Hybrid Probe Research Articles

A biodegradable porous silicon and polymeric hybrid probe for electrical neural signal recording

This paper reports on a novel design and fabrication strategy to develop a hybrid porous silicon (PSi)-polyimide-based neural interface probe. The PSi is soluble in the tissue matrix, leaving the flexible polyimide shank inside the tissue as a permanent probe-implant. The flexibility of the PSi-dissolved polyimide shank is possible to reduce the tissue response, resulting from the probe-implant. In-vitro degradation experiment was conducted in phosphate buffer solution (PBS), pH 7.4, at 37 °C. The PSi with over 50% porosity was dissolved in less than 6 days. The electrode contacts were made of Au or Pt; impedance measurements gave values of 6.83 MΩ and 656 kΩ @1 kHz, respectively. The minimum critical bucking force among the fabricated probes was tested to be 71 mN, corresponding to the highest porosities of 70% and 5 mm shank length. Assessment of the cell viability was done using mouse fibroblast cell line, indicating that there was no sign of obvious cell lysis and reduction of cell growth on the device surface. Lastly, the functionality of the probe was proved by successful implantation in rat cortex. The local field potential action spikes were recorded with ±40 μV peak and signal to noise ratio of 7.8.

Enzyme- and label-free electrochemical aptasensor for kanamycin detection based on double stir bar-assisted toehold-mediated strand displacement reaction for dual-signal amplification

It is critically important to detect antibiotic residues for monitoring food safety. In this study, an enzyme- and label-free electrochemical aptasensor for antibiotics, with kanamycin (Kana) as a typical analyte, was developed based on a double stir bar-assisted toehold-mediated strand displacement reaction (dSB-TMSDR) for dual-signal amplification. First, we modified two gold electrodes (E-1 and E-2) with different DNA probes (S1/S2 hybrid probe in E-1 and DNA fuel strand S3 in E-2). In the presence of Kana, an S1/S2 probe can be disassembled from E-1 to form an S2/Kana complex in supernatant. The S2/Kana could react with S3 on E-2 to form S2/S3 hybrid and release Kana through TMSDR. After then, the target recycling was triggered. Subsequently, the formed S2/S3 hybrid can also trigger a hybridization chain reaction (HCR). Consequently, the dual-signal amplification strategy was established, which resulted in many long dsDNA chains on E-2. The chains can associate with methylene blue (MB) as redox probes to produce a current response for the quantification of Kana. The assay exhibited high sensitivity and specificity with a detection limit at 16 fM Kana due to the dual-signal amplification. The double stir bars system can both increase phase separation and prevent leakage of DNA fuel to reduce background interference. Moreover, it allows flexible sequence design of the TMSDR probes. The assay was successfully employed to detect Kana residues in food and showed potential application value in food safety detection.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

Ratiometric detection of Zn2+ and Cd2+ based on self-assembled nanoarchitectures with dual emissions involving aggregation enhanced emission (AEE) and its application.

Glutathione-Au nanoclusters (GSH-AuNCs) are known as typical luminophores with aggregation-induced emission (AIE) features, which can be triggered by low temperature, low pH values, and metal ions. However, these strict "triggers" restrict the application of this AIE probe under mild and neutral conditions. In this work, fluorescent amino-modified silicon nanoparticles (SiNPs) and GSH-AuNCs can self-assemble into well-designed nanospheres through electrostatic interactions under physiological conditions. The hybrid probe of SiNPs@GSH-AuNCs exhibits dual emissions located at 450 nm from SiNPs and 570 nm originating from the aggregation enhanced emission (AEE) of GSH-AuNCs. Interestingly, the addition of Zn2+ or Cd2+ can connect the nanospheres to form large aggregates, so a unique second increase of AEE from GSH-AuNCs appears, while the emission of SiNPs remains constant to act as an internal reference. The fluorescence ratios (I570/I450) of SiNPs@GSH-AuNCs are positively correlated with Zn2+ or Cd2+ with the linear range from 1.5 μM to 500 μM. Most importantly, besides the advantages of dual emissions, high fluorescence brightness, and good stability, the SiNPs@GSH-AuNCs also exhibit excellent biocompatibility and low cytotoxicity, and have been successfully applied in zinc imaging in live cells. Furthermore, the proposed method is also used to measure Zn2+ and Cd2+ in real samples with satisfactory recoveries.

Organic-Dye-Modified Upconversion Nanoparticle as a Multichannel Probe To Detect Cu2+ in Living Cells.

The development of an inorganic-organic hybrid probe to more accurately detect ions in living systems is very challenging but highly desirable. Here we combined upconversion nanoparticles with the electrically active ferrocene group to detect Cu2+ in living cells. The as-prepared probe displays three different signal changes in absorption, emission, and electrochemical behavior, respectively, during Cu2+ ion detection. Moreover, this new probe has been demonstrated to show high stability and adaptability. In addition, bioimaging testing reveals that this probe is suitable for detecting and visualizing Cu2+ in A549 cells with low cytotoxicity.

Commercial glucometer as signal transducer for simple evaluation of DNA methyltransferase activity and inhibitors screening

DNA methyltransferase (MTase) plays an important role in many biological processes and has been recognized as a predictive cancer biomarker far before other signs of malignancy and a therapeutic target in cancer treatment. Thus simple and sensitive determination of DNA MTase activity is urgently required. The commercially available glucometer is considered as the most successful point-of-care (POC) sensor up to date, and researchers extend its application in monitoring different types of targets rather than only glucose. Here, we developed a simple strategy for the sensitive detection of the DNA MTase (using M.SssI as an example) activity by using a glucometer as the signal transducer. A S1/S2 hybrid probe was designed including a specific recognition sequence for both DNA MTase and restriction endonuclease, and a complementary sequence for biotin-S3. Firstly, the S1/S2 hybrid probe was self-assembled on the gold electrode and methylated by M.SssI MTase to form the methylated dsDNA. Then, HpaII endonuclease specifically cleaved the residue of the unmethylated dsDNA. Subsequently, biotin-S3 hybridized with the overhang sequence of the methylated dsDNA. Finally, the biotin tag was successively combined with streptavidin (STV) and biotin-invertase. The invertase efficiently catalyzed the hydrolysis of sucrose to generate abundant glucose, which led to an amplified response of glucometer. This strategy could detect DNA MTase activity as low as 0.3 U mL−1 with good selectivity against other two cytosine MTases (HaeIII MTase and AluI MTase), and be successfully applied for screening the DNA MTase inhibitors (5-azacytidine and 5-aza-2′-deoxycytidine), implying our proposed method holds great promising application in early cancer diagnosis and therapeutics.

Hybrid photonic-plasmonic near-field probe for efficient light conversion into the nanoscale hot spot.

In this Letter, we present a design and simulations of the novel hybrid photonic-plasmonic near-field probe. Near-field optics is a unique imaging tool that provides optical images with resolution down to tens of nanometers. One of the main limitations of this technology is its low light sensitivity. The presented hybrid probe solves this problem by combining a campanile plasmonic probe with the photonic layer, consisting of the diffractive optic element (DOE). The DOE is designed to match the plasmonic field at the broad side of the campanile probe with the fiber mode. This makes it possible to optimize the size of the campanile tip to convert light efficiently into the hot spot. The simulations show that the hybrid probe is ∼540 times more efficient compared with the conventional campanile on average in the 600-900nm spectral range.

Abordaje multimodal en la cirugía radioguiada de un caso de paraganglioma múltiple

The case involves a 34-year-old woman who underwent surgical removal of a retroperitoneal paraganglioma adjacent to the left kidney's lower pole, previously visualized by CT and MRI. The 123I-MIBG scan was positive for this lesion and, in addition, another uptake was observed located caudally at the level of L5 and of smaller size and less intensity. The second lesion was not considered for surgical removal, due to its lack of morphological definition. One week after surgical intervention, the patient presented difficult-to-control high blood pressure. A second 123I-MIBG scan was performed. The previously described second image was more intense in this study, and surgery was planned to remove it. A combination of techniques including freehand-SPECT and a portable hybrid gamma camera (with optical camera) were used pre-operatively to identify the location of the lesion. The combination of intra-operative laparoscopic gamma probe and portable hybrid gamma camera enabled the tumor to be located, excised, and its complete removal to be monitored. The histopathology analysis confirmed a second paraganglioma.

Feasibility study and cell design for performing local electrochemical impedance spectroscopy measurements using an atomic force microscopy set-up

This paper studies the feasibility of performing Local Electrochemical Impedance Spectroscopy (LEIS) and Atomic Force Microscopy (AFM) within a single set-up, using a hybrid probe. The geometry of the set-up along with the extremely small size of the probe used is expected to result in distorted impedances and very low potentials, respectively. Numerical simulations using a multi-ion transport and reaction model (MITReM), show that the distortions can be mathematically corrected for and that our electronic equipment is capable of measuring the minute potentials.

Flexible and stretchable nanowire-coated fibers for optoelectronic probing of spinal cord circuits.

Studies of neural pathways that contribute to loss and recovery of function following paralyzing spinal cord injury require devices for modulating and recording electrophysiological activity in specific neurons. These devices must be sufficiently flexible to match the low elastic modulus of neural tissue and to withstand repeated strains experienced by the spinal cord during normal movement. We report flexible, stretchable probes consisting of thermally drawn polymer fibers coated with micrometer-thick conductive meshes of silver nanowires. These hybrid probes maintain low optical transmission losses in the visible range and impedance suitable for extracellular recording under strains exceeding those occurring in mammalian spinal cords. Evaluation in freely moving mice confirms the ability of these probes to record endogenous electrophysiological activity in the spinal cord. Simultaneous stimulation and recording is demonstrated in transgenic mice expressing channelrhodopsin 2, where optical excitation evokes electromyographic activity and hindlimb movement correlated to local field potentials measured in the spinal cord.

Improving breast cancer diagnosis by reducing chest wall effect in diffuse optical tomography.

We have developed the ultrasound (US)-guided diffuse optical tomography technique to assist US diagnosis of breast cancer and to predict neoadjuvant chemotherapy response of patients with breast cancer. The technique was implemented using a hand-held hybrid probe consisting of a coregistered US transducer and optical source and detector fibers which couple the light illumination from laser diodes and photon detection to the photomultiplier tube detectors. With the US guidance, diffused light measurements were made at the breast lesion site and the normal contralateral reference site which was used to estimate the background tissue optical properties for imaging reconstruction. However, background optical properties were affected by the chest wall underneath the breast tissue. We have analyzed data from 297 female patients, and results have shown statistically significant correlation between the fitted optical properties ( ? a and ? s ? ) and the chest wall depth. After subtracting the background ? a at each wavelength, the difference of computed total hemoglobin (tHb) between malignant and benign lesion groups has improved. For early stage malignant lesions, the area-under-the-receiver operator characteristic curve (AUC) has improved from 88.5% to 91.5%. For all malignant lesions, the AUC has improved from 85.3% to 88.1%. Statistical test has revealed the significant difference of the AUC improvements after subtracting background tHb values.

Primer-Aided Truncation for the Creation of Hybrid Proteins.

Proteins frequently display modular architecture with several domains and segments connected by linkers. Proper protein functionality hinges on finely orchestrated interactions among these constituent elements. The underlying modularity lends itself to the engineering of hybrid proteins via modular rewiring; novel properties can thus be obtained, provided the linkers connecting the individual elements are conducive to productive interactions. As a corollary, the process of protein engineering often encompasses the generation and screening of multiple linker variants. To aid these steps, we devised the PATCHY method (primer-aided truncation for the creation of hybrid proteins) to readily generate hybrid gene libraries of predefined composition. We applied PATCHY to the mechanistic characterization of hybrid receptors that possess blue-light-regulated histidine kinase activity. Comprehensive sampling of linker composition revealed that catalytic activity and response to light are primarily functions of linker length. Variants with linkers of 7n residues mostly have light-repressed activity but those with 7n + 1 residues mostly have inverted, light-induced activity. We further probed linker length in the context of single residue exchanges that also lead to an inversion of the signal response. As in the original context, activity is only observed for certain periodic linker lengths. Taken together, these results provide mechanistic insight into signaling strategies employed by sensory photoreceptors and sensor histidine kinases. PATCHY represents an adequate and facile method to efficiently generate and probe hybrid gene libraries and to thereby identify key determinants for proper function.

Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes.

Photoswitchable fluorescent proteins with controllable light–dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light–dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

Ratiometric fluorescence nanosensor for selective and visual detection of cadmium ions using quencher displacement-induced fluorescence recovery of CdTe quantum dots-based hybrid probe

We report a ratiometric fluorescence nanosensor for selective and visual detection of Cd2+ using quencher displacement-induced fluorescence recovery of CdTe quantum dots (QDs)-based hybrid probe. In this design, the red emissive CdTe QDs (rQDs) embedded in silica sphere have been played the role of internal reference signal, while the green emissive CdTe QDs (gQDs) covalently bound on silica surface served as the specific recognition element. The complexation of 1,10-phenanthroline (Phen) with the ratiometric fluorescence probe resulted in the fluorescence quenching of gQDs while the fluorescence of the rQDs inner silica stayed constant. Upon exposure to different amounts of Cd2+, the fluorescence of gQDs could be selectively recovered to a certain extent due to the detachment of Phen ligands from gQDs surface. Based on the “off-on” green fluorescence of gQDs and the red background emitted by rQDs, a ratiometric fluorescence nanosensor for Cd2+ detection was successfully achieved in a wide concentration range of 0.5nM–2μM. More importantly, one can recognize for Cd2+ content by the naked eye with high resolution in a range of 2 nM–1μM. This nanosensor features to distinctly discriminate between Cd2+ and Zn2+, and succeeded in real tap water and rice samples.

Magnetic bead-liposome hybrids enable sensitive and portable detection of DNA methyltransferase activity using personal glucose meter

DNA methyltransferase (MTase) plays a critical role in maintaining genome methylation patterns, which has a close relationship to cancer and bacterial diseases. This encouraged the need to develop highly sensitive, simple, and robust assays for DNA MTase detection and inhibitor screening. Herein, a simple, sensitive, and specific DNA MTase activity assay was developed based on magnetic beads-liposome hybrids combined with personal glucose meter (PGM) for quantitative detection of DNA MTase and inhibitor screening. First, a magnetic beads-liposome hybrid probe is designed by the hybridization of p1DNA-functionalized magnetic bead with p2DNA-functionalized glucoamylase-encapsulated liposome (GEL). It integrates target recognition, magnetic separation and signal amplification within one multifunctional design. Then, in the presence of Dam MTase, the hybrids probe was methylated, and cleaved by methylation-sensitive restriction endonuclease Dpn I, making liposome separated from magnetic bead by magnetic separation. Finally, the separated liposome was decomposed, liberating the encapsulated glucoamylase to catalyze the hydrolysis of the signal substrate amylose with multiple turnovers, producing a large amount of glucose for quantitative readout by the PGM. In the proposed assay, the magnetic beads-liposome hybrids offered excellent sensitivity due to primary amplification via releasing numerous glucoamylase from a liposome followed by a secondary enzymatic amplification. The use of portable quantitative device PGM bypasses the requirement of complicated instruments and sophisticated operations, making the method simple and feasible for on-site detection. Moreover, the proposed assay was successfully applied in complex biological matrix and screen suitable inhibitor drugs for DAM for disease(s) treatment. The results reveal that the approach provides a simple, sensitive, and robust platform for DNA MTases detection and screening potential drugs in medical research and early clinical diagnostics.

Novel hybrid probe based on double recognition of aptamer-molecularly imprinted polymer grafted on upconversion nanoparticles for enrofloxacin sensing

A novel luminescent “double recognition” method for the detection of enrofloxacin (ENR) is developed to overcome some of the challenges faced by conventional molecularly imprinting. Biotinylated ENR aptamers immobilised on upconversion nanoparticles (UCNPs) surface are implemented to capture and concentrate ENR as the first imprinting recognition safeguard. After correct folding of the aptamer upon the existing targets, polymerization of methacrylic acid monomers around the ENR-aptamer complexes to interact with the residual functional groups of ENR by using molecularly imprinting techniques is the second imprinting recognition safeguard. The “double recognition” imprinting cavities are formed after removal of ENR, displaying recognition properties superior to that of aptamer or traditional molecularly imprinting alone. Another interest of this method is simultaneous molecular recognition and signal conversion by fabricating the “double recognition” receptor on to the surface of UCNPs to form a hybrid sensing system of apta-MIP/UCNPs. The proposed sensing method is applied to measure ENR in different fish samples. Good recoveries between 87.05% and 96.24%, and relative standard deviation (RSD) values in the range of 1.19–4.83% are obtained, with the limits of detection and quantification of 0.04 and 0.12ng/mL, respectively. It indicates that the sensing method is feasible for the quantification of target ENRs in real samples, and show great potential for wide-ranging application in bioassays.

Enzyme-free electrochemical aptasensor by using silver nanoparticles aggregates coupling with carbon nanotube inducing signal amplification through electrodeposition

A novel and enzyme-free electrochemical aptasensor based on magnetic separation platform was designed for highly sensitive determination of PDGF-BB by virtue of utilizing catalytic activity of silver nanoparticles (AgNPs) towards the reduction of o-nitrophenol (o-NP) to o-aminophenol (o-AP) by NaBH4, and the direct electric readout from the product of electrodeposition of o-AP and multiwalled carbon nanotubes (MWCNTs) on screen printed electrode (SPE). Aptamers functionalized magnetic beads (apt-MBs) were used as capture probe, while AgNPs modified with aptamers and hybrid probes (apt-AgNPs-A and apt-AgNPs-B) were used as tags. In the presence of PDGF-BB, the apt-MBs and the AgNPs aggregates which were induced by in-situ hybridization of apt-AgNPs-A and apt-AgNPs-B formed a sandwich-like complex, followed by adding of NaBH4 and o-NP. Owing to the amplified catalysis of AgNPs aggregates conjugated in the complex, the o-NP was reduced by NaBH4 to o-AP, which was electro-co-deposition with MWCNTs on the SPE, producing a conducting nano-composite. The signal readout in this aptasensor for PDGF-BB was obtained by direct measurement of the poly(o-AP-MWCNTs) film on the SPE. Using this signal transduction protocol, the analytical performance of the electrochemical aptasensor was studied in detail. Under the optimal conditions, the assay displayed a wide dynamic working range of between 0.1ngmL−1 and 100ngmL−1with a detection limit of 60pgmL−1 relative to target PDGF-BB. Incorporation of good catalytic ability of hybridization inducing AgNPs aggregates and excellent electrochemical property of poly(o-AP-MWCNTs) nanocomposite could result in the amplification of the electrochemical signal, owing to the high loading of catalytic AgNPs and electro-co-deposition of MWCNTs. This method would have a promising future for application in clinical diagnostics.

Graphene oxide/chitosan/Ag2S nanocomposite film sensing to toluene by using forster resonance energy transfer strategy through the conformation changes of chitosan

A convenient to prepare GO/CS/Ag2S nanocomposite film was successfully developed by in situ grown methods. The resultant heterostructures were characterised by Fourier transform infrared spectroscopy, scanning electron microscopy and differential scanning calorimetry. A hybrid probe of GO/CS/Ag2S nanocomposite film for detecting toluene using FRET technique is demonstrated through the fluorescence. The conformation of chitosan changes from the state of the extension to the state of the contraction by adding toluene to change the polarity of solvent, which decreases the distance between Ag2S and GO, enhancing the quenching effect between them. The fluorescence results reveal that the GO/CS/Ag2S nanocomposite film is sensitive to toluene. Notably, the fluorescence intensity of the composite film is quantitatively and reversibly controlled by toluene from 0 to 1.6 × 10−2 mol/L.

Conjugated Polymer-Based Hybrid Materials for Turn-On Detection of CO2 in Plant Photosynthesis.

Detection of carbon dioxide (CO2) is of fundamental importance in diverse applications ranging from environmental analysis to agricultural production. In this work, a hybrid probe based on guanidinium-pendent oligofluorene (G-OF) and water-soluble conjugated polythiophene (PTP) has been developed for the turn on detection of CO2 with low background signal, taking advantage of the efficient fluorescence quenching of the tight aggregate of G-OF/PTP. In the presence of CO2, the electrostatic repulsion between G-OF and PTP can be effectively enhanced through protonation of the side chains, leading to the disaggregation and thus the "turn-on" fluorescence. The strategy allows for the light-up visible detection of CO2 with high sensitivity. Importantly, this system is capable of sensitively monitoring the concentration changes of CO2 in the process of the photosynthesis, which represents a concept to monitor the photosynthesis based on water-soluble conjugated polymers.

Monitoring Nitric Oxide in Subcellular Compartments by Hybrid Probe Based on Rhodamine Spirolactam and SNAP-tag.

By connection of O(6)-benzylguanine (BG) to an "o-phenylenediamine-locked" rhodamine spirolactam responsive to nitric oxide (NO), a novel substrate (TMR-NO-BG) of genetically encoded SNAP-tag has been constructed. In living cells, labeling SNAP-tag fused proteins with TMR-NO-BG will in situ generate corresponding probe-protein conjugates (TMR-NO-SNAP) that not only inherit high NO sensitivity from the small-molecule parent but also guarantee the site-specificity to the designated subcellular compartments such as the mitochondrial inner membrane, nucleus, and cytoplasm. In two representative cellular processes, TMR-NO-BG demonstrates its applicability to monitor endogenous subcellular NO in the activated RAW264.7 cells stimulated by lipopolysaccharide and in the apoptotic COS-7 cells induced by etoposide.