Platform For Protein Detection Research Articles

Molecularly imprinted polymer film interfaced with Surface Acoustic Wave technology as a sensing platform for label-free protein detection

Molecularly imprinted polymer (MIP)-based synthetic receptors integrated with Surface Acoustic Wave (SAW) sensing platform were applied for the first time for label-free protein detection. The ultrathin polymeric films with surface imprints of immunoglobulin G (IgG-MIP) were fabricated onto the multiplexed SAW chips using an electrosynthesis approach. The films were characterized by analyzing the binding kinetics recorded by SAW system. It was revealed that the capability of IgG-MIP to specifically recognize the target protein was greatly influenced by the polymer film thickness that could be easily optimized by the amount of the electrical charge consumed during the electrodeposition. The thickness-optimized IgG-MIPs demonstrated imprinting factors towards IgG in the range of 2.8–4, while their recognition efficiencies were about 4 and 10 times lower toward the interfering proteins, IgA and HSA, respectively. Additionally, IgG-MIP preserved its capability to recognize selectively the template after up to four regeneration cycles. The presented approach of the facile integration of the protein-MIP sensing layer with SAW technology allowed observing the real-time binding events of the target protein at relevant sensitivity levels and can be potentially suitable for cost effective fabrication of a biosensor for analysis of biological samples in multiplexed manner.

Detection of folic acid protein in human serum using reduced graphene oxide electrodes modified by folic-acid.

The detection of disease markers is considered an important step for early diagnosis of cancer. We design in this work a novel electrochemical sensing platform for the sensitive and selective detection of folic acid protein (FP). The platform is fabricated by electrophoretic deposition (EPD) of reduced graphene oxide (rGO) onto a gold electrode and post-functionalization of rGO with folic acid. Upon FP binding, a significant current decrease can be measured using differential pulse voltammetry (DPV). Using this scheme, a detection limit of 1pM is achieved. Importantly, the method also allows the detection of FP in serum being thus an appealing approach for the sensitive detection of biomarkers in clinical samples.

Branched zinc oxide nanorods arrays modified paper electrode for electrochemical immunosensing by combining biocatalytic precipitation reaction and competitive immunoassay mode.

Branched zinc oxide nanorods (BZR) arrays, an array with high charge carries collection efficiency and specific surface area, are grown on the reduced graphene oxide-paper working electrode for the first time to construct a paper-based electrochemical (EC) immunosensor. Typically, the BZR are fabricated via a simple hydrothermal process, which can provide abundant sites for antibodies loading. By combining the large surface area of porous zinc oxide (PZS) and good biocompatibility of gold nanoparticles (AuNPs), PZS-AuNPs (PZS@Au) nanocomposites are designed to label horseradish peroxide (HRP) and antigens. After a competitive reaction between antigens and PZS@Au nanocomposites labeled antigens, the signal labels are introduced into the immunosensor, in which, HRP participate in biocatalytic precipitation process. The produced precipitate reduces the electrode surface area and hinders the electron transfer. With the increase of concentration of antigens, the signal labels introduced into the sensor decrease, thus, a signal-on immunoassay for α-fetoprotein detection is constructed. The proposed paper-based EC immunosensor combines enzymatic biocatalytic precipitation reaction and competitive immunoassay mode for the first time, and possesses a wide linear range from 0.2 pg mL(-1) to 500 ng mL(-1) with a detection limit of 0.08 pg mL(-1). In addition, the proposed method is simple, sensitive and specific and can be a promising platform for other protein detection.

A highly sensitive fluorescence turn-on platform with silver nanoparticles aptasening for human platelet-derived growth factor-BB

In this paper, we demonstrated a simple and highly sensitive fluorescence platform for protein detection. Silver nanoparticles (AgNPs) worked as carriers and quenchers for FAM labeled aptamers (FAM-apt). Biotin labeled aptamers (Bio-apt), FAM-apt functionalized AgNPs (Ag-FAM-apt), and a target protein, human platelet-derived growth factor-BB (PDGF-BB) could form a sandwich-type complex. Once the etching solvents were added, AgNPs were dissolved and the fluorescence resonance energy transfer (FRET) between AgNPs and FAM was broken. FAM-apt were no longer quenched and released into the solution in the 96-well microplates, so the fluorescence signal would turn from “off” state to “on” state. This method had possessed several advantages: Firstly, increased specificity which was contributed by the sandwich binding of aptamers; Secondly, quenching ability of AgNPs which was utilized to make signal turn-on; Thirdly, high throughout in which 96 samples could be detected simultaneously. The results showed a linear relationship between fluorescence intensity and PDGF-BB concentration (10ngmL−1–100ngmL−1), and the detection limit was 7ngmL−1. This simple and sensitive method would have a promising future for development and application.

A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins

An amplified colorimetric method has been developed for the detection of protein and cancer cells based on the assembly of nucleic acids and proteins for the first time. In this process, the assembly of nucleic acids was triggered by a biotinylated DNA strand after a sandwich immunoreaction. The biotinylated DNA strand and sandwich immunocomplex were connected by streptavidin. Then, the assembly of biotinylated bovine serum albumin (Biotin-BSA) and streptavidin-horseradish peroxidase (SA-HRP) occurred at a node of the assembled products of nucleic acids through the biotin-streptavidin reaction. Under the catalysis of horseradish peroxidase, 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized by H2O2 and the oxidized product was analyzed by its UV–vis absorbance signal and sensitive colorimetric detection. This colorimetric sensor could not only achieve the quantitative determination of protein by UV–vis absorbance but could also be applied for semiquantitative determination by digital visualization. Using alpha-fetoprotein (AFP) as the model target, this proposed colorimetric method showed a wide linear range from 5pg/mL to 1ng/mL with a detection limit of 1.95pg/mL by the instrument, and even 5pg/mL target protein could be distinguished simply by the naked eye. This approach was then expanded to detect cancer cells based on the recognition of folic acid receptors that were over-expressed on the cancer cells by folic acid-tethered DNA. More importantly, this strategy can be further used as a universal colorimetric method for the determination of viruses or other proteins by changing the corresponding antibodies.

Ultrasensitive biosensing platform based on the luminescence quenching ability of plasmonic palladium nanoparticles.

An ultrasensitive biosensing platform for DNA and protein detection is constructed based on the luminescence quenching ability of plasmonic palladium nanoparticles (PdNPs). By growing the particles into large sizes (ca. 30 nm), the plasmonic light absorption of PdNPs is broadened and extended to the visible range with extinction coefficients as high as 10(9) L mol(-1) cm(-1) , enabling complete quenching of fluorescent dyes that emit at diverse ranges and that are tagged to bioprobes. Meanwhile the nonspecific quenching of the dyes (not bound to probes) is negligible, leading to extremely low background signal. Utilizing the affinity of PdNPs towards bioprobes, such as single-stranded (ss) DNA and polypeptide molecules, which is mainly assigned to the coordination interaction, nucleic acid assays with a quantification limit of 3 pM target DNA and protein assay are achieved with a simple mix-and-detect strategy based on the luminescence quenching-and-recovery protocol. This is the first demonstration of biosensing employing plasmonic absorption of nanopalladium, which offers pronounced sensing performances and can be reasonably expected for wide applications.

Detection of thrombin with an aptamer-based macromolecule biosensor using bacterial ghost system.

A rapid on-site detection of exogenous proteins without the need for equipped laboratories or skilled personnel would benefit many areas. We built a rapid protein detection platform based on aptamer-induced inner-membrane scaffolds dimerization by virtue of bacterial ghost system. When the detection platform was coincubated with two kinds of aptamers targeting two different sites of thrombin, green fluorescence or β-lactamase activity were yielded with two different designs. The latter was detected by commercially available testing strips.

Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016.

Preparation of Conjugated Polymer Dots as a Fluorescence Turn-On Assay for Bovine Serum Albumin by Interaction with Graphene Oxide

Fluorescent conjugated polymers with various monomer compositions were synthesized via Suzuki coupling polymerization and spherically-shaped conjugated polymer dots (CPdot) with uniform size were prepared via conventional reprecipitation technique. Chemically modified graphene oxide (GO) was prepared to use as a component of sensor platform for protein detection. GO showed an excellent interaction with CPdot on its surface via hydrophobic interaction, which, in turn, induced the quenching of the fluorescence of CPdot. Upon exposure to bovine serum albumin (BSA), the quenched fluorescence was recovered, resulting from the release of CPdot from the complex with GO, as BSA adsorbed preferentially on the GO surface.

4-Fluoro-3-nitrophenyl grafted gold electrode based platform for label free electrochemical detection of interleukin-2 protein

A new platform based on 4-Fluoro-3-nitrophenyl (FNP) grafted gold disk electrode prepared via electrochemical reduction of 4-fluoro-3-nitrobenzene diazonium ion has been developed and utilized for biosensor fabrication. Anti-interleukin-2 (anti-IL2) antibody has been covalently immobilized onto FNP/Au surface and utilized for label free electrochemical impedance based detection of cytokine IL2. FNP acts as a bridge (cross-linker) between gold surface and anti-IL2, where fluoro group of FNP undergoes nucleophilic substitution by amino group of biomolecule and results in its covalent immobilization. The immobilization process and fabricated electrode have been characterized using contact angle (CA) measurements, cyclic voltammetry (CV) and electrochemical impedance (EIS) technique. CV studies show that FNP grafted surface provides conductive surface for anti-IL2 immobilization. The EIS response of studies as a function of IL2 concentrations exhibits a detection in linear range from 1pgml−1 to 10ngml−1 with minimum detectable concentration of 1pgml−1. The electrode has been found to be selective against other cytokine molecules.

Exonuclease III-Aided Autocatalytic DNA Biosensing Platform for Immobilization-Free and Ultrasensitive Electrochemical Detection of Nucleic Acid and Protein

Homogenous electrochemical biosensor has attracted substantial attention owing to its simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensor, but the relatively low detection sensitivity and the limited detection analytes prohibit its potential applications. To address these issues, herein, a simple, rapid, isothermal, and ultrasensitive homogeneous electrochemical DNA biosensing platform for target DNA and protein detection has been developed on the basis of an exonuclease III (Exo III)-aided autocatalytic target recycling strategy. A ferrocene-labeled hairpin probe (HP1) is ingeniously designed, which contains a protruding DNA fragment at 3'-termini as the recognition unit for target DNA. Also, the DNA fragment that could be used as secondary target analogue was introduced, but it was caged in the stem region of HP1. In the presence of target DNA, its recognition with the protruding fragment of HP1 triggered the Exo III cleavage process, accompanied with the target recycling and autonomous generation of secondary target analogues. This accordingly resulted into the autonomous accumulation of ferrocene-labeled mononucleotide, inducing a distinct increase in the electrochemical signal owing to its elevated diffusivity toward indium tin oxide (ITO) electrode surface. The autocatalytic biosensing system was further extended for protein detection by advising an aptamer hairpin switch with the use of thrombin as a model analyte. The current developed autocatalytic and homogeneous strategy provided an ultrasensitive electrochemical detection of DNA and thrombin down to the 0.1 and 5 pM level, respectively, with a high selectivity. It should be further used as a general autocatalytic and homogeneous strategy toward the detection of a wide spectrum of analytes and may be associated with more analytical techniques. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

The systematic study of circulating tumor cell isolation using lithographic microfilters

Circulating tumor cells (CTCs) disseminated into peripheral blood from a primary, or metastatic, tumor can be used for early detection, diagnosis and monitoring of solid malignancies. CTC isolation by size exclusion techniques have long interested researchers as a simple broad based approach, which is methodologically diverse for use in both genomic and protein detection platforms. Though a variety of these microfiltration systems are employed academically and commercially, the limited ability to easily alter microfilter designs has hindered the optimization for CTC capture. To overcome this problem, we studied a unique photo-definable material with a scalable and mass producible photolithographic fabrication method. We use this fabrication method to systematically study and optimize the parameters necessary for CTC isolation using a microfiltration approach, followed by a comparison to a "standard" filtration membrane. We demonstrate that properly designed microfilters can capture MCF-7 cancer cells at rate of 98 ± 2% if they consist of uniform patterned distributions, ≥160 000 pores, and 7 μm pore diameters.

Site-specific immobilization of proteins on non-conventional substrates via solvent-free initiated chemical vapour deposition (iCVD) process

Currently, the development of protein chips faces new requirements of improved environmental stability, disposability, and cost-effectiveness for their wide applicability in our daily life. Here, we demonstrate a new protein detection platform on low-cost plastic and paper substrates. To achieve this platform, a highly selective thiol–ene photochemical reaction between vinyl functionality on the surface and cysteine-linked protein was utilized to immobilize the relevant proteins on the disposable substrates. First, the surface of the flexible substrates was modified with a vinyl-containing polymer, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4), via a solvent-free initiated chemical vapour deposition (iCVD) process. Next, on the vinyl functionalized surface, cysteine-linked proteins were covalently immobilized by a UV-assisted thiol–ene click reaction. The solvent-free characteristic of iCVD enabled us to directly pattern the functionality on the substrate via shadow mask patterning. On the patterned substrate, selective immobilization of the proteins was possible. With four model proteins, including two fluorescent proteins, single-chain variable fragment (scFv), and non-immunoglobulin protein scaffold (human kringle domain), we successfully demonstrated the immobilization of proteins on various substrates including polyethylene (PE) film and chromatography paper.

Alginate and alginate-titanium dioxide nanocomposite as electrode materials for anti-myelin basic protein immunosensing

An electrochemical immunosensing platform for direct protein detection has been developed by immobilization of myelin basic protein (MBP), a multiple sclerosis (MS) auto-antigen, as a model biomarker on an alginate and alginate-titanium dioxide (TiO2) nanocomposite film modified platinum electrode. Modification steps were evaluated using scanning electron microscopy (SEM). Atomic force microscopy (AFM) was used to investigate the surface topographies of the films. Fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analysis (TGA) were used as proof for success of nanocomposite material preparation in addition to SEM. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed in determination of the electrode response capability and applicability. The formal potentials and the peak separations showed the electron transfer reaction to be quasireversible. Alginate-MBP and alginate-TiO2-MBP immunosensors revealed a detection limit of 0.25ngmL−1 and 0.18ngmL−1, respectively. The method employed is both sensitive and practical and the importance and uniqueness of this study lies in the fact that the immunosensor can determine Anti-MBP levels directly from blood samples, without the need for cerebrospinal fluid (CSF).

A Universal DNA-Based Protein Detection System

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

Coffee Ring Aptasensor for Rapid Protein Detection

We introduce a new biosensing platform for rapid protein detection that combines one of the simplest methods for biomolecular concentration, coffee ring formation, with a sensitive aptamer-based optical detection scheme. In this approach, aptamer beacons are utilized for signal transduction where a fluorescence signal is emitted in the presence of the target molecule. Signal amplification is achieved by concentrating aptamer-target complexes within liquid droplets, resulting in the formation of coffee ring "spots". Surfaces with various chemical coatings were utilized to investigate the correlation among surface hydrophobicity, concentration efficiency, and signal amplification. On the basis of our results, we found that the increase in the coffee ring diameter with larger droplet volumes is independent of surface hydrophobicity. Furthermore, we show that highly hydrophobic surfaces produce enhanced particle concentration via coffee ring formation, resulting in signal intensities 6-fold greater than those on hydrophilic surfaces. To validate this biosensing platform for the detection of clinical samples, we detected α-thrombin in human serum and 4-fold-diluted whole blood. Coffee ring spots from serum and blood produced detection signals up to 40 times larger than those from samples in liquid droplets. Additionally, this biosensor exhibits a lower limit of detection of 2 ng/mL (54 pM) in serum, and 4 ng/mL (105 pM) in blood. On the basis of its simplicity and high performance, this platform demonstrates immense potential as an inexpensive diagnostic tool for the detection of disease biomarkers, particularly for use in developing countries that lack the resources and facilities required for conventional biodetection practices.

Vertical nanowire arrays as a versatile platform for protein detection and analysis

Protein microarrays are valuable tools for protein assays. Reducing spot sizes from micro- to nano-scale facilitates miniaturization of platforms and consequently decreased material consumption, but faces inherent challenges in the reduction of fluorescent signals and compatibility with complex solutions. Here we show that vertical arrays of nanowires (NWs) can overcome several bottlenecks of using nanoarrays for extraction and analysis of proteins. The high aspect ratio of the NWs results in a large surface area available for protein immobilization and renders passivation of the surface between the NWs unnecessary. Fluorescence detection of proteins allows quantitative measurements and spatial resolution, enabling us to track individual NWs through several analytical steps, thereby allowing multiplexed detection of different proteins immobilized on different regions of the NW array. We use NW arrays for on-chip extraction, detection and functional analysis of proteins on a nano-scale platform that holds great promise for performing protein analysis on minute amounts of material. The demonstration made here on highly ordered arrays of indium arsenide (InAs) NWs is generic and can be extended to many high aspect ratio nanostructures.

Enzyme-free colorimetric bioassay based on gold nanoparticle-catalyzed dye decolorization

A novel, enzyme-free and aptamer-based colorimetric platform for protein detection has been developed, which takes advantage of aptamer-functionalized magnetic beads (MBs) for target capture, concentration and separation, and aptamer-conjugated gold nanoparticle (AuNP)-catalyzed color bleaching reaction of methyl orange (MO) to generate the colorimetric signals. It was demonstrated that the proposed colorimetric sensing strategy enables simple, cost-effective, sensitive and specific thrombin detection without the use of any enhancing solutions and enzymes. Herein, by naked eye observation, we can detect the human thrombin with a detection limit of approximately 320 pM, which can be further decreased to 30 pM with the help of a UV-vis instrument. In addition, this method also works for targets with two or more binding sites.

Ultrasensitive and fast fluorescent bioassay based on fluorescence enhancement of silver nanoparticles

An ultrasensitive, fast and specific fluorescent platform for protein detection is developed. In this protocol, silver nanoparticles were conjugated with paramagnetic particles (MPs-Ag) for target capture, concentration and separation; fluorescent dyes functionalized silver nanoparticles (Tag) for generating signals. The presented method is highly sensitive and specific with a detection limit of 2.2 pM for thrombin, and no significant interference was observed for other proteins such as human serum albumin (HSA), lysozyme and IgG. This novel approach combining the magnetic separation and concentration of MPs-Ag, aptamer recognition and fluorescence enhancement of Tag, can be successfully used to enhance the sensitivity of detecting ultra-low levels of target proteins or biomolecules.

Simultaneous Analysis of HER2 Gene and Protein on a Single Slide Facilitates HER2 Testing of Breast and Gastric Carcinomas

This study sought to evaluate a new combined gene and protein detection platform in the context of HER2 evaluation in breast and gastric carcinomas. HER2 immunohistochemistry (IHC) and dual color in situ hybridization (Dual ISH) were combined on a single slide. Results were compared with conventional HER2 IHC and fluorescence ISH. Results from the gene and protein assay were reliable and highly reproducible for both breast and gastric carcinomas. Concordance was found between conventional HER2 IHC and ISH testing and the gene and protein assay in the same laboratory (>95% for Dual ISH; lower for IHC because of different antibody clones), between IHC and Dual ISH performed on the same slide (>92%), and in the gene and protein assays between laboratories (>96%). This cost- and time-effective method provides fast and definitive results (IHC confirmed by means of Dual ISH) to aid in rapid treatment decisions. It can also be applied to other gene and protein combinations.