Detection Of Target Molecules Research Articles

Genetically functionalized ferritin nanoparticles with a high-affinity protein binder for immunoassay and imaging

Molecular detection of target molecules with high sensitivity and specificity is of great significance in bio and medical sciences. Here, we present genetically functionalized ferritin nanoparticles with a high-affinity protein binder, and their utility as a signal generator in a variety of immunoassays and imaging. As a high-affinity protein binder, human IgG-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was used. The repebody was genetically fused to the N-terminal heavy-chain ferritin, and the resulting subunits were self-assembled to the repebody-ferritin nanoparticles composed of 24 subunits. The repebody-ferritin nanoparticles were shown to have a three-order of magnitude higher binding affinity toward human IgG than free repebody mainly owing to a decreased dissociation rate constant. The repebody-ferritin nanoparticles were conjugated with fluorescent dyes, and the resulting nanoparticles were used for western blotting, cell imaging, and flow cytometric analysis. The dye-labeled repebody-ferritin nanoparticles were shown to generate about 3-fold stronger fluorescent signals in immunoassays than monovalent repebody. The repebody-functionalized ferritin nanoparticles can be effectively used for sensitive and specific immunoassays and imaging in many areas.

LncRNA GHET1 activated by H3K27 acetylation promotes cell tumorigenesis through regulating ATF1 in hepatocellular carcinoma

BackgroundGHET1 is one of tumor-related lncRNAs. We aimed to explore the functional involvement of GHET1 in hepatocellular carcinoma (HCC). MethodsIn this study, HCC tissues and the paired normal tissues were collected for the detection of target molecules. The expression level of target molecules in HCC tissues or cell lines was determined by qRT-PCR and western blot, respectively. The expression of endogenous GHET1 and ATF1 was modulated by using cell transfection. RNA pull down assay was performed to examine the interaction between GHET1 and ATF1. ChIP assay was conducted to determine the H3K27Ac acetylation of GHET1 promoter. ResultsH3K27 acetylation activated-GHET1 was upregulated in HCC tissues and cell lines. Moreover, GHET1 silencing could inhibit the proliferation, migration, invasion and EMT of HCC cells in vitro. GHET1 could regulate the expression of ATF1 mRNA and protein; RNA pull-down assays supported that GHET1 could bind to ATF1 protein. Furthermore, overexpression of ATF1 almost completely reversed the GHET1 knockdown mediated inhibition on the proliferation, migration, invasion and EMT of HCC cells. ConclusionLncRNA GHET1 was intimately involved in the occurrence and development of HCC through regulating ATF1.

Graphene for Biomedical Applications: A Review

Since its discovery in 2004, graphene has enticed engineers and researchers from various fields to explore its possibilities to be incepted into various devices and applications. Graphene is deemed a ‘super’ material by researchers due to its extraordinary strength, extremely high surface-to-mass ratio and superconducting properties. Nonetheless, graphene has yet to find plausible footing as an electronics material. In biomedical field, graphene has proved useful in tissue engineering, drug delivery, cancer teraphy, as a component in power unit for biomedical implants and devices and as a vital component in biosensors. Graphene is used as scaffolding for tissue regeneration in stem cell tissue engineering, as active electrodes in supercapacitor for powering wearable and implantable biomedical devices and as detectors in biosensors. In tissue engineering, the extreme strength of monolayer graphene enables it to hold stem cell tissues as scaffold during in-vitro cell regeneration process. In MEMS supercapacitor, graphene’s extremely high surface-to-mass ratio enables it to be used as electrodes in order to increase the power unit’s energy and power densities. A small yet having high energy and power densities cell is needed to power often space constrainted biomedical devices. In FET biosensors, graphene acts as detector electrodes, owing to its superconductivity property. Graphene detector electrodes is capable of detecting target molecules at a concentration level as low as 1 pM, making it the most sensitive biosensor available today. Graphene continues to envisage unique and exciting applications for biomedical field, prompting continuous research which results and implementation could benefit the general public in decades to come.

Recent developments on application of nanometal-oxide based gas sensor arrays

It is shown that the sensor arrays can acquire more information on a given sample than an individual sensor. The main classes of gas-sensing materials include metal-oxide semiconductors. Gas sensors based on chemiresistive semiconducting metal-oxides have many potential benefits including, their very low cost, fast response, recovery time, simple electronic interface, ease of use, and ability to detect a large analytes. Recent advances in gas sensor arrays have shown the capability to incorporate nanomaterial based cross-reactive array. In this way it is possible to increase the surface/volume ratio of the sensing layer. Therefore the surface scattering is better influenced by adsorbed species and change in sensor conductivity is higher. Also the sensitivity of semiconductor oxide materials can be improved by using of nanoparticles. Gas sensor arrays based on nanotechnology can rapidly, sensitively, and selectively detect target molecules.

Plasmonic nanoparticles-decorated diatomite biosilica: extending the horizon of on-chip chromatography and label-free biosensing.

Diatomite consists of fossilized remains of ancient diatoms and is a type of naturally abundant photonic crystal biosilica with multiple unique physical and chemical functionalities. In this paper, we explored the fluidic properties of diatomite as the matrix for on-chip chromatography and, simultaneously, the photonic crystal effects to enhance the plasmonic resonances of metallic nanoparticles for surface-enhanced Raman scattering (SERS) biosensing. The plasmonic nanoparticle-decorated diatomite biosilica provides a lab-on-a-chip capability to separate and detect small molecules from mixture samples with ultra-high detection sensitivity down to 1 ppm. We demonstrate the significant potential for biomedical applications by screening toxins in real biofluid, achieving simultaneous label-free biosensing of phenethylamine and miR21cDNA in human plasma with unprecedented sensitivity and specificity. To the best of our knowledge, this is the first time demonstration to detect target molecules from real biofluids by on-chip chromatography-SERS techniques.

A highly sensitive EDTA-based senor for detection of disease biomarker and drug

In this study, a fascinating photocatalytic property of EDTA under light irradiation was observed, much the same as that of natural horseradish peroxidase. The small EDTA molecules enable it easily attaches to amine terminal on antibody surface, an ELISA-like assay may be fabricated through combining with gold nanoparticles (AuNPs) in suspensions can exhibit various colors. The labeled EDTA on the proteins which may catalyze decomposition of hydrogen peroxide and in situ mediate the growth of hydrogen peroxide-induced formation of AuNPs, displaying various color nanoparticle dispersion as a read-out means. A straightforward plasmonic sensor was accordingly designed to enable naked-eye observation and quantitative determination of ultra-trace target analytes based on peroxidase-like EDTA. The highly sensitive assay was utilized to determine cancer antigen 15-3 (CA15-3, a breast cancer biomarker) and methamphetamine, and their limit of detection CA15-3 and methamphetamine are 7.5×10−15U/mL and 2.8×10−20mg/mL by naked eyes, respectively. Furthermore, practical applications for detection of CA15-3 and MA in human blood samples were also carried out. The results of detecting CA15-3 of breast cancer serum samples are close agreement with those given by hospital, implying this sensor with high accuracy. This assay has a great potential to be developed into a platform to quantitatively determine analytes as long as the specific antibodies against them were available. Its versatile features will broaden the applicability toward the ultrasensitive detection of target molecules in clinical diagnosis, environmental monitoring, and food quality control only using naked eyes.

Electrochemical Redox Cycling Realized by Chromatography Paper-based Sensor

In this work, we demonstrated that enhancement of electrochemical current due to redox cycling could be accomplished by paper-based biosensor without any expensive micro-fabrication process. The paper-based sensor had layered structure to generate higher current than a conventional one. We took advantage of the fact that the paper thickness was micrometer-sized (180um), and it defined the distance between two electrochemical electrodes on both sides of the paper. Experimental results showed signatures of the redox cycling, where the electrochemical current from low concentration molecules could be arbitrarily increased by decreasing the distance between electrodes. Such a structure was advantageous for detecting target molecules at very low concentration, proposing a low-cost highly-sensitive biochemcal sensor.

Multicolor Functional Carbon Dots via One-Step Refluxing Synthesis.

Carbon dots are admirable fluorescent nanomaterials due to their low cost, high photostability, excellent biocompatibility, and environmental friendliness. Most conventional carbon dot fabrication approaches produce single-colored fluorescent material in the preparation process; different methods are therefore required to synthesize distinct carbon dots for specific optical applications. In this study, carbon dots carrying different emission colors are prepared through a one-step refluxing process. The emission of these materials can be well-tuned by sodium hydroxide content in the precursor solution. The carbon dots produced are used as sensing probes based on the spectrofluorometric inner filter effect for target molecule detection. Three sensing categories that combine carbon dots and inner filter effect are demonstrated, including direct, metal nanoparticle-assisted, and enzymatic reaction-supported detection. Caffeine, melamine, and fenitrothion are selected as targets to demonstrate the strategies, respectively. These multifunctional carbon dot-based sensors achieve comparable sensitivity toward analytes with a much more convenient preparation route.

A route to new colorimetric pH sensors

Abstract The detection of target molecules via luminescence changes of appropriate sensors is a powerful tool to determine the presence of analytes qualitatively and quantitatively. Therefore, the exploration of synthetic routes to new sensors is important to facilitate the recognition of a large range of analytes. In this report we describe such a new route to sensors and discuss the behavior of the synthesized sensors in the presence of different analytes. The prepared anthracene derivatives belong to two classes of substances. On the one hand imines were synthesized wherein the isomerization of their inherent carbon nitrogen double bond provides an effective non-radiative relaxation pathway. On the other hand amines were received by reduction of the mentioned imines. Their intramolecular charge transfer (ICT) based sensing mechanism allows the detection of protons in a reversible colorimetric fashion. Furthermore, the addition of zinc cations in the aprotic solvent DCM provides the first example showing that the detection of these colorimetric pH sensors is not limited to protons.

Solid-phase extraction based on a molecularly imprinted polymer nanoshell at the surface of silica nanospheres for the specific enrichment and identification of alkaloids from Crinum asiaticum L. var. sinicum.

A molecularly imprinted nanoshell on the surface of silica nanospheres was prepared for specific enrichment and identification of alkaloids from Crinum asiaticum L. var. sinicum. The nanoshell was synthesized by surface polymerization using lycorine as the template, acrylamide as the functional monomer, ethylene glycol dimethacrylate as the cross-linker, 2',2-azobisisobutyronitrile as the initiator and acetonitrile as the pore-forming agent. The core-shell nanospheres were characterized by transmission electron microscopy and infrared spectroscopy, and the results show that the nanoshell layer was homogeneously attached to the surface of vinyl-modified SiO2 nanospheres. The adsorption capacity of the nanospheres was estimated by binding equilibrium and adsorption kinetics experiments. The maximum adsorption amount of lycorine on the nanospheres was 6.68 μmol/g and the imprinting factor was nearly 2.5, indicating a good imprinting effect. The nanospheres were successfully applied in solid-phase extraction for lycorine from Crinum asaticum L. var. sinicum and detection of target molecule in rat metabolites. The average recoveries of lycorine in Crinum asaticum L. var. sinicum extraction and rat metabolites were 93.5 ± 0.6% (n = 3) and 91.6 ± 1.9% (n = 3), respectively. This work provides a simple approach for the fabrication of a molecularly imprinted nanoshell at the surface of silica nanospheres-based solid-phase extraction for drug analysis.

Development of a simple and quick immunochromatography method for detection of anti-HPV-16/-18 antibodies.

Immunochromatography (IC) is widely used to detect target molecules in biological fluids. Since this method can be performed without a special technique or device, IC is a convenient way to assess the existence of antibodies or pathogens such as viruses and bacteria, simply and quickly. In this study, we established an IC method to detect serum antibodies against oncogenic human papillomavirus (HPV)-16 and HPV-18 L1 proteins using recombinant L1 proteins produced by silkworms as antigens. Infection of oncogenic HPVs is a major risk factor of cervical cancer, which is one of the most common cancers in women worldwide. We first measured blood sera of two groups by magnetic beads enzyme-linked immunosorbent assay (MB-ELISA). For the first group, sera were collected prospectively from young women who planned to receive HPV vaccination. The second group consisted of children under 20 years of age, non-vaccinated healthy women, vaccinated healthy women, dysplasia, cervical intraepithelial neoplasia III, and cervical cancer patients. We confirmed that standard vaccination doses significantly increased serum HPV antibody concentrations, and the level was sustained at least more than 30 months after vaccination. In contrast, an increase in antibody concentration was not observed in patients with precancerous cervical changes and cervical cancer. We next measured the samples in both groups using the IC method we originally developed, and found that the measurement values of IC highly correlated with those of MB-ELISA. The simple and quick IC method would be a useful tool for rapid monitoring of L1 specific antibody levels in a non-laboratory environment. With less than one drop of serum, our IC can easily detect serum HPV-16/-18 antibodies within 15 minutes, without the need for electronic devices or techniques.

Imaging Single Molecules vs. Single-Molecule Imaging: Counting Single Molecules using Dextran-Streptavidin-Antibody Clusters (DSA)

Single-molecule imaging using TIRF has become a routine technique in many biophysical applications. However, a complex instrument setup, cumbersome surface treatment procedures and stringent requirements for sample preparation pose a challenge. For the case of digital counting, some single-molecule imaging approaches can be achieved using tools tuned for single-molecule detection. Here, we demonstrate a simple method for imaging a single target molecule using an epi-fluorescence wide-field microscope. Polystyrene magnetic microparticles instead of a specially treated glass were used as the surface for target immobilization. Such antibody-coated particles are easy to manipulate and can be stored for an extended period of time. Conventionally, detection of target molecules is performed with fluorescently-labeled reporter antibody. However, the autofluorescence of many commercially available microparticles dominates over even the brightest fluorophores and interferes with imaging. To overcome this limitation, we enhance the brightness of the reporter by preparing biotinylated antibodies complexed with a dextran-streptavidin scaffold. Subsequently, we detect this antibody assembly using multiple streptavidin-phycoerythrin (PE) conjugates. The resulting signal increase, driven by numerous PE molecules, overcomes the background and allows for direct detection of a single target molecule. Thus, imaging of a single antibody sandwich can be performed on microparticles using multi-molecular fluorescent conjugates.

Fluorescence properties and analytical applications of covalent organic frameworks

Fluorescent COFs with large π-conjugated building units and inherent rigid structure have important potential for chemosensing detection of target molecules or ions based on turn-on and turn-off modes.

Magnetic Resonance Imaging of Neurotransmitter-Related Molecules.

Molecular imaging implies the method capable of pictorially displaying distribution of target molecules and their relative concentration in space. In clinical medicine, where non-invasiveness is mandatory, diagnostic molecular imaging has been considered virtually identical to positron emission tomography (PET). However, there is another powerful, apparently underutilized molecular imaging, namely, proton magnetic resonance spectroscopic imaging (1H-MRSI). The technique can detect target molecules endogenous in brain in virtue of their own specific resonance frequencies (chemical shift) and can create quantitative images of each molecule. 1H-MRSI is conventionally utilized for imaging relatively easily detectable molecules such as N-acetyl-aspartate or lactate. More recently, however, the method is extended into imaging of more challenging molecules such as glutamate or γ-aminobutyric acid (GABA). In this small review, we summarize basic concept of 1H-MRSI and introduce an advanced technique, i.e. chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), which made realistic glutamate imaging in vivo possible.

Offset-Free Gigahertz Midinfrared Frequency Comb Based on Optical Parametric Amplification in a Periodically Poled Lithium Niobate Waveguide

Compact laser frequency combs covering the midinfrared ``fingerprint'' spectral region are desirable light sources for high-sensitivity, high-resolution detection of target molecules. The authors leverage the nonlinear properties of chip-scale Si${}_{3}$N${}_{4}$ and periodically poled LiNbO${}_{3}$ waveguides to obtain a tunable, energy-efficient mid-IR frequency comb. Here the pulse energy needed for a given power per comb line is nearly two orders of magnitude lower than for conventional systems. These results are promising for applications in environmental, medical, and industrial diagnostics.

Controlling the Cross-Sensitivity of Carbon Nanotube-Based Gas Sensors to Water Using Zeolites.

Carbon nanotube-based gas sensors can be used to detect harmful environmental pollutants such as NO2 at room temperature. Although they show promise as low-powered, sensitive, and affordable monitoring devices, cross-sensitivity of functionalized carbon nanotubes to water vapor often obscures the detection of target molecules. This is a barrier to adoption for monitoring of airborne pollutants because of the varying humidity levels found in real world environments. Zeolites, also known as molecular sieves because of their selective adsorption properties, are used in this work to control the cross-sensitivity of single-walled carbon nanotube (SWCNT)-based sensors to water vapor. Zeolites incorporated into the sensing layer are found to reduce interference effects that would otherwise obscure the identification of NO2 gas, permitting repeatable detection over a range of relative humidities. This significant improvement is found to depend on the arrangement of the SWCNT-zeolite layers in the sensing device, as well as the hydrophilicity of the chosen zeolite.

Chiral Sensing of Glucose Using Porous Pt-Modified Electrodes Exposing Pt High-Index Planes

The discrimination and detection of chiral molecules, or chiral sensing, is an extremely important technique in organic chemistry, pharmacology and living body-related sciences. Earlier studies have shown that high-index surfaces of metals possess chirality in its atomic arrangements, and that the surfaces are able to discriminate chirality of various organic molecules. Recently, we reported the enantioselectivity of porous platinum electrode with a large number of atomic steps bearing high-index surfaces, for the redox reaction process of 3, 4-dihydroxyphenyl alanine, glucose, and so on (1). However, for medical diagnosis, the chiral sensor is expected to develop to detect target molecules in a wider concentration range. In this study, we performed chiral sensing using various types of porous platinum-modified electrodes to develop the sensitivity of sensing. And, the dependence of concentration of glucose on the sensing signal, or the peak current density in cyclic voltammogram, was investigated to evaluate the performance for the chiral sensing. As electrodes with high-index surfaces, a porous platinum electrode, a platinum black-modified electrode, porous platinum particles-modified electrodes were investigated. Porous platinum electrode was prepared by electrodeposition from the plating bath including a surfactant Brij58(2), and porous platinum particles was prepared using chemical synthesis method(3). The detail preparation methods will be explained at the presentation. The chiral discrimination was investigated by cyclic voltammetry in the acidic glucose solution. Cyclic voltammetry was performed using the three-electrode cell: Ag/AgCl electrode as the reference electrode, platinum wire as the counter electrode and the various electrodes as working electrodes. We succeeded in discriminating chirality of glucose with all electrodes. The relation between the glucose concentration and the peak current density at around 0.35 V vs Ag/AgCl is shown in Figure. Here, the weight of Pt is standardized to be about 6.8×10-4g for all the samples. For all electrodes, as the glucose concentration increases, the peak current density increase, and the tendency emerged most conspicuously for L-form. In addition, the difference of the peak current densities between L- and D-form become the largest for a porous platinum particles-modified electrode. This is considered to be mainly due to the large specific surface area of high-index surfaces of the electrode. The detail examination and the detection limit of the concentration will be discussed at the presentation. 1 "Chiral Discrimination of 3,4-Dihydroxyphenylalanine by Electrodes Modified with Mesoporous Pt Film with a Large Number of Atomic Steps," Mariko Matsunaga, Kaji Kohei, 64th Annual Meeting of the International Society of Electrochemistry, Santiago de Queretaro, Mexico, Sep. 2013. 2 “Synthesis of Mesoporous Pt Films with Tunable Pore Sizes from Aqueous Surfactant Solutions,” H. J. Wang, L. Wang, T. Sato, Y. Sakamoto, S. Tominaka, K. Miyasaka, N. Miyamto, Y. Nemoto, O. Terasaki and Y. Yamauchi, Chem. Mater., 2012, 24, 1591. 3 “Multimetallic Mesoporous Spheres Through Surfactant-Directed Synthesis,” B. Jiang, C. L. Li, M. Imura, J. Tang and Y. Yamauchi, Adv Sci, 2015, 2. Figure 1

Flexible and Adhesive Surface Enhance Raman Scattering Active Tape for Rapid Detection of Pesticide Residues in Fruits and Vegetables.

The efficient extraction of targets from complex surfaces is vital for technological applications ranging from environmental pollutant monitoring to analysis of explosive traces and pesticide residues. In our present study, we proposed a proof-of-concept surface enhance Raman scattering (SERS) active substrate serving directly to the rapid extraction and detection of target molecules. The novel substrate was constructed by decorating the commercial tape with colloidal gold nanoparticles (Au NPs), which simultaneously provides SERS activity and "sticky" of adhesive. The utility of SERS tape was demonstrated by directly extracting pesticide residues in fruits and vegetables via a simple and viable "paste and peel off" approach. The obtained strong and easily distinguishable SERS signals allow us to detect various pesticide residues such as parathion-methyl, thiram, and chlorpyrifos in the real samples with complex surfaces including green vegetable, cucumber, orange, and apple.

Double-Fluorescent-Labeled Single-Chain Antibodies Showing Antigen-Dependent Fluorescence Ratio Change

Abstract Fluorescence ratio probes are useful tools for quantitative detection of target molecules even if the concentration of probe molecules is unknown. However, a general and widely applicable method for design and synthesis of fluorescence ratio probes has never been established. Here, we developed double-labeled single-chain antibody fragment (scFv) derivatives showing antigen-dependent fluorescence ratio change based on fluorescence resonance energy transfer (FRET) and antigen-dependent fluorescence quenching. Double-labeled scFvs were synthesized by incorporating fluorescent nonnatural amino acids labeled with TAMRA and Rhodamine Green (RhG) as a FRET donor and acceptor pair into N- and C-termini of scFv, respectively, using four-base and amber codons in a cell-free translation system. The resulting double-labeled antibody fragments showed significant fluorescence ratio change upon the antigen-binding. This result was explained by FRET occurring from RhG to TAMRA but TAMRA being quenched by Trp residues in the absence of antigen. The binding of the antigen canceled the quenching of TAMRA without changing FRET efficiency. Double-labeled antibody fragments developed here will be useful as diagnostic and imaging tools.

Molecularly Imprinted Tailor-Made Functional Polymer Receptors for Highly Sensitive and Selective Separation and Detection of Target Molecules

Molecularly imprinted polymers (MIPs) are functional materials capable of molecular recognition and other biorelevant functions and can be prepared with ease in a tailor-made fashion by copolymerization in the presence of a crosslinker and a template molecule (the target molecule or its analog) conjugated with a functional monomer(s). These robust and affordable synthetic polymer receptors are highly accepted as favorable alternatives to biomacromolecules such as antibodies and enzymes. This article provides a critical review of the present state of MIPs to establish perspectives on this technique via a survey of early to contemporary work, mainly conducted by the authors, covering new principles and methodology to generate MIPs. These include the design and synthesis of new functional monomers and crosslinkers to develop and/or introduce new functionalities, new polymerization methods to improve the imprint effect, and highly sensitive MIP-based assays and sensors.