Label-free Protein Detection Research Articles

Microneedle Biosensor: A Method for Direct Label-free Real Time Protein Detection.

Here we present the development of an array of electrical micro-biosensors in a microfluidic channel, called microneedle biosensors. A microneedle biosensor is a real-time, label-free, direct electrical detection platform, which is capable of high sensitivity detection, measuring the change in ionic current and impedance modulation, due to the presence or reaction of biomolecules such as proteins and nucleic acids. In this study, we successfully fabricated and electrically characterized the sensors and demonstrated successful detection of target protein. In this study, we used biotinylated bovine serum albumin as the receptor and streptavidin as the target analyte.

A close analysis of metal-enhanced fluorescence of tryptophan induced by silver nanoparticles: wavelength emission dependence

Abstract In the last few years, silver nanoparticles have been proposed as a promising alternative for the label-free detection of proteins via metal-enhanced fluorescence. Generally, the aromatic amino acid tryptophan is most frequently used in this type of studies, because the intrinsic fluorescence of proteins is usually dominated by tryptophan emissions. In the present study, we evaluated the fluorescence behavior of tryptophan in the presence of a silver colloid with nanoparticles of 100 nm in diameter. The results showed that a nanoparticles concentration of 32 mg L−1 induced maximum fluorescence enhancement. However, the metal-enhanced fluorescence was dependent on the emission wavelength of tryptophan, and this phenomenon was closely related to the metal surface reabsorption process (inner filter effect), suggesting that the plasmon resonance reabsorption effect should be taken into account in analyses involving protein studies by metal-enhanced fluorescence.

Reduced graphene oxide field-effect transistor for label-free femtomolar protein detection

We report reduced graphene oxide field effect transistor (R-GO FET) biosensor for label-free ultrasensitive detection of a prostate cancer biomarker, prostate specific antigen/α1-antichymotrypsin (PSA-ACT) complex. The R-GO channel in the device was formed by reduction of graphene oxide nanosheets networked by a self-assembly process. Immunoreaction of PSA-ACT complexes with PSA monoclonal antibodies on the R-GO channel surface caused a linear response in the shift of the gate voltage, Vg,min, where the minimum conductivity occurs. The R-GO FET can detect protein-protein interactions down to femtomolar level with a dynamic range over 6-orders of magnitude in the Vg,min shift as a sensitivity parameter. High association constants of 3.2nM−1 and 4.2nM−1 were obtained for the pH 6.2 and pH 7.4 analyte solutions, respectively. The R-GO FET biosensor showed a high specificity to other cancer biomarker in the phosphate buffered saline solutions as well as in the human serum.

Label-Free Detection of Prion Protein with Its DNA Aptamer through the Formation of T-Hg2+-T Configuration

Though rapid tests were developed for mass screening of prion diseases in the last century, bovine spongiform encephalopathy (BSE) was still epidemic in some European countries. The main reason is that the sensitivity of such tests is insufficient for detecting animals that are incubating with prion diseases at the presymptomatic stage. Driven by this, in this contribution, we developed a novel sensitive label-free method taking advantage of DNA aptamer for prion proteins (PrP) detection through the formation of T-Hg(2+)-T configuration. In the presence of Hg(2+) ions, double-strand structures formed due to the strong binding affinity of Hg(2+) ions to the T bases of DNA aptamer, which dramatically enhanced the fluorescence of Syber Green I, a double-strand indicator. With the addition of prion protein, however, the specific interaction between prion protein and its aptamer forced the destruction of the double-strand structures, and thus the fluorescence of Syber Green I decreased. It was found that there is a linear relationship between the decreased fluorescence intensities and prion protein concentration ranging from 13.0 to 156.0 nmol/L. Compared with other methods, the method presented here holds the advantages of being label-free, rapid, highly sensitive, and selective, which shows great promise for clinical application.

Rational design of modular allosteric aptamer sensor for label-free protein detection

An aptamer can be redesigned to new functional molecules by conjugating with other oligonucleotides. However, it requires experimental trials to optimize the conjugating module with the sensitivity and selectivity toward a target. To reduce these efforts, we report rationally-designed modular allosteric aptamer sensor (MAAS), which is composed of coupled two aptamers and the regulator. For label-free protein detection, the protein–aptamer was conjugated with the malachite green (MG) aptamer for signaling. The MAAS additionally has the regulator domain which is designed to hybridize to a protein binding domain. The regulator makes MAAS to be inactive by destructing the original structure of the two aptamers. However, its conformation becomes active by dissociating the hybridization from the protein recognition signal, thereby inducing the binding of MG emitting the enhanced fluorescence. The design of regulator is based on the thermodynamic energy difference by the RNA conformational change and protein–aptamer affinity. Here we first demonstrated the MAAS for hepatitis C helicase and replicase. The target proteins were detected up to 250nM with minimized blank signals and displayed high specificities 10-fold greater than in non-specific proteins. The MAAS provides valuable tools that can be adapted to a wide range of configurations in bioanalytical applications.

An optimised electrochemical biosensor for the label-free detection of C-reactive protein in blood

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. There exists, in particular, a great deal of interest in the correlation between blood serum levels and the severity of risk for cardiovascular disease. A sensitive, label-free, non-amplified and reusable electrochemical impedimetric biosensor for the detection of CRP in blood serum was developed herein based on controlled and coverage optimised antibody immobilization on standard polycrystalline gold electrodes. Charge transfer resistance changes were highly target specific, linear with logCRPconcentration across a 0.5–50nM range and associated with a limit of detection of 176pM. Significantly, the detection limits are better than those of current CRP clinical methods and the assays are potentially cheap, relatively automated, reusable, multiplexed and highly portable. The generated interfaces were capable not only of comfortably quantifying CRP across a clinically relevant range of concentrations but also of doing this in whole blood serum with interfaces that were, subsequently, reusable. The importance of optimising receptor layer resistance in maximising assay sensitivity is also detailed.

Ultra-Sensitive Label-Free Detection of Proteins by Chemically Derived Graphene Based Field-Effect Transistor

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Label-free detection of C-reactive protein using reflectometric interference spectroscopy-based sensing system

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique, and suitable for detecting antibody–antigen interaction. This work describes a continuous flow biosensor for C-reactive protein (CRP), involving an effective immobilization method of a monoclonal antibody against CRP (anti-CRP) to achieve highly sensitive RIfS-based detection of CRP. The silicon nitride-coated silicon chip (SiN chip) for the RIfS sensing was first treated with trimethylsilylchloride (TMS), followed by UV-light irradiation to in situ generation of homogeneous silanols on the surface. Following amination by 3-aminopropyltriethoxysilane, carboxymethyldextran (CMD) was grafted, and subsequently, protein A was immobilized to create the oriented anti-CRP surface. The immobilization process of protein A and anti-CRP was monitored with the RIfS system by consecutive injections of an amine coupling reagent, protein A and anti-CRP, respectively, to confirm the progress of each step in real time. The sensitivity was enhanced when all of the processes were adopted, suggesting that the oriented immobilization of anti-CRP via protein A that was coupled with the grafted CMD on the aminated surface of TMS-treated SiN chip. The feasibility of the present sensing system was demonstrated on the detection of CRP, where the silicon-based inexpensive chips and the simple optical setup were employed. It can be applied to other target molecules in various fields of life science as a substitute of surface plasmon resonance-based expensive sensors.

Label-Free Fluorescent Detection of Ions, Proteins, and Small Molecules Using Structure-Switching Aptamers, SYBR Gold, and Exonuclease I

We have demonstrated a label-free sensing strategy employing structure-switching aptamers (SSAs), SYBR Gold, and exonuclease I to detect a broad range of targets including inorganic ions, proteins, and small molecules. This nearly universal biosensor approach is based on the observation that SSAs at binding state with their targets, which fold into secondary structures such as quadruplex structure or Y shape structure, show more resistance to nuclease digestion than SSAs at unfolded states. The amount of aptamer left after nuclease reaction is proportional to the concentrations of the targets and in turn is proportional to the fluorescence intensities from SYBR Gold that can only stain nucleic acids but not their digestion products, nucleoside monophosphates (dNMPs). Fluorescent assays employing this mechanism for the detection of potassium ion (K(+)) are sensitive, selective, and convenient. Twenty μM K(+) is readily detected even at the presence of a 500-fold excess of Na(+). Likewise, we have generalized the approach to the specific and convenient detection of proteins (thrombin) and small molecules (cocaine). The assays were then validated by detecting K(+), cocaine, and thrombin in urine and serum or cutting and masking adulterants with good agreements with the true values. Compared to other reported approaches, most limited to G-quadruplex structures, the demonstrated method has less structure requirements of both the SSAs and their complexes with targets, therefore rending its wilder applications for various targets. The detection scheme could be easily modified and extended to detection platforms to further improve the detection sensitivity or for other applications as well as being useful in high-throughput and paralleled analysis of multiple targets.

Label-free specific detection of immunoglobulin G antibody using nanoporous hydrogel photonic crystals

Photonic crystal (PC) based biosensors have been given attention because of their obvious advantages such as sensitivity, simple and low-cost realization. Many kinds of PC based biosensors have been reported, for example, holographic PCs, porous silicon PCs, PC fibers and colloidal crystal film. Among these PC based sensors, we have interested on an inverse opal structure film in order to apply to specific and label-free detection of proteins. This label-free PC bioassay is more attractive than labeling detection because target molecules can be detected in their nature forms, truly portable point-of-care testing is possible, and quantitative measurement of molecule interaction is allowable. In this study, we propose a specific and label-free detection method for immunoglobulin G (IgG) antibody using nanoporous hydrogel photonic crystals with simple process and cost-effective way. The hydrogels used as a backbone of PC in this study, which provide rather homogeneous water surrounding, are ideal substrates for biomolecular screening. Also, three-dimensional hydrogel structures show high capacity for biomolecule immobilization, greater probability of interacting with the target ligand, high S/N, and sensitivity. Finally, the film type biosensor was fabricated using photo-curable hydrogels with colloidal templating methods. The reflective color of the fabricated sample is changed from green to dark orange with the bandgap shift of 50nm, when the IgG concentration was 10mg/ml. Furthermore, our device showed different bandgap shifts according to the level of antibody concentration, so that it can be used to measure quantitative molecular interactions. This designed label free detection system has great potential for next generation of bioassay platform for diagnosis of pathogens such as virus, bacteria, and fungi.

Surface Plasmon Resonance Biosensors Incorporating Gold Nanoparticles

SPR biosensing is increasingly popular for the detection of a multitude of biomolecules. It offers label-free detection and study of proteins, nucleic acids, and other biomolecules in real time. A recent trend involves incorporation of AuNPs, either within the sensing surface itself or as signal enhancing tagging molecules. The importance of AuNP and detecting agent spacing is described and techniques using macromolecular spacing aids are highlighted. Recent methods to enhance SPR detection capabilities using gold nanoparticles are reviewed, as well as device fabrication and the results of incorporation. SPR detection is a highly versatile method for the detection of biomolecules and, with the incorporation of AuNPs, shows promise in extending it to a number of new applications.

Fluorescence-based multiplex protein detection using optically encoded microbeads.

Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.

Adsorptive Stripping Voltammetry of Hen-Egg-White-Lysozyme via Adsorption–Desorption at an Array of Liquid–Liquid Microinterfaces

Electrochemical adsorption and voltammetry of hen-egg-white-lysozyme (HEWL) was studied at an array of microinterfaces between two immiscible electrolyte solutions (μITIES). Adsorption of the protein was achieved at an optimal applied potential of 0.95 V, after which it was desorbed by a voltammetric scan to lower potentials. The voltammetric peak recorded during the desorption scan was dependent on the adsorption time and on the aqueous phase concentration of HEWL. The slow approach to saturation or equilibrium indicated that protein reorganization at the interface was the rate-determining step and not diffusion to the interface. For higher concentrations and longer adsorption times, a HEWL multilayer surface coverage of 550 pmol cm(-2) was formed, on the basis of the assumption that a single monolayer corresponded to a surface coverage of 13 pmol cm(-2). Implementation of adsorption followed by voltammetric detection as an adsorptive stripping voltammetric approach to HEWL detection demonstrated a linear dynamic range of 0.05-1 μM and a limit of detection of 0.03 μM, for 5 min preconcentration in unstirred solution; this is a more than 10-fold improvement over previous HEWL detection methods at the ITIES. These results provide the basis for a new analytical approach for label-free protein detection based on adsorptive stripping voltammetry.

Label-free detection of proteins from dried-suspended droplets using surface enhanced Raman scattering

A simple sample preparation method to obtain rich and reproducible surface-enhanced Raman scattering (SERS) spectra from proteins regardless of their surface properties and dimensions for label-free detection and identification is reported. The method uses colloidal silver nanoparticles (AgNPs) as substrates and is based on suspending the droplet of a mixture containing AgNPs and proteins from a hydrophobic surface. Drying the droplet at this suspended configuration allows the accumulation and packing of AgNPs and protein molecules in the middle of the droplet area rather than getting jammed at the edges of drying droplets as the solvent evaporates. A detection limit of down to 0.05 μg mL(-1) for some of the model proteins used in this study is obtained with this simple approach. The advantage of this method is its simplicity and improved sensitivity over other approaches reported in the literature.

Application of a new microcantilever biosensor resonating at the air–liquid interface for direct insulin detection and continuous monitoring of enzymatic reactions

Here we describe the application of a recently developed high-resolution microcantilever biosensor resonating at the air-liquid interface for the continuous detection of antigen-antibody and enzyme-substrate interactions. The cantilever at the air-liquid interface demonstrated 50% higher quality factor and a 5.7-fold increase in signal-to-noise-ratio (SNR) compared with one immersed in the purified water. First, a label-free detection of a low molecular weight protein (insulin, 5.8 kDa) in physiological concentration was demonstrated. The liquid facing side of the cantilever was functionalized by coating its surface with insulin antibodies, while the opposite side was exposed to air. The meniscus membrane at the micro-slit around the cantilever sustained the liquid in the microchannel. After optimizing the process of surface functionalization, the resonance frequency shift was successfully measured for insulin solutions of 0.4, 2.0, and 6.3 ng ml(-1). To demonstrate additional application of the device for monitoring enzymatic protein degradation, the liquid facing microcantilever surface was coated with human recombinant SOD1 (superoxide dismutase 1) and exposed to various concentrations of proteinase K solution, and the kinetics of the SOD1 digestion was continuously monitored. The results showed that it is a suitable tool for sensitive protein detection and analysis.

Integrated Microfluidic Environment for Solid-state Nanopore Sensors

Abstract The micro- and nanotechnology based biosensing principles open up new possibilities towards the development and realisation of robust, user-friendly and cost-effective in-vitro diagnostic platforms. Furthermore, label-free and multi- analyte detection is envisioned to allow more accuracy and higher throughput in clinical diagnostic. The bioanalytical systems are expected to integrate the nanoscale transducers with interface chemistry, and bio-receptors as well as the microfluidics, control software and hardware. The final goal of this work is to develop nanopore based multi- parametric biosensing platform, applicable for label-free detection of blood marker proteins of cardiovascular symptoms.

A sensitive strategy for label-free and time-resolved fluorescence assay of thrombin using Tb-complex and unmodified gold nanoparticles

Gold nanoparticles (GNPs) can effectively differentiate the unfolded and folded aptamer, and quench the fluorescence of terbium ternary complexes (Tb-complexes), thus the authors herein report a sensitive strategy for protein detection, using label-free aptamer, Tb-complexes and GNPs. In the presence of thrombin, the aptamer is inclined to form G-quartet, and the folded aptamer cannot adsorb on the surface of GNPs, inducing the GNPs aggregation in the presence of 0.5 mol L(-1) salt. After centrifugation at low speed to remove the aggregated GNPs, the quenching capability of the supernatant for Tb-complexes is decreased. The fluorescence intensity of Tb-complexes increases as the concentration of thrombin increases. Due to the highly specific recognition ability of the aptamer for thrombin and the strong quenching property of GNPs for Tb-complexes, the proposed protocol has good selectivity and high sensitivity for thrombin. Under the optimum conditions, a linear range from 1.0 × 10(-9) M to 1.0 × 10(-8) M is obtained with a detection limit of 0.14 nM, which is much lower than those commonly obtained for colorimetric sensors and some fluorescent sensors. The signal of Tb-complexes can be measured by time-resolved manner which made most of the unspecific fluorescent background signals be eliminated. The proposed sensor has been successfully applied in complicated biological samples for thrombin detection, and it can provide a promising potential for label-free aptamer-based protein detection.

Configuration change of liquid crystal microdroplets coated with a novel polyacrylic acid block liquid crystalline polymer by protein adsorption

4-cyano-4'-pentylbiphenyl (5CB) microdroplets coated with PAA-b-LCP were successfully made via microfluidics. Their functionalized interfaces were examined for lysozyme and bovine serum albumin detection, which could be easily visualized with an optical microscope under crossed polarizers by the radial-to-bipolar configurational change of the 5CB. This transition was strongly dependent on the electrostatic states of the proteins and PAA chains, which were controlled by the pH of the water. This study demonstrated a simple approach for the development of a liquid crystal-based sensor for label-free detection of proteins, which may have diverse applications in various biomedical fields.

Direct-writing colloidal photonic crystal microfluidic chips by inkjet printing for label-free protein detection

Integrating photonic crystals (PC) into microfluidic systems has attracted immense interest for its novel functions. However, it is still a great challenge to fabricate PC microfluidic chips rapidly with complex functions. In this work, a direct-writing colloidal PC microchannel was firstly achieved by inkjet printing and was used for the surface-tension-confined microfluidic immune assay. PC channels with different structure colors have been successfully integrated on one chip. The fabricated chip has the advantages of rapid fabrication, quick fluidic transport and can monitor the fluidic fluxion using the naked eye. Utilizing this PC microfluidic chip, a colorimetric label-free immune assay was realized without nonspecific adsorption interference of the target.

Gold nanoparticles as a label-free probe for the detection of amyloidogenic protein

Because amyloidogenic proteins, such as prion protein, β-amyloid peptide and α-synuclein, are associated with a variety of diseases, methods for their detection are important. Recombinant prion protein (rPrP) can selectively induce aggregation of dihydrolipoic acid capped gold nanoparticles (DHLA-AuNPs), which reduces the absorbance of the DHLA-AuNPs and changes their color from red to blue. These changes were used for label-free qualitative and quantitative detection of amyloidogenic protein. The addition of NaCl improved the detection sensitivity considerably, and the detection limit was as low as 33pmol/L.