.     

Fluorometric aptamer based determination of adenosine triphosphate based on deoxyribonuclease I-aided target recycling and signal amplification using graphene oxide as a quencher

Microchimica Acta - The authors describe an aptamer based fluorometric assay for the determination of ATP. It is based on deoxyribonuclease I-aided target recycling and signal amplification. The...     

Ultrasensitive amperometric determination of PSA based on a signal amplification strategy using nanoflowers composed of single-strand DNA modified fullerene and Methylene Blue,and an improved surface-initiated enzymatic polymerization

The authors describe a signal amplification strategy for highly sensitive detection of the prostate-specific antigen (PSA). This is accomplished by a combination of two methods, viz. (a) improved surface-initiated enzymatic polymerization (SIEP), and (b) the use of nanoflowers prepared from C₆₀ fullerene and Methylene Blue (C₆₀/MB) modified with a long single-strand DNA. C₆₀/MB acts as a novel electrochemical indicator. The C₆₀/MB nanoflowers improve the load of MB and promote the electron transfer. The integration of the SIEP technique and the C₆₀/MB nanomaterial also results in improved loading of MB on the nucleic acid. Ultimately, dual cascade signal amplification is accomplished. The biosensor was constructed as follows: (a) Gold nanospheres were modified with antibody 2 (Ab₂) and a thiolated oligonucleotide (referred to as S₀). (2) S₀ is then extended by the SIEP reaction. (3) The redox indicator C₆₀/MB is then connected to the extended guanine-rich ssDNA which then yields the amperometric signal. (4) A sandwich immunoassay is performed by capturing the nanoprobe oy type Ab₂-Au-S₀ on the gold electrode modified with multi-walled carbon nanotubes (MWCNTs) and protein A. Current is measured by using differential pulse voltammetry (DPV). The synergic effect of the biofunctional nanomaterial and the signal amplification strategy greatly improves the performance of this immunoassay. Under optimized conditions and at a working voltage of typically ?0.18 V (vs Ag/AgCl), the assay has a linear range that extends from 15 pg·mL^?1 to 8 ng·mL^?1 of PSA. The detection limit is as low as 1.7 pg·mL^?1 (at an S/N ratio of 3). In our perception, this dual amplification scheme has a wide scope in that it may become applicable to numerous other immunoassays.

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Graphical abstract C₆₀/Methylene blue nanoflowers, a novel electrochemical indicator, connect with the long single-stranded DNA (ssDNA) extended by the improved surface-initiated enzymatic polymerization method. This amplification strategy is utilized to construct a sandwich prostate-specific antigen (PSA) immunosensor.

     

A novel fluorescent aptasensor based on silica nanoparticles,PicoGreen and exonuclease III as a signal amplification method for ultrasensitive detection of myoglobin

Measurement of myoglobin (Mb) in human blood serum is of great interest for quick diagnosis of acute myocardial infarction (AMI). In this study, a novel fluorescent aptasensor was designed for ultrasensitive and selective detection of Mb, based on target-induced high fluorescence intensity, complementary strand of aptamer (CS), PicoGreen (PG) dye, exonuclease III (Exo III) and silica nanoparticles coated with streptavidin (SNPs-Streptavidin). The developed aptasensor obtains characteristics of SNPs as enhancers of fluorescence intensity, Exo III as an enzyme which selectively digests the 3'-end of double-stranded DNA (dsDNA), PG as a fluorescent dye which could selectively bind to dsDNA and high selectivity and sensitivity of aptamer (Apt) toward its target. In the absence of Mb, no free CS remains in the environment of SNPs-Streptavidin, resulting in a weak fluorescence emission. In the present of Mb, dsDNA-modified SNPs-Streptavidin complex forms, leading to a very strong fluorescence emission. The developed fluorescent aptasensor exhibited high specificity toward Mb with a limit of detection (LOD) as low as 52 pM. In addition, the designed fluorescent aptasensor was efficiently used to detect Mb in human serum.     

Colorimetric determination of Hg(II) based on a visually detectable signal amplification induced by a Cu@Au-Hg trimetallic amalgam with peroxidase-like activity

We report on a method for highly sensitive and selective colorimetric determination of Hg(II) via a signal amplification strategy. Cu@Au nanoparticles (NPs) are found to exhibit intrinsic peroxidase-like activity and can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine by H₂O₂. This is accompanied by a solution color change from colorless to green (with an absorption peak at 655 nm). The catalytic capability of the Cu@Au NPs (pale green) is strongly enhanced by a Cu@Au-Hg trimetallic amalgam (bluish), and this effect can be applied directly to the determination of Hg(II). The limit of detection as observed with the unaided eye is 10 nM, which is at least one order of magnitude lower than that of the known AuNP-based colorimetric assay. Due to excellent specificity of the amalgamation process, the assay is highly selective for Hg(II) and is not interfered by other metal ions in up to 0.5 μM concentrations. This assay was successfully applied to the determination of Hg(II) in tap water. In view of these advantages, we expect this colorimetric method to become an attractive tool for the quantitation of Hg(II) in biological, environmental, and food samples.

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Graphical Abstract Cu@Au nanoparticles (NPs) exhibit intrinsic peroxidase-like activity and can catalyze the oxidation of tetramethylbenzidine (TMB) by H₂O₂. This is accompanied by a color change of the solution from colorless to green.

     

Interfacial properties of a synthetic peptide derived from hepatitis G virus E2 protein: interaction with lipid monolayers

A useful approach to get information about the potential fusogenic ability of virus synthetic peptides is the study of its interfacial properties and subsequent study in mono- and bilayers. In this work, we have characterized by means of physicochemical tools (i.e. compression isotherms and surface activity) the sequence 267-284, LLGTEVSEVLGGAGLTGG, derived from the E2 structural protein of HGV/GBV-C. The adsorption of the peptide at the air/water interface was monitored by following the increase in surface pressure as a function of time at two different pH values: 5 and 7. Parameters such as surface excess or molecular area were calculated from the equation of Gibbs. The peptide showed a tendency to migrate to the surface of a saline-buffered solution. It formed stable monolayers at the air/water interface giving a compression isotherm with a shape consistent with that of some alpha-helical peptide conformations. Brewster angle microscopy (BAM) showed that through compression the peptide formed multilayers. The studies with lipid monolayers (DPMC, DMPC/DMPG, and DMPC/DMTAP) showed that the peptide interacts with all the lipids assayed producing a marked disrupting effect upon them. In these effects electrostatic interactions seem to have some participation.     

Tobacco Mosaic Virus as a Carrier for Small Molecules. II. Cooperative affinity labeling of membrane vesicles with a TMV angiotensin conjugate. Preliminary communication

A (3-mercapto-1-succinyl)-TMV ?-maleimidohexanoyl-[1-asparagine, 5-valine]-angiotensin II conjugate was prepared and used for cooperative affinity labeling of bovine adrenal cortex cell membrane vesicles containing angiotensin-binding sites. The labeling was demonstrated by electron microscopy and by CsCl and sucrose density gradient centrifugation Preliminary evidence for specific binding and for the postulated cooperative affinity interaction is produced.     

Conformation and Interaction of a D,L-alternating peptide with a bilayer membrane: x-ray reflectivity, CD, and FTIR spectroscopy.

Peptides with alternating amino acid configuration provide helical secondary structures that are especially known from the membrane channel and pore-forming gramicidin A. In analogy to this natural D,L-alternating pentadecapeptide, the potential of D,L-alternating peptides for membrane insertion is investigated using the model dodecamer peptide H-(Phe-Tyr)(5)-Trp-Trp-OH. This aromatic peptide is introduced as a novel pore-forming synthetic analogue of gramicidin A. It forms a well-organized homodimer similar to one of the gramicidin A transmembrane motifs. X-ray reflectivity measurements are performed on solid-supported peptide-lipid complexes to obtain information about the influence of the artificial dodecamer peptide on the bilayer parameters. In addition, Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopic studies determine the conformational state of H-(Phe-Tyr)(5)-Trp-Trp-OH within the model membrane. Site-specific iodine labeling assists in determining the topology of the membrane-embedded peptide by pinpointing the position of the iodine label within the bilayers.