Sensitive Detection Of Biomolecules Research Articles

A microchip electrophoresis-based fluorescence signal amplification strategy for highly sensitive detection of biomolecules.

We have developed a microchip electrophoresis (MCE)-based fluorescence signal amplification strategy as a universal MCE method for the detection of trace biomolecules. This strategy exhibits high sensitivity and specificity for target molecules, and has been applied for the detection of interferon-gamma (IFN-γ) in human plasma with satisfactory results.

Sensitive detection of biomolecules and DNA bases based on graphene nanosheets

High surface area electrode materials are of interest for the application of electrochemical sensors. Currently, chemical vapor deposition (CVD) graphene-sensing electrodes are scarce. Herein, for the first time, a graphene based on a Ta wire support was prepared using the CVD method to form a highly electroactive biosensing platform. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV) were utilized to characterize the morphology and investigate the electrochemical properties of the CVD graphene electrodes. The resulting CVD graphene electrode exhibited good electrocatalytic activity and had a prominent response effect on dopamine, uric acid, guanine, and adenine. Standing graphene nanosheets have rich catalytic sites such as the edges, the defect levels of the plane, and porous network structures between the graphene nanosheets. These catalytic sites prompt the adsorption and resolution for the four species and the strong electron transport capability of the CVD graphene, which effectively improved the electrical signals for response to four species. Moreover, the graphene electrode is a promising candidate in electrochemical sensing and other electrochemical device applications.

Quantum dots bilayers/Au@Ag-based electrochemiluminescence resonance energy transfer for detection of thrombin by autocatalytic multiple amplification strategy

A novel CdSe quantum dots (QDs) bilayers/Au@Ag electrochemiluminescence (ECL) resonance energy transfer (ERET) system was designed for sensitive determination of thrombin by autocatalytic multiple amplification strategy. In this protocol, the graded-gapquantum dots (QDs) bilayers were fabricated by layer-by layer assembly of differently sized CdSe-NH2 and CdSe-COOH QDs on a Au-graphite oxide (Au-GO) nanocomposite modified electrode. A surprising ECL enhancement effect in the QDs bilayers was observed, ECL signal from the QDs bilayers was enhanced 3-foldcompared to that of the monolayer CdSe-NH2 QDs. Furthermore, a new Au@Ag core−shell nanoparticle (NP) was synthesized, the ECL signal from the bilayers QDs was efficiently quenched by the Au@Ag NPs. Based on the ECL energy transfer between the bilayers QDs and the Au@AgNPs, we designed a new ECL biosensor for thrombin detection by using multiple nuclease-assisted autocatalytic amplification strategy. Importanly, the layered QDs with highly intense ECL could provide a promising candidate for the development of a sensitive ECL biosensor, and it is for the first time that layered QDs were used as ECL probe and combined with recycling amplification for highly sensitive detection of biomolecules. The proposed scheme may open a new QDs-based ERET system for highly sensitive detection of versatile target biomolecules, and has a good prospect for clinical diagnosis in the future.

Silicon-Based BioFETs with 3-D Nanostructure: Easy integration, precise control of nanostructure, and a low device-to-device variation

Biosensors enable the quantitative detection of target biomolecules in a sample. A biosensor is composed of two parts: the receptor and the transducer. The detection mechanism of biosensors is typically based on a lockand-key approach, also known as the affinity-based approach. A receptor, such as deoxyribonucleic acid or an antibody, captures the target molecules by selectively binding with them for the purpose of detection. The transducer can convert the biobinding event into a measureable signal. For example, a cantilever transduces the mass of biomolecules into a change in resonance frequency, and electrochemical sensors transduce the charge of biomolecules into a change in current. Well-developed transducers are critical for accurate and sensitive detection of biomolecules.

(Invited) Electrical Detection of Biomolecules Using Graphene-Based Devices

Label-free electrical monitoring of biorecognition events provides a promising platform, which is simpler, less expensive and requires less energy. Rapid testing of different proteins is required in various applications, including clinical diagnostics, environmental testing, food analysis, bioterrorism detection technologies, etc. Graphene, a single honeycomb-like sheet of carbon atoms, have been intensively investigated in most recent owing to its extraordinary high mobility even at room temperature [1]. Since graphene has a perfect two-dimensional structure, electrical characteristics in graphene devices are very sensitive for modulation of surface potentials in graphene channels. In this study, we have demonstrated highly sensitive electrical detection of biological molecules based on graphene devices.A monolayer graphene was obtained by mechanical cleavage of graphite or CVD-based graphene on SiO2 films. Graphene devices were fabricated by conventional lithography and lift-off method. A silicone rubber was placed on the graphene devices so that the graphene channel was immersed in a buffer solution. An Ag/AgCl reference electrode was used as the top-gated electrode to minimize environmental effects.Solution-gated graphene devices showed good transfer characteristics in buffer solution [2]. To selectively detect targets, receptors were modified on graphene channels [3-7]. For highly sensitive detection of biomolecules, small receptors such as aptamer or antigen-binding fragment (Fab) were utilized. Atomic force microscopy images revealed that receptors were successfully immobilized on the graphene channel. The receptor-modified graphene devices showed electrical detection of targets with concentration from few hundred pM to several hundred nM. Moreover, for development of highly sensitive influenza-virus sensors, glycan-modified graphene devices were also fabricated. The biological molecules were detected with high sensitivity based on graphene devices. When a different kind of receptor is functionalized on each graphene channel in the graphene-device array, many kinds of biomolecules can be electrically detected simultaneously. Therefore, the graphene-device array can be useful for the fabrication of multiplex hand-held chemical and biological sensors for home medical care. K. Novoselov, A. Geim, S. Morozov, D. Jiang, Y. Zhang, S. Dubonos, I. Grigorieva and A. Firsov, Science 306, 666 (2004). Y. Ohno, K. Maehashi, and K. Matsumoto, Biosens. Bioelectron. 26 , 1727 (2010). Y. Ohno, K. Maehashi, Y. Yamashiro, and K. Matsumoto, Nano Lett. 9, 3318 (2009). K. Maehashi, Y. Sofue, S. Okamoto, Y. Ohno, K. Inoue and K. Matsumoto, Sensors and Actuators B 187, 45 (2013). Y. Ohno, K. Maehashi, and K. Matsumoto, J. Am. Chem. Soc. 132, 18012 (2010). S. Okamoto, Y. Ohno, K. Maehashi, K. Inoue, and K. Matsumoto, Jpn. J. Appl. Phys. 51, 06FD08 (2012). N. B. M. Zaifuddin, S. Okamoto, T. Ikuta, Y. Ohno, K. Maehashi, M. Miyake, P. Greenwood, K. B. K. Teo, and K. Matsumoto, Jpn. J. Appl. Phys. 52, 06GK04 (2013).

Polystyrene latex particles containing europium complexes prepared by miniemulsion polymerization using bovine serum albumin as a surfactant for biochemical diagnosis

Luminescent particles have been attracting significant attention because they can be used in biochemical applications, such as detecting and imaging biomolecules. In this study, luminescent polystyrene latex particles were prepared through miniemulsion polymerization of styrene with dissolved europium complexes in the presence of bovine serum albumin (BSA) and poly(ethylene glycol) monomethoxy methacrylate as surfactants. The solubility of the europium complex in styrene has a strong effect on the yield of the particle. Europium tris(2-thenoyl trifluoroacetonate) di(tri-n-octyl phosphine oxide), which has a high solubility in styrene, was sufficiently incorporated into the polystyrene particles compared to europium tris(2-thenoyl trifluoroacetonate), which has a low solubility in styrene. The luminescence property of the europium complex could remain intact even after its incorporation through the miniemulsion polymerization. In the aqueous dispersion, the resulting particles could emit strong luminescence, which is a characteristic of the europium complex. The antibody fragments were covalently attached to BSA-covered particles after a reaction with a bifunctional linker, N-(6-maleimidocaproyloxy)succinimide. The time-resolved fluoroimmunoassay technique showed that 3.3pg/mL of human α-fetoproteins (AFP) can be detected by using the resulting luminescent particles. An immunochromatographic assay using the resulting particles was also performed as a convenient method to qualitatively detect biomolecules. The detection limit of AFP measured by the immunochromatographic assay was determined to be 2000pg/mL. These results revealed that the luminescent particles obtained in this study can be utilized for the highly sensitive detection of biomolecules and in vitro biochemical diagnosis.

Optical Detection of Biological Activity, One Molecule at a Time

Molecular recognition is central to the design of therapeutics, chemical catalysis and sensor platforms, with the most common mechanisms involving biological structures such as protein antibodies. However, identifying and isolating an antibody for a particular biological molecule is often costly and time-consuming, and their use is limited to physiological conditions in which protein antibodies remain stable. Furthermore, most metabolites lack molecular recognition elements. Therefore, the biggest limitation to metabolite detection has been the selective and sensitive detection of biomolecules that have no naturally-occurring molecular detection counterpart. Nanomaterials are of a similar size to the molecular entities that govern life within cells, and are therefore well-suited to studying life at the sub-cellular scale.This work reports the development of a synthesis platform for the design of synthetic sensors, based on the principle of polymer corona phases surrounding near-infrared optically active nanoparticles. The design of such sensors is based on the adsorptive properties of engineered heteropolymers to carbon nanotubes, which create a unique corona with molecular recognition properties for a target analyte. This phenomenon is shown to be generic, with new recognition complexes demonstrated for riboflavin, l-thyroxine, and estradiol, and confirmed with single-molecule imaging of the nanotube and the corona phase. The immediate utility of this platform was confirmed in its application to discover the first optical sensor for neurotransmitter dopamine, and was applied to monitor vitamin trafficking in living macrophage cells for over an hour. Furthermore, this platform has most recently been used in the scope of “plant nanobionics”: the detection of molecular pollutants inside living plants. The analyte library for these sensors could be amplified to identify synthetic sensors for a variety of different molecules for which there currently exists no detection method.

A nanoporous gold membrane for sensing applications

Design and fabrication of three-dimensionally structured, gold membranes containing hexagonally close-packed microcavities with nanopores in the base, are described. Our aim is to create a nanoporous structure with localized enhancement of the fluorescence or Raman scattering at, and in the nanopore when excited with light of approximately 600nm, with a view to provide sensitive detection of biomolecules. A range of geometries of the nanopore integrated into hexagonally close-packed assemblies of gold micro-cavities was first evaluated theoretically. The optimal size and shape of the nanopore in a single microcavity were then considered to provide the highest localized plasmon enhancement (of fluorescence or Raman scattering) at the very center of the nanopore for a bioanalyte traversing through. The optimized design was established to be a 1200nm diameter cavity of 600nm depth with a 50nm square nanopore with rounded corners in the base. A gold 3D-structured membrane containing these sized microcavities with the integrated nanopore was successfully fabricated and ‘proof of concept’ Raman scattering experiments are described.

Fluorescent aptamer-based assay for thrombin with large signal amplification using peroxidase mimetics

This article describes an aptamer-based thrombin assay using a hemin-based peroxidase mimetic for signal amplification. Thrombin is recognized by an immobilized primary aptamer (G1-quadruplex). The G1-quadruplex/hemin complex formed quenches the fluorescence of CdTe quantum dots (QDs) due to photoinduced electron transfer (PET). In the next step, thrombin is associated with a secondary aptamer (G2-quadruplex) consisting of Pt nanoparticles (NPs), G2-quadruplex and hemin to form a sandwich structure. Both the G1-quadruplex/hemin complex and the Pt NPs/G2-quadruplex/hemin complex associated with thrombin act as enzyme mimetics and catalyze the oxidation of hydroquinone by H2O2 to form 2-hydroxy-p-benzoquinone (HPB). The HPB produced quenches the fluorescence of the CdTe QDs via a PET and an inner filter effect, thus causing large signal amplification. The effects were exploited to design a highly sensitive and selective thrombin assay. Under optimized conditions, a linear fluorescence response is achieved in the 0.05 pmol·L−1 to 10 nmol·L−1 concentration range, with a lower detection limit of 15 fmol·L−1. This approach, in our perception, represents a promising platform for sensitive detection of biomolecules for which appropriate aptamers can be found.

Sensitive biomolecule detection in lateral flow assay with a portable temperature–humidity control device

Lateral flow assays (LFAs) have currently attracted broad interest for point-of-care (POC) diagnostics, but their application has been restricted by poor quantification and limited sensitivity. While the former has been currently solved to some extent by the development of handheld or smartphone-based readers, the latter has not been addressed fully, particularly the potential influences of environmental conditions (e.g., temperature and relative humidity (RH)), which have not yet received serious attention. The present study reports the use of a portable temperature–humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification by using a smartphone. We found that a RH beyond 60% with temperatures of 55–60°C and 37–40°C produced optimum nucleic acid hybridization and antigen–antibody interaction in LFAs, respectively representing a 10-fold and 3-fold signal enhancement over ambient conditions (25°C, 60% RH). We envision that in the future the portable device could be coupled with a fully integrated paper-based sample-to-answer biosensor for sensitive detection of various target analytes in POC settings.

A plasmonic colorimetric strategy for biosensing through enzyme guided growth of silver nanoparticles on gold nanostars

A plasmonic colorimetric strategy was designed for sensitive detection of biomolecules through enzyme guided silver nanoparticles (AgNPs) growth on gold nanostars (AuNS). The growth of AgNPs on AuNS led to a substantial blue shift of the localized surface plasmon resonance (LSPR) peak and the color change of AuNS from blue to dark blue, purple and ultimately orange. Both the LSPR blueshift wavelength and the color of detection solution containing AuNS, Ag+ and ascorbic acid 2-phosphate (AAP) depend on the amount of enzyme that catalyzed the dephosphorylation of AAP to reduce Ag+ on AuNS surface. Thus this strategy could be used for LSPR and naked-eye detections of both the enzyme such as alkaline phosphatase (ALP) and other biomolecules involved in biorecognition events using ALP as a tag. The LSPR detection method for ALP showed a linear range from 1.0pM to 25nM with a detection limit of 0.5pM. Using DNA as a mode target molecule, this technique showed a detection range from 10fM to 50pM DNA with a detection limit of 2.6fM through the convenient combination with hybridization chain reaction amplification. The proposed plasmonic colorimetric strategy could be extended as a general analytical platform for design of immunosensors and aptasensors with ALP as a label.

Desalting by crystallization: detection of attomole biomolecules in picoliter buffers by mass spectrometry.

Sensitive detection of biomolecules in small-volume samples by mass spectrometry is, in many cases, challenging because of the use of buffers to maintain the biological activities of proteins and cells. Here, we report a highly effective desalting method for picoliter samples. It was based on the spontaneous separation of biomolecules from salts during crystallization of the salts. After desalting, the biomolecules were deposited in the tip of the quartz pipet because of the evaporation of the solvent. Subsequent detection of the separated biomolecules was achieved using solvent assisted electric field induced desorption/ionization (SAEFIDI) coupled with mass spectrometry. It allowed for direct desorption/ionization of the biomolecules in situ from the tip of the pipet. The organic component in the assistant solvent inhibited the desorption/ionization of salts, thus assured successful detection of biomolecules. Proteins and peptides down to 50 amol were successfully detected using our method even if there were 3 × 10(5) folds more amount of salts in the sample. The concentration and ion species of the salts had little influence on the detection results.

CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum.

Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive visualization of metabolically labeled molecules in biological systems. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Termed the CalFluors, these probes possess emission maxima that range from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash conditions. We used the CalFluor probes to image various alkyne-labeled biomolecules (glycans, DNA, RNA, and proteins) in cells, developing zebrafish, and mouse brain tissue slices.

A novel label-free biosensor based on self-assembled aptamer/GO architecture for sensitive detection of biomolecules

We developed a novel label-free biosensor for biomolecule detection based on the thioflavin T (ThT)-induced conformational change of guanine-rich oligonucleotides and self-assembled aptamer/GO nanosheet architecture.

A WS2 nanosheet based chemiluminescence resonance energy transfer platform for sensing biomolecules.

We developed a WS2 nanosheet based chemiluminescence resonance energy transfer (CRET) platform for sensing biomolecules. This platform exhibits high detection sensitivity and high specificity for target molecules.

Two-dimensional layered MoS₂ biosensors enable highly sensitive detection of biomolecules.

We present a MoS2 biosensor to electrically detect prostate specific antigen (PSA) in a highly sensitive and label-free manner. Unlike previous MoS2-FET-based biosensors, the device configuration of our biosensors does not require a dielectric layer such as HfO2 due to the hydrophobicity of MoS2. Such an oxide-free operation improves sensitivity and simplifies sensor design. For a quantitative and selective detection of PSA antigen, anti-PSA antibody was immobilized on the sensor surface. Then, introduction of PSA antigen, into the anti-PSA immobilized sensor surface resulted in a lable-free immunoassary format. Measured off-state current of the device showed a significant decrease as the applied PSA concentration was increased. The minimum detectable concentration of PSA is 1 pg/mL, which is several orders of magnitude below the clinical cut-off level of ~4 ng/mL. In addition, we also provide a systematic theoretical analysis of the sensor platform – including the charge state of protein at the specific pH level, and self-consistent channel transport. Taken together, the experimental demonstration and the theoretical framework provide a comprehensive description of the performance potential of dielectric-free MoS2-based biosensor technology.

Active accumulation of very diluted biomolecules by nano-dispensing for easy detection below the femtomolar range.

Highly sensitive detection of biomolecules is of paramount interest in many fields including biomedicine, safety and eco-pollution. Conventional analyses use well-established techniques with detection limits ~1 pM. Here we propose a pyro-concentrator able to accumulate biomolecules directly onto a conventional binding surface. The operation principle is relatively simple but very effective. Tiny droplets are drawn pyro-electro-dynamically and released onto a specific site, thus increasing the sensitivity. The reliability of the technique is demonstrated in case of labelled oligonucleotides diluted serially. The results show the possibility to detect very diluted oligonucleotides, down to a few hundreds of attomoles. Excellent results are shown also in case of a sample of clinical interest, the gliadin, where a 60-fold improved detection limit is reached, compared with standard ELISA. This method could open the way to a mass-based technology for sensing molecules at very low concentrations, in environmental as well as in diagnostics applications.

Sensitive Detection of Human Insulin Using a Designed Combined Pore Approach

A unique combined pore approach to the sensitive detection of human insulin is developed. Through a systematic study to understand the impact of pore size and surface chemistry of nanoporous materials on their enrichment and purification performance, the advantages of selected porous materials are integrated to enhance detection sensitivity in a unified two-step process. In the first purification step, a rationally designed large pore material (ca. 100 nm in diameter) is chosen to repel the interferences from nontarget molecules. In the second enrichment step, a hydrophobically modified mesoporous material with a pore size of 5 nm is selected to enrich insulin molecules. A low detection limit of 0.05 ng mL(-1) in artificial urine is achieved by this advanced approach, similar to most antibody-based analysis protocols. This designer approach is efficient and low cost, and thus has great potential in the sensitive detection of biomolecules in complex biological systems.

Carbon nanoparticle-protected aptamers for highly sensitive and selective detection of biomolecules based on nuclease-assisted target recycling signal amplification.

Based on the protective properties of carbon nanoparticles for aptamers against the digestion of nuclease, we have developed a nuclease-assisted target recycling signal amplification method for highly sensitive detection of biomolecules, such as ATP and kanamycin. The high binding specificity between aptamers and targets leads to excellent selectivity of the assay.

High sensitivity molecule detection by plasmonic nanoantennas with selective binding at electromagnetic hotspots

We report a highly sensitive biomolecule detection by plasmonic nanoantenna arrays with selective binding at the optical hotspots. The plasmonic nanoantennas consist of two separated Au nanorods with a thin Ti disk placed in between. By using selective surface modification chemistry, controlled binding occurs only in the gaps between the plasmonic nanoantennas, which ensures a high detection sensitivity. Both optical characterization using a dark field microscope and the FDTD simulation show that after the streptavidin binding, the signal increases with decreasing gap size. Compared to a single nanorod, the signal obtained per bound molecule in the nanoantennas increases by a factor of six, which is promising with respect to the future detection of single molecules.