Binary Probe Research Articles

Sensitive and Specific Y-Shaped Ratio Biosensor for Detecting Serum miR-18a: Potential Early Scanning Tool for Non-Small Cell Lung Cancer

AbstractmiR-18a has been identified as a significantly expressed microRNA (miRNA) in non-small cell lung cancer (NSCLC) and plays a vital role in cancer cell transformation, metastasis, and carcinogenesis. Herein, a pair of binary probes from numerous probe pairs based on single nucleotide polymorphism analyses of miR-18a and miR-18b was first designed and screened to develop a Y-shaped ratio biosensor for accurate detection of serum miR-18a in NSCLC. The special structure of the binary probes combined with the hairpin showed strong specificity for miR-18a, which was confirmed by polyacrylamide gel electrophoresis assay and square wave voltammetry assay. Furthermore, it is beneficial to immobilize single-stranded DNA (ssDNA) probes due to the large specific surface area of nanoporous gold, thereby improving the sensitivity of the biosensor. The Y-shaped ratio biosensor exhibited a wide detection range and can quantify the concentration of miR-18a in the range of 10 fmol/L–100 pmol/L, with a limit of detection of 0.211 fmol/L (S/N = 3). Moreover, it exhibits excellent detection capabilities in serum samples since the biosensor showed a high selectivity toward the coexistence of miR-18a and miR-18b. Therefore, the prepared Y-shaped ratio biosensor is a highly sensitive and specific miR-18a detection tool, capable of identifying microscopic amounts of miR-18a in serum samples, providing great potential for early screening of NSCLC.

Efficient binary sequence set designs for MIMO PMCW radar

Multiple-input multiple-output (MIMO) phase-modulated continuous wave (PMCW) radar systems have major advantages for mutual-interference mitigation over their linear frequency-modulated continuous wave (LFMCW) counterparts. To attain a virtual array length of M×M̃ with M transmit and M̃ receive antennas, a probing sequence set with both low auto-correlation sidelobes and low cross-correlations is needed for MIMO PMCW radar. To reduce the hardware cost of sequence generation, binary probing sequence (BPS) sets are desirable. We present herein four efficient computational algorithms to generate BPS sets with desirable properties, and arbitrary sequence lengths and arbitrary number of sequences in the set are allowed. When initialized by random BPS sets, they can be used to obtain many different BPS sets with similar properties, i.e., with large diversity. Moreover, fast Fourier transform (FFT) are exploited, making the algorithms computationally efficient. They can be used to design long BPS sets that are hardly possible using existing methods in the literature. The convergence properties and the relationships among the proposed algorithms are also analyzed under a unified framework. Numerical examples are included to illustrate the effectiveness of our designs.

Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular "Click" Approach.

Fluorescent DNA probes were prepared in a modular approach using the “click” post-synthetic modification strategy. The new glycol-based module and DNA building block place just two carbons between the phosphodiester bridges and anchor the dye by an additional alkyne group. This creates a stereocenter in the middle of this artificial nucleoside substitute. Both enantiomers and a variety of photostable cyanine–styryl dyes as well as thiazole orange derivatives were screened as “clicked” conjugates in different surrounding DNA sequences. The combination of the (S)-configured DNA anchor and the cyanylated cyanine–styryl dye shows the highest fluorescence light-up effect of 9.2 and a brightness of approximately 11,000 M–1 cm–1. This hybridization sensitivity and fluorescence readout were further developed utilizing electron transfer and energy transfer processes. The combination of the hybridization-sensitive DNA building block with the nucleotide of 5-nitroindole as an electron acceptor and a quencher increases the light-up effect to 20 with the DNA target and to 15 with the RNA target. The fluorescence readout could significantly be enhanced to values between 50 and 360 by the use of energy transfer to a second DNA probe with commercially available dyes, like Cy3.5, Cy5, and Atto590, as energy acceptors at the 5′-end. The latter binary probes shift the fluorescent readout from the range of 500–550 nm to the range of 610–670 nm. The optical properties make these fluorescent DNA probes potentially useful for RNA imaging. Due to the strong light-up effect, they will not require washing procedures and will thus be suitable for live-cell imaging.

Fluorescence ratiometric DNA detection by peptide nucleic acid-pyrene binary probes

We developed a method for detecting DNA by excimer fluorescence from two peptide nucleic acids (PNAs) modified with a pyrene (Pyr). The two PNA-Pyr probes were prepared by solid-phase peptide synthesis, and we assessed fluorescence from the mixture of probes with DNA. From the results, excimer fluorescence derived from the two PNA-Pyr probes forming hybrids with the complementary DNA was observed, and the two probes showed the maximum excimer/monomer ratio when the probes and DNA were hybridized at a 1:1:1 ratio, indicating that the PNA-Pyr probes can detect target DNA. Furthermore, we adjusted the spatial arrangement between the two PNA-Pyr hybrids formed on the DNA to promote optimal excimer formation. As a result, optimal excimer formation was achieved by spacing the two nucleobases between the formed two hybrids and further inserting a hexamethylene linker (C6) between the PNA and Pyr of the PNA-Pyr probe on one side.

Ultrasensitive Detection of Amyloid β Oligomers Based on the "DD-A" FRET Binary Probes and Quadrivalent Cruciform DNA Nanostructure-Mediated Cascaded Amplifier.

The reported donor donor-acceptor ("DD-A") fluorescence resonance energy transfer (FRET) was typically achieved through random collisions and interactions of DNA molecules in the bulk solution, which has inevitable defects, including weak biological stability, slow reaction kinetics, and low hybridization efficiency. In order to overcome these deficiencies, this work developed a quadrivalent cruciform DNA nanostructure (qCDN)-mediated cascaded catalyzed hairpin assembly (CHA) amplifier for the fluorescence detection of amyloid β oligomer species (AβOs). First, four H1 and four H2 hairpins were assembled on one qCDN to obtain qCDNH1 and qCDNH2, respectively. In the presence of AβOs, strand C was released from the P1-C hybrid hairpin and then alternately opened qCDNH1 and qCDNH2 to trigger the qCDN-mediated CHA. As a result, double donors in H1 and one acceptor in H2 were mutually closed, and the porous DNA nanonet with a high loading of "DD-A" FRET binary probes was formed. The FRET efficiency was approximately 78%, and the initial reaction rate was 25-fold faster than the conventional CHA. The detection limit of AβOs was as low as 0.69 pM. The combination of the "DD-A" FRET binary probes and qCDN-mediated cascaded amplifier exhibited great promise for detecting biomarkers with trace levels.

Detection of average methylation level of specific genes by binary-probe hybridization

We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.

Intermolecular dark resonance energy transfer (DRET): upgrading fluorogenic DNA sensing.

The sensitivity of FRET-based sensing is usually limited by the spectral overlaps of the FRET donor and acceptor, which generate a poor signal-to-noise ratio. To overcome this limitation, a quenched donor presenting a large Stokes shift can be combined with a bright acceptor to perform Dark Resonance Energy Transfer (DRET). The consequent fluorogenic response from the acceptor considerably improves the signal-to-noise ratio. To date, DRET has mainly relied on a donor that is covalently bound to the acceptor. In this context, our aim was to develop the first intermolecular DRET pair for specific sensing of nucleic acid sequences. To this end, we designed DFK, a push–pull probe based on a fluorenyl π-platform that is strongly quenched in water. DFK was incorporated into a series of oligonucleotides and used as a DRET donor with Cy5-labeled complementary sequences. In line with our expectations, excitation of the dark donor in the double-labeled duplex switched on the far-red Cy5 emission and remained free of cross-excitation. The DRET mechanism was supported by time-resolved fluorescence measurements. This concept was then applied with binary probes, which confirmed the distance dependence of DRET as well as its potency in detecting sequences of interest with low background noise.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

Завадостійка система імпульсної лазерної дальнометрії

Pulsed laser rangefinders prove to be cost-effective and practical devices when used at distances of several tens of kilometers due to their compactness, portability and energy efficiency. However, the measurement accuracy is significantly reduced by the presence of pulsed interference affecting the input of the optical receiver both during the sensing period and when the reflected signal is being received. Using the algorithms with the accumulation and subsequent processing of the results of several successive measurements reduces the speed of decision-making and does not guarantee the convergence of the results to the real value of the distance. The paper proposes a structural diagram of a laser rangefinder with the ability to detect pulsed interference in the range interval and correct errors that occur in the structure of the signal reflected from the target. The basis of the rangefinder circuit is a logical consistent filter, the structure of which contains multipliers (multiplication operations). The following requirements were formulated for the structure of the probe signal: — the first element should always be set to +1 to synchronize the receiver decider; — the weight of the coding sequence is equal to half its length; — the length of the coding sequence is even. Based on the requirements for coding sequences, the optimal structures of binary probing signals of length 8 were found, providing the best corrective ability. Comparison of the correlation properties of the found sequences and the sequences that are constructed using the Walsh functions showed the advantage of the optimal sequences by the criterion of the minimum level of the ACF side lobes. The simulation of the rangefinder under pulsed noise conditions has shown that the logical filter is advisable to use for those cases when the duration of the obstacle does not exceed 1/3 of the duration of the probing signal.

DNA-Silver Nanocluster Binary Probes for Ratiometric Fluorescent Detection of HPV-related DNA

Herein, we described a ratiometric strategy based on “chameleon” DNA-silver nano-clusters(DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus(HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas the red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. The ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 nmol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in human serum, illustrating its potential promise for clinical diagnosis.

A 365 nm UV LED-excitable antenna ligand for switchable lanthanide luminescence.

The time-resolved luminescence of lanthanide complexes is a highly sensitive and widely used bioassay technology for clinical diagnostics. With the time-resolved luminescence detection the naturally occurring autofluorescence of biological matrices, solid supports and plastics can be avoided. A major drawback of the current technique is that the luminescent lanthanide labels require ultraviolet (UV) excitation, typically shorter than 360 nm, which is strongly absorbed and can damage living biological systems. The lack of cost-efficient high power solid state excitation light sources for UV excitation further limits the development of low-cost and more compact measurement instruments for time-resolved luminescence and the potential use of lanthanide luminescence in different applications. Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in a separation-free detection of targets. The intrinsically luminescent lanthanide chelate is split into two nonluminescent moieties, a lanthanide ion carrier chelate and a light harvesting antenna ligand, each of which can be attached to a separate molecular probe. A luminescent lanthanide complex is formed only when the two probes bind adjacently to the target molecule. Herein we describe a new 365 nm excitable antenna ligand (AL360) for switchable lanthanide luminescence of europium(iii) (EuIII) that would enable the use of 365 nm light emitting diodes (LEDs) as an excitation light source for time-resolved fluorescence imaging and detection. With the acquired subpicomolar assay sensitivity it would be applicable for solution or surface arrays and UV LED microscopy.

MnO2 nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

The donor donor-acceptor (DD-A) FRET model has proven to have a higher FRET efficiency than donor-acceptor acceptor (D-AA), donor-acceptor (D-A), and donor donor-acceptor acceptor (DD-AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the "DD-A" FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary "D-A" model. Furthermore, MnO2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn2+ ions by intracellular GSH. The results indicated that the MnO2 nanosheet mediated "DD-A" FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO2 nanosheet mediated "DD-A" FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

Switchable lanthanide luminescent binary probes in efficient single nucleotide mismatch discrimination

Switchable lanthanide luminescence is a novel proximity-based binary probe technology wherein two oligonucleotide probes are labelled either with lanthanide ion carrier chelate or light-absorbing antenna ligand. A highly luminescent complex is formed when the two label moieties are brought in close contact by adjacent hybridization of the labelled probes to a target sequence. Herein we report how hybridization distance between the labelled probes and single nucleotide mismatches in the target sequence affect to the luminescence intensity of the complex formed from the label moieties. The highest luminescence intensity was observed when the probes were hybridized to full matched target at a distance of 4–10 nucleotides, a fact that indicates the lanthanide complex requires a certain space to be formed. At optimal hybridization distance between the labelled probes the maximal discrimination efficiency between a full matched and a single mismatched target was 1400-fold. Furthermore, computer-based modelling of the luminescent complex formation was in good agreement with the experimental results.

An integrated closed-tube 2-plex PCR amplification and hybridization assay with switchable lanthanide luminescence based spatial detection.

Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in separation-free detection of DNA targets. A luminescent lanthanide complex is formed only when the two probes hybridize adjacently to their target DNA. We have now further adapted this technology for the first time in the integration of a 2-plex polymerase chain reaction (PCR) amplification and hybridization-based solid-phase detection of the amplification products of the Staphylococcus aureus gyrB gene and an internal amplification control (IAC). The assay was performed in a sealed polypropylene PCR chip containing a flat-bottom reaction chamber with two immobilized capture probe spots. The surface of the reaction chamber was functionalized with NHS-PEG-azide and alkyne-modified capture probes for each amplicon, labeled with a light harvesting antenna ligand, and covalently attached as spots to the azide-modified reaction chamber using a copper(i)-catalyzed azide-alkyne cycloaddition. Asymmetric duplex-PCR was then performed with no template, one template or both templates present and with a europium ion carrier chelate labeled probe for each amplicon in the reaction. After amplification europium fluorescence was measured by scanning the reaction chamber as a 10 × 10 raster with 0.6 mm resolution in time-resolved mode. With this assay we were able to co-amplify and detect the amplification products of the gyrB target from 100, 1000 and 10,000 copies of isolated S. aureus DNA together with the amplification products from the initial 5000 copies of the synthetic IAC template in the same sealed reaction chamber. The addition of 10,000 copies of isolated non-target Escherichia coli DNA in the same reaction with 5000 copies of the synthetic IAC template did not interfere with the amplification or detection of the IAC. The dynamic range of the assay for the synthetic S. aureus gyrB target was three orders of magnitude and the limit of detection of 8 pM was obtained. This proof-of-concept study shows that the switchable lanthanide luminescent probes enable separation-free array-based multiplexed detection of the amplification products in a closed-tube PCR which can enable a higher degree of multiplexing than is currently feasible by using different spectrally separated fluorescent probes.

G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food

In this paper, a novel label-free G-quadruplex DNAzyme sensor has been proposed for colorimetric identification of GMO using CaMV 35S promoter sequence as the target. The binary probes can fold into G-quadruplex structure in the presence of DNA-T (Target DNA) and then combine with hemin to form a DNAzyme resembling horseradish peroxidase. The detection system consists of two G-rich probes with 2:2 split mode by using the absorbance and color of ABTS2− as signal reporter. Upon the addition of a target sequence, two probes both hybridize with target and then their G-rich sequences combine to form a G-quadruplex DNAzyme, and the DNAzyme can catalyze the reaction of ABTS2− with H2O2. Then the linear range is from 0.05 to 0.5μM while detection limit is 5nM. These results demonstrate that the proposed G-quadruplex DNAzyme method could be used as a simple, sensitive and cost-effective approach for assays of GMO.

Real-Time mRNA Measurement during an in Vitro Transcription and Translation Reaction Using Binary Probes

In vitro transcription and translation reactions have become popular for a bottom-up approach to synthetic biology. Concentrations of the mRNA intermediate are rarely determined, although knowledge of synthesis and degradation rates could facilitate rational engineering of in vitro systems. We designed binary probes to measure mRNA dynamics during cell-free protein synthesis by fluorescence resonance energy transfer. We tested different mRNA variants and show that the location and sequence environment of the probe target sites are important parameters for probe association kinetics and output signal. Best suited for sensitive real-time quantitation of mRNA was a target site located in the 3' untranslated region, which we designed to reduce secondary structure. We used this probe-target pair to refine our knowledge of mRNA dynamics in the commercially available PURE cell-free protein synthesis system and characterized the effect of TetR repressor on mRNA synthesis rates from a T7 promoter.

Selective alignment of brain responses by task demands during semantic processing

The way the brain binds together words to form sentences may depend on whether and how the arising cognitive representation is to be used in behavior. The amplitude of the N400 effect in event-related brain potentials is inversely correlated with the degree of fit of a word's meaning into a semantic representation of the preceding discourse. This study reports a double dissociation in the latency characteristics of the N400 effect depending on task demands. When participants silently read words in a sentence context, without issuing a relevant overt response, greater temporal alignment over recording sites occurs for N400 onsets than peaks. If however a behavior is produced – here pressing a button in a binary probe selection task – exactly the opposite pattern is observed, with stronger alignment of N400 peaks than onsets. The peak amplitude of the N400 effect correlates best with the latency characteristic showing less temporal dispersion. These findings suggest that meaning construction in the brain is subtly affected by task demands, and that there is complex functional integration between semantic combinatorics and control systems handling behavioral goals.

Multiplex analysis of tumor multidrug-resistance genes expression with photonic suspension array

An easy-operated suspension array based on silica colloidal crystal beads is developed for multiplex analysis of tumor multidrug-resistance genes expression, such as multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1), and potentially single nucleotide polymorphism. In order to obtain high fluorescence intensity, controlled PCR was used to amplify targets at the samples pretreatment stage. By optimizing the conditions a hybridization procedure, which is similar to nucleic acids analysis with binary probes, was established. Small amounts of analytes 10(-19) M could be detected by the method. The K562 cell, human myeloma cell, and its multidrug-resistance string, adriamycin-selected P-glycoprotein-overexpressed K562/A02, were analyzed by using an established procedure to validate feasibility. Clinical blood samples were detected by our method and real-time PCR simultaneously to validate accuracy. Moreover, when combined with multiplex controlled PCR, the method successfully meets the requirements of multiplex analysis. Hence, the method presented is a good method for multiplex analysis of tumor multidrug-resistance genes expression.

Fluorescent hybridization probes for nucleic acid detection.

Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

Homogeneous Detection of Avidin Based on Switchable Lanthanide Luminescence

We have developed switchable lanthanide luminescence-based binary probe technology for homogeneous detection of avidin, which is a tetrameric protein. Two different nonluminescent label moieties--a light-absorbing antenna ligand and a lanthanide ion carrier chelate--were conjugated to separate biotins, which is known as avidin's natural ligand. The assay was based on binding of the two differently labeled biotins on separate binding sites on the target protein and consequent self-assembly of a luminescent complex from the two label moieties. Specific luminescence signal was observed only at the presence of the target protein. The characteristics of the switchable lanthanide luminescence assay were compared to the reference assay, based on lanthanide resonance energy transfer. Both assays had a limit of detection in the low-picomolar concentration range; however, the lanthanide chelate complementation-based assay had wider dynamic range and its optimization was more straightforward. The switchable lanthanide luminescence technology could be further applied to generic protein detection, using reagents that are analogous to the proximity ligation assay principle.