Strategy For Protein Detection Research Articles

Proximity Ligation Assay to Detect the Proximity Between Host Proteins and Viral Proteins of HIV-1.

The study of host-pathogen interaction often requires interrogating the protein-protein interactions and examining post-translational modifications of the proteins. Traditional protein detection strategies are limited in their sensitivity, specificity, and multiplexing capabilities. The Proximity Ligation Assay (PLA), a versatile and powerful molecular technique, can overcome these limitations. PLA blends the specificity of antibodies, two antibodies detecting two different epitopes on the same or two different proteins, with the amplification efficiency of a polymerase to allow highly specific and sensitive detection of low-abundant proteins, protein-protein interactions, or protein modifications. In this protocol, we describe the application of PLA to detect the proximity between HIV-1 Tat with one of its cellular partners, p65, in an infected host cell. The protocol could be applied to any other context with slight modifications. Of note, PLA can only confirm the physical proximity between two epitopes or proteins; however, the proximity need not necessarily allude to the functional interaction between the two proteins.

Novel quantitative immunohistochemical analysis for evaluating PD-L1 expression with phosphor-integrated dots for predicting the efficacy of patients with cancer treated with immune checkpoint inhibitors.

Programmed cell death ligand 1 (PD-L1) expression in tumor tissues is measured as a predictor of the therapeutic efficacy of immune checkpoint inhibitors (ICIs) in many cancer types. PD-L1 expression is evaluated by immunohistochemical staining using 3,3´-diaminobenzidine (DAB) chronogenesis (IHC-DAB); however, quantitative and reproducibility issues remain. We focused on a highly sensitive quantitative immunohistochemical method using phosphor-integrated dots (PIDs), which are fluorescent nanoparticles, and evaluated PD-L1 expression between the PID method and conventional DAB method. In total, 155 patients with metastatic or recurrent cancer treated with ICIs were enrolled from four university hospitals. Tumor tissue specimens collected before treatment were subjected to immunohistochemical staining with both the PID and conventional DAB methods to evaluate PD-L1 protein expression. PD-L1 expression assessed using the PID and DAB methods was positively correlated. We quantified PD-L1 expression using the PID method and calculated PD-L1 PID scores. The PID score was significantly higher in the responder group than in the non-responder group. Survival analysis demonstrated that PD-L1 expression evaluated using the IHC-DAB method was not associated with progression-free survival (PFS) or overall survival (OS). Yet, PFS and OS were strikingly prolonged in the high PD-L1 PID score group. Quantification of PD-L1 expression as a PID score was more effective in predicting the treatment efficacy and prognosis of patients with cancer treated with ICIs. The quantitative evaluation of PD-L1 expression using the PID method is a novel strategy for protein detection. It is highly significant that the PID method was able to identify a group of patients with a favorable prognosis who could not be identified by the conventional DAB method.

A Single-Component Luminescent Biosensor for the SARS-CoV-2 Spike Protein.

Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of Escherichia coli and Saccharomyces cerevisiae. This biosensor is deployed in multiple homogeneous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an unaugmented cell phone camera. Binding-activated tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.

Ultrasensitive electrochemical biosensor for protein detection based on target-triggering cascade enzyme-free signal amplification strategy

Herein, a cost-effective, simple and sensitive electrochemical sensing platform was established based on aptamer - target recognition and target-triggering signal amplification strategy for protein detection. Due to the high affinity between the aptamer and target, the assistant DNA1 (a1) could release from a1-aptamer duplex and trigger the following DNA circuits. The strand displacement and branch migration reaction brought assistant DNA3 (a3) released. Eventually, a large number of duplex structures of a3−Hairpin DNA3 were formed on the surface of electrode. Consequently, the capture DNA on the surface of platinum nanoparticles could hybridize with the unfolded DNA fragment of Hairpin DNA3 on the sensor surface, resulting in the electrochemical signal readout of H2O2 reduction. Using thrombin as a model target, under the optimal conditions, this method exhibited a linear detection range from 0.5 pM to 300 nM with a detection limit of 0.17 pM. The proposed detection strategy was enzyme-free and exhibited good selectivity and sensitivity for a variety of protein targets detection by using corresponding DNA-based affinity probes, which makes it possible to apply the sensor for sensitivity detection of analytes in bioassays.

A Simple Visual Strategy for Protein Detection Based on Oxidase-Like Activity of Silver Nanoparticles

In this paper, we propose a simple and sensitive colorimetric sensing strategy for protein identification. The sensing array utilizes 3,3′,5,5′ tetramethylbenzidine (TMB) as the colorimetric sensing probe and three single-stranded (ss) DNA strands (15A, 15C, and 15T) as sensing elements. After adding protein, various interactions between DNA and protein result in differences in the amount of ssDNA remaining on the silver nanoparticles (AgNPs). It is well known that AgNPs have a strong catalytic ability toward the reaction between TMB and H2O2. Different amounts of ssDNA strands bound to AgNPs lead to diverse changes in the catalytic activity of AgNPs, which leads to different color and absorbance variations of the oxidized TMB solutions. These colorimetric responses as “fingerprints” were processed by linear discriminant analysis (LDA) to identify these target proteins. The sensing array can quantitatively identify lysozyme (Lys), Y-globin (Y-Glb), horseradish peroxidase (HRP), bovine liver peroxidase (Gat), trypsin (Try), human serum protein (HSA), pepsin (Pep), and ovalbumin (Alb) in the 1–400 nM, 1–1000 nM, 1–1000 nM, 50–700 nM, 1–400 nM, 1–400 nM, 1–700 nM, and 50–700 nM concentration ranges, respectively.

A novel aptasensor strategy for protein detection based on G-quadruplex and exonuclease III-aided recycling amplification

The detection of biomarkers is of great significance in the diagnosis of numerous diseases, especially cancer. Herein, we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads, DNA G-quadruplex, and exonuclease III (Exo III). In the presence of a target protein, a label-free single strand DNA (ssDNA) hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target. Subsequently, ssDNA initiates the Exo III-aided recycling to amplify the fluorescence signal, which was caused by N-methylmesoporphyrin IX (NMM) insertion into the G-quadruplex structure. This proposed strategy combines the excellent specificity between the aptamer and target, high sensitivity of the fluorescence signal by G-quadruplex and Exo III-aided recycling amplification. We selected (50–1200 nmol/L) MUC1, a common tumor biomarker, as the proof-of-concept target to test the specificity of our aptasensor. Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection (LODs) of 3.68 and 12.83 nmol/L in buffer solution and 10% serum system, respectively. The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.

C60@C3N4 nanocomposites as quencher for signal-off photoelectrochemical aptasensor with Au nanoparticle decorated perylene tetracarboxylic acid as platform

Herein, a novel signal-off photoelectrochemical (PEC) aptasensor was proposed for sensitive detection of thrombin on the basis of C60@C3N4 nanocomposites as quencher and Au nanoparticles (depAu) decorated perylene tetracarboxylic acid (PTCA) as sensing platform. Owing to the excellent membrane-forming of PTCA and superior conductivity of depAu, the PTCA between two depAu layers can simply and effectively produce an extremely high initial photocurrent to afford a precondition for sensitive biodetection. Thereafter, the assembly of C60@C3N4 nanocomposites on electrode via typical sandwich reaction enabled the generation of a significantly decreased photocurrent. Here, the C3N4 with high surface area not only provided massive binding sites for C60 immobilization, but also partly competed with PTCA in light absorption for producing a significantly smaller photocurrent in the presence of electron donor ascorbic acid (AA). Additionally, both the C3N4 and C60 have the poor conductivity, which could inhibit the electron transfer to achieve a further decreased photocurrent, effectively improving the sensitivity of proposed biosensor. As a result, the PEC biosensor in a “signal-off” mode showed an extremely low detection limit down to 1.5 fM, providing a sensitive and universal strategy for protein detection.

Plasmon‐Enhanced Fluorescent Sensor based on Aggregation‐Induced Emission for the Study of Protein Conformational Transformation

AbstractThe alteration in protein conformation not only affects the performance of its biological functions, but also leads to a variety of protein‐mediated diseases. Developing a sensitive strategy for protein detection and monitoring its conformation changes is of great significance for the diagnosis and treatment of protein conformation diseases. Herein, a plasmon‐enhanced fluorescence (PEF) sensor is developed, based on an aggregation‐induced emission (AIE) molecule to monitor conformational changes in protein, using prion protein as a model. Three anthracene derivatives with AIE characteristics are synthesized and a water‐miscible sulfonate salt of 9,10‐bis(2‐(6‐sulfonaphthalen‐2‐yl)vinyl)anthracene (BSNVA) is selected to construct the PEF–AIE sensor. The sensor is nearly non‐emissive when it is mixed with cellular prion protein while emits fluorescence when mixed with disease‐associated prion protein (PrPSc). The kinetic process of conformational conversion can be monitored through the fluorescence changes of the PEF–AIE sensor. By right of the amplified fluorescence signal, this PEF–AIE sensor can achieve a detection limit 10 pM lower than the traditional AIE probe and exhibit a good performance in human serum sample. Furthermore, molecular docking simulations suggest that BSNVA tends to dock in the β‐sheet structure of PrP by hydrophobic interaction between BSNVA and the exposed hydrophobic residues.

Colorimetric detection of protein via the terminal protection of small-molecule-linked DNA and unmodified gold nanoparticles.

A novel colorimetric strategy for protein detection was developed based on unmodified gold nanoparticles (AuNPs) and terminal protection from a target protein. A hairpin-structured DNA probe with a biotin modification at the 3′ terminus combined with streptavidin (SA) by the high affinity between biotin and SA and resisted degradation from exonucleases III (Exo III), which selectively digested double-stranded DNA from the 3′-terminus. The remaining hairpin DNA lost the capacity to protect AuNPs from salt-induced aggregation, resulting in a color change of AuNPs from red to blue. In the absence of SA, Exo III degraded the hairpin DNA into single-stranded DNA, which adsorbed on the surfaces of AuNPs and prevented aggregation, keeping AuNPs with a red color. The state of AuNP aggregation can be discriminated by colorimetry and also can be measured by UV-vis spectroscopy. Our proposed method provided great selectivity and high sensitivity with a detection limit of 0.24 pmol for SA detection. Moreover, the assay avoided modification, enzymatic amplification and special equipment, and also offered the advantages of being simple, cost-effective, and sensitive. This novel strategy holds great promise for the detection of other proteins by small molecule interactions with proteins.

Aptamer-based rolling circle amplification coupled with graphene oxide-based fluorescence resonance energy transfer for sensitive detection of cardiac troponin I

Combining the high affinity and specificity of an aptamer for a protein, a highly sensitive strategy for protein detection was developed based on rolling circle amplification (RCA) and graphene oxide based fluorescence resonance energy transfer (FRET).

Doping control analysis at the Rio 2016 Olympic and Paralympic Games.

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America. Copyright © 2017 John Wiley & Sons, Ltd.

A one-pot strategy for the detection of proteins based on sterically and allosterically tunable hybridization chain reaction

In this work, we report a facile one-pot strategy for protein detection based on sterically and allosterically tunable hybridization chain reaction (HCR). In our strategy, DNA hairpins H1 and H2 are dual-labeled with pyrene moieties through a six-carbon-atom spacer at each end; and a single-stranded DNA primer is designed to contain two small molecules near each end. In the absence of target protein, the primer can trigger HCR events between alternating H1 and H2 hairpins to form a nicked double-helix. As a result, the pyrene excimers are formed to emit at approximately 485nm. On the contrary, upon binding of the specific target protein onto the primer through the protein-small molecule interaction, the HCR will be inhibited due to the steric and allosteric effect. The changes of the fluorescent signals of pyrene excimers are in response to the concentration of target protein, so that the detection of protein can be realized. We have demonstrated the feasibility of this strategy by using streptavidin (SA) and folate receptor (FR) as model targets. Results show that both of them can be well detected with a detection limit of 1.07nM and 2.7nM, respectively. The developed method for protein assay is flexible, so we infer that the one-pot strategy holds great potential for the detection of other proteins.

Recognition-induced covalent capturing and labeling as a general strategy for protein detection

In this work we have developed a peptide-based method for protein detection, termed as “Recognition-induced Covalent Capturing and Labeling” (RCCL). In this method, upon binding of the peptide with the target protein, electrochemically controlled and metal catalyzed oxidative cross-linking can be induced between the peptide and the target protein. Specifically, the peptide and the target protein are cross-linked by the formation of dityrosine between tyrosine moieties of the two molecules. Meanwhile, the dityrosine formed in this manner also has fluorescent signal readout. Therefore, the proposed method needs only one probe for the target protein, and the initial non-covalent molecular recognition can be finalized by cross-linking between the peptide and the target, while the dityrosine formed between peptide and protein can also act as a signal reporter, thereby greatly simplifying the design. Moreover, the robust covalent capturing via RCCL also enables detection in complex biological and clinical samples. These results point to the prospect of using RCCL as a promising method in protein detection in the future.

Surface charge modulated aptasensor in a single glass conical nanopore.

In this work, we have proposed a label-free nanopore-based biosensing strategy for protein detection by performing the DNA-protein interaction inside a single glass conical nanopore. A lysozyme binding aptamer (LBA) was used to functionalize the walls of glass nanopore via siloxane chemistry and negatively charged recognition sites were thus generated. The covalent modification procedures and their recognition towards lysozyme of the single conical nanopore were characterized via ionic current passing through the nanopore membrane, which was measured by recording the current-voltage (I-V) curves in 1mM KCl electrolyte at pH=7.4. With the occurring of recognition event, the negatively charged wall was partially neutralized by the positively charged lysozyme molecules, leading to a sensitive change of the surface charge-dependent current-voltage (I-V) characteristics. Our results not only demonstrate excellent selectivity and sensitivity towards the target protein, but also suggest a route to extend this nanopore-based sensing strategy to the biosensing platform designs of a wide range of proteins based on a charge modulation.

A supramolecular dissociation strategy for protein sensing.

We report a simple, robust, and general strategy for protein detection based on supramolecular dissociation. The simplicity of the design is exemplified by the fact that the host assemblies can be widely varied and that these assemblies can be achieved from commercially available surfactants. An operating mechanism that is consistent with all the data has been proposed.

Gold nanoparticles enhanced electrochemiluminescence of graphite-like carbon nitride for the detection of Nuclear Matrix Protein 22

A novel label-free electrochemiluminescence (ECL) immunosensor was developed for the detection of Nuclear Matrix Protein 22 (NMP 22) based on the graphite-like carbon nitride (g-C3N4) and gold nanoparticles (AuNPs). Graphite-like carbon nitride was firstly combined with AuNPs, which promoted the electrons transfer and enhanced the ECL intensity of g-C3N4 largely. Then, anti-NMP 22 was immobilized onto the electrode through Au–NH2 without the usage of crosslinking agents. The connection between AuNPs and antibody influenced the conductive performance of AuNPs, leading to some decrease of ECL intensity. Then, bovine serum albumin (BSA) solution was dropped on the surface to block nonspecific binding sites. Finally, after the specific immunoreaction between NMP 22 and anti-NMP 22, the ECL intensity decreased further, because the protein on the electrode hindered the diffusion of luminescent reagents and electrons toward the electrode surface. Therefore, a linear relation between ECL intensity and the logarithm of NMP 22 concentration was obtained from 50.0pgmL−1 to 2.0ng/mL (R2=0.988) with the detection limit of 10.0pgmL−1. The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay strategy for protein detection, which might be promising for clinical application.

Target-Induced Conjunction of Split Aptamer Fragments and Assembly with a Water-Soluble Conjugated Polymer for Improved Protein Detection

Rapid and sensitive detection of proteins is crucial to biomedical research as well as clinical diagnosis. However, so far, most detection methods rely on antibody-based assays and are usually laborious and time-consuming, with poor sensitivity. Herein, we developed a simple and sensitive fluorescence-based strategy for protein detection by using split aptamer fragments and a water-soluble polycationic polymer (poly{[9,9-bis(6'-(N,N,N-diethylmethylammonium)hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis(3'-(N,N,N-diethylmethylammonium)-1'-oxapropyl)-1,4-phenylene] tetraiodide} (PFEP)). The thrombin-binding DNA aptamer was split into two fragments for target recognition. The PFEP with high fluorescence emission was used as energy donor to amplify the signal of dye-labeled DNA probe. In the absence of target, three DNA/PFEP complexes were formed via strong electrostatic interactions, resulting in efficient Föster resonance energy transfer (FRET) between two fluorophores. While the presence of target induces a conjunction of two split aptamer fragments to form G-quadruplex, and subsequent assemble with PFEP leading to the formation of G-quadruplex/thrombin/PFEP complex. The distance between the PFEP and dye increased due to protein's large size, leading to a remarkable decrease of the FRET signal. Compared with the intact aptamer, the use of shorter split aptamer fragments increases the possibility of forming G-quadruplex upon target. Thus, the rate of change of FRET signal before and after the addition of target improved significantly and a higher sensitivity (limit of detection (LOD) = 2 nM) was obtained. This strategy is superior in that it is rapid, has low cost and homogeneous detection, and does not need heating to avoid an unfavorable secondary structure of DNA probe. With further efforts, this method could be extended to a universal way for simple and sensitive detection of a variety of biomolecules.

Quantitative and sensitive protein detection strategies based on aptamers

Aptamers are functional oligonucleotides of single-stranded RNA or DNA that can selectively recognize their targets with high affinity. Hence, they have been widely developed for analytical, diagnostic, and therapeutic applications. In this review, we have summarized recent advances in the development of aptamer-based detection systems. Aptamers can be amplified exponentially by PCR, which is one of the advantages of aptamers over antibodies. Recently, we have developed immuno-aptamers that bind to mouse or rabbit IgG and constructed a novel sensitive detection system based on a conventional ELISA, called the immuno-aptamer PCR assay. In this article, the aptamer-based ready-to-use sensors and another PCR-based aptamer assays are also described; moreover, we have discussed highly sensitive aptamer-based detection systems.

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.

Label-free biosensor by protein grating coupler on planar optical waveguides

We report a diffraction-based, label-free biosensor on the surface of a planar optical waveguide. By selectively immobilizing proteins on an optical waveguide, the proteins can form the ridge/valley of a latent image optical grating. Binding events with patterned surface proteins dynamically increase grating height and cause an increase in light coupling out of the waveguide. We demonstrate the efficacy of this strategy for protein detection and present the design and fabrication of a device.