Label-free Protein Detection Research Articles

Influence of intermediate aminodextran layers on the signal response of surface acoustic wave biosensors

Surface acoustic wave (SAW) devices based on horizontally polarized surface shear waves enable direct and label-free detection of proteins in real time. Binding reactions on the sensor surface are detected by determining changes in surface wave velocity caused mainly by mass adsorption or change of viscoelasticity in the sensing layer. Intermediate hydrogel layers have been proven to be useful to immobilize capture molecules or ligands corresponding to the analyte. However, the SAW signal response strongly depends on the morphology of the hydrogel due to different relative changes of its acoustomechanical parameters such as viscoelasticity and density. In this work five aminodextrans (AMD) and one diamino polyethylene glycol (DA-PEG) were used as intermediate hydrogel layers. Sensors with immobilized streptavidin and samples containing biotinylated bovine serum albumin were used to exemplify affinity assays based on immobilized capture molecules for protein detection. The effects of the three-dimensional AMDs and the two-dimensional (2D) DA-PEG on the SAW signal response were investigated. The signal height decreased with increasing molar mass and increasing amount of immobilized AMD. Consequently, thin hydrogel layers are ideal to obtain optimum signal responses in this type of assay, whereas it is not necessarily a 2D hydrogel that gives the best results.

Label-free aptasensor for platelet-derived growth factor (PDGF) protein

A label-free aptasensor for platelet-derived growth factor (PDGF) protein is reported. The aptasensor uses mixed self-assembled monolayers (SAMs) composed of a thiol-modified PDGF binding aptamer and 6-mercaptohexanol (MCH) on a gold electrode. The SAMs were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) before and after binding of the protein using [Fe(CN) 6] 3−/4−, a redox marker ion as an indicator for the formation of a protein–aptamer complex. The CVs at the PDGF modified electrode showed significant differences, such as changes in the peak currents and peak-to-peak separation, before and after binding of the target protein. The EIS spectra, in the form of Nyquist plots, were analyzed with a Randles circuit while the electron transfer resistance R ct was used to monitor the binding of the target protein. The results showed that, without any modification to the aptamer, the target protein can be recognized effectively at the PDGF binding aptamer SAMs at the electrode surface. Control experiments using non-binding oligonucleotides assembled at the electrode surfaces also confirmed the results and showed that there was no formation of an aptamer–protein complex. The DPV signal at the aptamer functionalized electrode showed a linearly decreased marker ion peak current in a protein concentrations range of 1–40 nM. Thus, label-free detection of PDGF protein at an aptamer modified electrode has been demonstrated.

Label-Free SERS Detection of Small Proteins Modified to Act as Bifunctional Linkers

Two double-cysteine mutants of a small protein judiciously modified so that the cysteines appear at axially opposite sides of the native fold were prepared such that different axes were defined in the two mutants. Upon reduction, the disulfide bonds are broken, and the proteins act as bifunctional ligands toward Ag nanoparticles, encouraging their assembly into nanoparticle dimers and small aggregates such that, when excited with laser light, the proteins are automatically located at electromagnetic hot spots within the aggregates. Because the protein molecules are small (~2.3 nm) and because the electromagnetic energy at a hot spot tends to increase as the size of the interparticle gap decreases, this nanoparticle-protein-nanoparticle geometry significantly enhances the Raman emission at the metallic surface. Exploiting this effect, we have recorded surface-enhanced Raman spectra (SERS) of the proteins at near-single-molecule level. The observed SERS spectra were dominated by the vibrations of molecular groups near the anchor points of the proteins.

Analytical Technique for Label-Free Multi-Protein Detection Based on Western Blot and Surface-Enhanced Raman Scattering

We have developed a new analytical procedure for label-free protein detection designated "Western SERS", consisting of protein electrophoresis, Western blot, colloidal silver staining, and surface-enhanced Raman scattering (SERS) detection. A novel method of silver staining for Western blot that uses a silver colloid, an excellent SERS-active substrate, is first proposed in the present study. During the process of silver staining, interactions between proteins and silver nanoparticles result in the emergence of SERS of proteins. In the present study, we use myoglobin (Mb) and bovine serum albumin (BSA) as model proteins. From different protein bands on a nitrocellulose (NC) membrane, we have observed surface-enhanced resonance Raman scattering (SERRS) spectra of Mb and SERS spectra of BSA. The proposed technique offers dual advantages of simplicity and high sensitivity. On one hand, after the colloidal silver staining, we can detect label-free multi-proteins directly on a NC membrane without digestion, extraction, and other pretreatments. On the other hand, the detection limit of the Western SERS is almost consistent with the detection limit of colloidal silver staining, and the SERRS detection limit of Mb is found to be 4 ng/band. This analytical method, which combines the technique of protein separation with SERS, may be a powerful protocol for label-free protein detection in proteomic research.

A Novel Sensor Platform Based on Aptamer‐Conjugated Polypyrrole Nanotubes for Label‐Free Electrochemical Protein Detection

We first present a simple yet versatile strategy for the functionalization of polymer nanotubes in a controlled fashion. Carboxylic-acid-functionalized polypyrrole (CPPy) nanotubes were fabricated by using cylindrical micelle templates in a water-in-oil emulsion system, and the functional carboxyl groups were effectively incorporated into the polymer backbone during the polymerization by using pyrrole-3-carboxylic acid (P3CA) as a co-monomer without a sophisticated functionalization process. It was noteworthy that the chemical functionality of CPPy nanotubes was readily controlled in both qualitative and quantitative aspects. On the basis of the controlled functionality of CPPy nanotubes, a field-effect transistor (FET) sensor platform was constructed to detect specific biological entities by using a buffer solution as a liquid-ion gate. The CPPy nanotubes were covalently immobilized onto the microelectrode substrate to make a good electrical contact with the metal electrodes, and thrombin aptamers were bonded to the nanotube surface via covalent linkages as the molecular recognition element. The selective recognition ability of thrombin aptamers combined with the charge transport property of CPPy nanotubes enabled the direct and label-free electrical detection of thrombin proteins. Upon exposure to thrombin, the CPPy nanotube FET sensors showed a decrease in current flow, which was probably attributed to the dipole-dipole or dipole-charge interaction between thrombin proteins and the aptamer-conjugated polymer chains. Importantly, the sensor response was tuned by adjusting the chemical functionality of CPPy nanotubes. The efficacy of CPPy nanotube FET sensors was also demonstrated in human blood serum; this suggests that they may be used for practical diagnosis applications after further optimization.

A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays.

We describe a novel scanning optical microscope based on a polarization-modulated nulling ellipsometry. The new microscope employs a combination of scanning mirror and sample translation and thus enables high-throughput label-free detection of biomolecular microarrays with more than 10 000 protein or small-molecule targets. For illustration, we show the image of a 2760-spot protein microarray on a functionalized glass slide obtained with such a microscope. The new scanning microscope is also capable of determining, in parallel, the real-time binding kinetics of multiple molecular species under aqueous conditions.

Resonant Cavity Imaging: A Means Toward High-Throughput Label-Free Protein Detection

The resonant cavity imaging biosensor (RCIB) is an optical technique for detecting molecular binding interactions label free at many locations in parallel that employs an optical resonant cavity for high sensitivity. Near-infrared light centered at 1512.5 nm couples resonantly through a Fabry-Perot cavity constructed from dielectric reflectors (Si/SiO(2)), one of which serves as the binding surface. As the wavelength is swept using a tunable laser, a near-infrared digital camera monitors cavity transmittance at each pixel. A wavelength shift in the local resonant response of the optical cavity indicates binding. Positioning the sensing surface with respect to the standing wave pattern of the electric field within the cavity controls the sensitivity with which the presence of bound molecules is detected. Transmitted intensity at thousands of pixel locations is recorded simultaneously in a 10 s, 5 nm scan. An initial proof-of-principle setup has been constructed. A test sample was fabricated with 25, 100-mum wide square features, each with a different density of 1-mum square depressions etched 12 nm into the SiO(2) surface. The average depth of each etched region was found with 0.05 nm rms precision. In a second test, avidin, bound selectively to biotin conjugated bovine serum albumin, was detected.

An Overview of Label-free Electrochemical Protein Sensors.

Electrochemical-based protein sensors offer sensitivity, selectivity and reliabilityat a low cost, making them very attractive tools for protein detection. Although the sensorsuse a broad range of different chemistries, they all depend on the solid electrode surface,interactions with the target protein and the molecular recognition layer. Traditionally, redoxenzymes have provided the molecular recognition elements from which target proteins haveinteracted with. This necessitates that the redox-active enzymes couple with electrodesurfaces and usually requires the participation of added diffusional components, or assemblyof the enzymes in functional chemical matrices. These complications, among many others,have seen a trend towards non-enzymatic-based electrochemical protein sensors. Severalelectrochemical detection approaches have been exploited. Basically, these have fallen intotwo categories: labeled and label-free detection systems. The former rely on a redox-activesignal from a reporter molecule or a label, which changes upon the interaction of the targetprotein. In this review, we discuss the label-free electrochemical detection of proteins,paying particular emphasis to those that exploit intrinsic redox-active amino acids.

Label-Free Detection of Protein−Protein Interactions at the GaAs/Water Interface through Surface Infrared Spectroscopy: Discrimination between Specific and Nonspecific Interactions by Using Secondary Structure Analysis

Here, we propose a label-free detection of protein-protein interactions that enables simultaneous qualitative analysis of target proteins by using Fourier transform infrared (FTIR) absorption spectroscopy in multiple internal reflection geometry (MIR-FTIR). Using this method, the target proteins were detected based on the peak height of the amide I and amide II bands, while discrimination of specific and nonspecific signals is made based on the secondary structure of the analytes, which is determined through second-derivative analysis of the amide I band. As a model system, an antigen peptide was immobilized on the surface of GaAs, which was transparent to mid-infrared light, and the interaction with its antibody was examined in aqueous media. We demonstrated that the binding of the antibody to the antigen immobilized on a GaAs surface selectively gave rise to beta-sheet amide I vibrations (1639 and 1690 cm-1), while no structurally related signals were induced by nonspecifically adsorbed proteins. The peak height of the beta-peak (1639 cm-1) in the amide I band linearly increased with the antiserum concentration as well as that of the amide II band. The detection limit (S/N = 3) was a 1:36 000 dilution for the amide I signal. In addition, through the use of surface-sensitive MIR-FTIR, the present sensor selectively detected the antigen-antibody interactions at the surfaces without being affected by the presence of bulk species, enabling rapid and wash-free detection. Our method provides not only rapid label-free detection of protein-protein interactions but a more accurate discrimination between specific and nonspecific interactions through the use of the secondary structure of the target proteins as a measure for the specific signals.

A universal biosensing platform based on optical micro-ring resonators

The use of optical micro-ring resonators as a platform for quantitative and qualitative biosensing applications was explored. Vertically coupled, high refractive index micro-ring resonators, used as sensing elements, were fabricated on silicon chips by photolithographic techniques. An optical reader system consisting of a near-infrared broad band light source and an optical spectrum analyzer were employed for data acquisition. Micro-ring resonator surfaces were modified with specific target receptors, including antibodies and single-stranded DNA oligonucleotides. The system was successfully used for label-free, specific, and rapid detection of whole bacterial cells, proteins and nucleic acids.

Integrated Label-Free Protein Detection and Separation in Real Time Using Confined Surface Plasmon Resonance Imaging

We demonstrate a label-free protein detection and separation technology for real-time monitoring of proteins in micro/nanofluidic channels, confined surface plasmon resonance imaging (confined-SPRi). This was achieved by fabricating ultrathin fluidic channels (500 nm high, 500 microm wide) directly on top of a specialized SPRi sensor surface. In this way, SPRi is uniquely used to detect proteins deep into the fluidic channel while maintaining high lateral accuracy of separated products. The channel fluid and proteins were driven electrokinetically under an external electric field. For this to occur, the metallic SPR sensor (46 nm of Au on 2 nm of Cr) was segmented into an array of squares (each 200 microm x 200 microm in size and spaced 8 microm apart) and coated with 30 nm of CYTOP polymer. In this work, we track label-free protein separation in real time through a simple cross-junction fluidic device with an 8-mm separation channel length under 30 V/cm electric field strength.

Analysis of recombinant protein expression using localized surface plasmon resonance (LSPR)

The localized surface plasmon resonance (LSPR)-based optical biosensor using nano-structures of noble metals has been considered as a useful tool for label-free detection of DNA hybridization and protein–protein interactions. We fabricated LSPR-based optical biosensors using gold nano-islands (nominal thickness; 75 Å) on glass substrates that were easily made using the conventional fabrication methods. The formation of gold nano-islands on glass substrates was realized by heat treatment of thin gold film deposited with a low deposition rate (∼0.05 Å/s). The morphologies of sensor surfaces composed of gold nano-islands were observed using an atomic force microscope (AFM) with a non-contact mode. To investigate the sensing capacity of the gold nano-island sensor for the binding of proteins by affinity interactions, the streptavidin and biotin interaction was used as a model system. In addition, detection of recombinant glutathione- S-transferase (GST)-tagged human interleukin-6 (hIL6) expressed in Escherichia coli was carried out by LSPR. It is expected that the LSPR sensors composed of gold nano-islands can be an alternative to traditional methods such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for fast analysis of protein expression.

Peptide Aptamers in Label-Free Protein Detection: 1. Characterization of the Immobilized Scaffold

Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be KD = 1.47 +/- 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.

Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis

Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.

Electrochemical strategies for the label-free detection of amino acids, peptides and proteins

Electrochemical methods for the detection of amino acids, peptides, and proteins in a variety of media are reviewed. Label-free strategies in which the detection is based on the inherent electrochemical properties of the analyte are discussed. Various processes such as direct or mediated (in solution or immobilised) redox processes and interfacial ion transfers have been employed for the electrochemical detection and determination of the target analytes. The various methods covered encompass voltammetry at uncoated and modified electrodes and at immiscible liquid-liquid interfaces, potentiometry at polymer membrane electrodes and electrochemical impedance spectroscopy. The determination of the target analytes in complex biological matrices is discussed. The various approaches highlighted here illustrate the rich capabilities of electrochemical methods as simple, low-cost, sensitive tools for the determination of these important biological analytes at trace and ultra-trace levels.

Label-Free Detection of Single Native Proteins: Ultimate Sensitivity and Convenience

Label-Free Detection of Single Native Proteins: Ultimate Sensitivity and Convenience

Multiple Label-Free Detection of Antigen−Antibody Reaction Using Localized Surface Plasmon Resonance-Based Core−Shell Structured Nanoparticle Layer Nanochip

In this research, a localized surface plasmon resonance (LSPR)-based bioanalysis method for developing multiarray optical nanochip suitable for screening bimolecular interactions is described. LSPR-based label-free monitoring enables to solve the problems of conventional methods that require large sample volumes and time-consuming labeling procedures. We developed a multiarray LSPR-based nanochip for the label-free detection of proteins. The multiarray format was constructed by a core-shell-structured nanoparticle layer, which provided 300 nanospots on the sensing surface. Antibodies were immobilized onto the nanospots using their interaction with Protein A. The concentrations of antigens were determined from the peak absorption intensity of the LSPR spectra. We demonstrated the capability of the array measurement using immunoglobulins (IgA, IgD, IgG, IgM), C-reactive protein, and fibrinogen. The detection limit of our label-free method was 100 pg/mL. Our nanochip is readily transferable to monitor the interactions of other biomolecules, such as whole cells or receptors, with a massively parallel detection capability in a highly miniaturized package. We anticipate that the direct label-free optical immunoassay of proteins reported here will revolutionize clinical diagnosis and accelerate the development of hand-held and user-friendly point-of-care devices.

Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices

Single cell analytics is a key method in the framework of proteom research allowing analyses, which are not subjected to ensemble-averaging, cell-cycle or heterogeneous cell-population effects. Our previous studies on single cell analysis in poly(dimethylsiloxane) microfluidic devices with native label-free laser induced fluorescence detection [W. Hellmich, C. Pelargus, K. Leffhalm, A. Ros, D. Anselmetti, Electrophoresis 26 (2005) 3689] were extended in order to improve separation efficiency and detection sensitivity. Here, we particularly focus on the influence of poly(oxyethylene) based coatings on the separation performance. In addition, the influence on background fluorescence is studied by the variation of the incident laser power as well as the adaptation of the confocal volume to the microfluidic channel dimensions. Last but not least, the use of carbon black particles further enhanced the detection limit to 25 nM, thereby reaching the relevant concentration ranges necessary for the label-free detection of low abundant proteins in single cells. On the basis of these results, we demonstrate the first electropherogram from an individual S podoptera frugiperda (Sf9) cell with native label-free UV-LIF detection in a microfluidic chip.

Label-Free Electrochemical Detection of Protein Based on a Ferrocene-Bearing Cationic Polythiophene and Aptamer

Two label-free electrochemical methods for the detection of human alpha-thrombin using a water-soluble, ferrocene-functionalized polythiophene transducer and a single-stranded oligonucleotide aptamer probe are described. The first approach is a direct method in which the recorded current decreases upon addition of the targeted protein. The second one requires more steps and the additional utilization of PNA probes and nuclease enzyme. This indirect method leads to an increase of the electrical signal as a function of the concentration of human alpha-thrombin with a detection limit of 75 fmol.

Label-free detection of proteins using SERS-based immuno-nanosensors

This paper describes the development and optimization of a novel class of SERS-based immuno-nanosensors for the label-free detection of specific proteins in complex environments (e.g., cell culture matrices and intracellular environments). These SERS-based nanosphere sensors are fabricated by depositing multiple layers of silver on silica nanospheres, followed by binding of the antibody of interest to the silver surface via a short rigid crosslinker. In these studies, several different crosslinkers were characterized and evaluated for optimal nanosensor activity. The crosslinkers evaluated contained either thiol or isothiocyanate functionalities, which bind to the silver surface on one end, while the other end of the crosslinker contained either a carboxylic or primary amine group, which reacts readily with the antibodies. These SERS-based nanosensors were also optimized for underlying silica sphere diameters, silica sphere coating conditions during silver deposition, number of silver layers applied, and silver surface coverage with crosslinkers. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens (e.g., human insulin or interleukin II) in complex environments.