Biotin-streptavidin Interaction Research Articles

Hybridization Chain Reaction-Based Electrochemical Biosensors by Integrating the Advantages of Homogeneous Reaction and Heterogeneous Detection.

The conventional hybridization chain reaction (HCR)-based electrochemical biosensors usually require the immobilization of probes on the electrode surface. This will limit the applications of biosensors due to the shortcomings of complex immobilization processes and low HCR efficiency. In this work, we proposed astrategy for the design of HCR-based electrochemical biosensors by integrating the advantages of homogeneous reaction and heterogeneous detection. Specifically, the targets triggered the autonomous cross-opening and hybridization oftwobiotin-labeled hairpin probes to form long-nicked dsDNA polymers. The HCR products with many biotin tags were then captured by a streptavidin-covered electrode, thus allowing for the attachment of streptavidin-conjugated signal reporters through streptavidin-biotin interactions. By employing DNA and microRNA-21 as the model targets and glucose oxidase as the signal reporter, the analytical performances of the HCR-based electrochemical biosensors were investigated. The detection limits of this method were found to be 0.6 fM and 1 fM for DNA and microRNA-21, respectively. The proposed strategy exhibited good reliability for target analysis in serum and cellular lysates. The strategy can be used to develop various HCR-based biosensors for a wide range of applications because sequence-specific oligonucleotides exhibit high binding affinity to a series of targets. In light of the high stability and commercial availability of streptavidin-modified materials, the strategy can be used for the design of different biosensors by changing the signal reporter and/or the sequence of hairpin probes.

An ultra-sensitive SPR immunosensor for quantitative determination of human cartilage oligomeric matrix protein biomarker

This paper reports the development of a novel surface plasmon resonance (SPR) immunosensor for ultra-sensitive quantitative determination of human articular cartilage oligomeric matrix protein (COMP), a major component of the extracellular matrix and an exploratory biomarker. Capture antibodies against human COMP (anti-COMP16F12) were covalently immobilized on an 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayer (SAM)-coated SPR sensor disk and a dual sandwich-type signal amplification strategy using biotinylated detection antibodies against COMP (anti-COMP17C10-biot) and streptavidin-conjugated quantum dots (SAv‒QDs) were used for the development of an immunosensor. The binding of high-mass SAv‒QDs via biotin–streptavidin interaction to the surface of the immunosensor resulted in a drastic increase in the sensitivity. The developed immunosensor was able to detect concentrations of COMP in a range from 2.80 to 680.54 fM with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.15 and 0.50 fM, respectively. The immunosensor exhibited good repeatability (relative standard deviation (RSD) 8.05%) and reproducibility (RSD 9.88%) as well as excellent operational stability (2.14 % decrease in SPR signal after 13 days). In addition, the analysis of secretomes of human knee articular cartilage explants from patients with osteoarthritis revealed that the immunosensor has good accuracy (analytical error less than 5 %). These results indicate that the immunosensor developed may be suitable for quantitative determination of COMP derived from articular cartilage and other synovial joint tissues in clinical studies.

Enhanced electrochemiluminescence immunoassay: 2. Enabling signal detection at an early stage of incubation for rapid point-of-care testing

Signal detection in a label-based immunoassay is performed normally when the antigen/antibody binding reaction reaches the equilibrium state during the incubation period of an assay process. Shortening the incubation period in an assay helps reduce the turnaround time and is particularly valuable for point-of-care testing, but the cost is the reduction of signal level and, possibly, measurement precision as well. This work demonstrates that the signal loss could be offset by the stronger emission of an electronically neutral ruthenium(II) complex label, Ru(2, 2′-bipyridine) (bathophenanthroline disulfonate)[4-(2, 2′-bipyridin-4-yl)butanoic acid], used in the electrochemiluminescence (ECL) immunoassay. Combined with the uniquely well-established flow-through washing process in the automated ECL analyzers and the precise control over liquid handling, the assays performed with a 5-minute incubation period showed the same signal level and measurement precision as those of conventional ECL assays. Additionally, the absence of biotin and streptavidin components in the reagent formulation avoids the biotin-streptavidin interaction during assay incubation and fundamentally eliminates the interference of biotin, especially when used in some high-dose therapies. The results obtained from the procalcitonin prototype kit and the supporting evidence from other preliminary reagents (for SARS-CoV-2 N protein and troponin T) are general. The nonequilibrium detection, along with the downsized instrument design, makes the enhanced ECL (EECL) technology a fast high-performance POCT platform that provides the same high-quality data as those generated from the widely deployed [Ru(bpy)3]2+based laboratorial ECL systems.The anticipated regulatory approval and follow-up clinical implementation will beasignificant stride in the decade-long pursuit of novel ECL labels.

Digital Magnetic Detection of Biomolecular Interactions with Single Nanoparticles.

Biomolecular interactions compose a fundamental element of all life forms and are the biological basis of many biomedical assays. However, current methods for detecting biomolecular interactions have limitations in sensitivity and specificity. Here, using nitrogen-vacancy centers in diamond as quantum sensors, we demonstrate digital magnetic detection of biomolecular interactions with single magnetic nanoparticles (MNPs). We first developed a single-particle magnetic imaging (SiPMI) method on 100 nm-sized MNPs with negligible magnetic background, high signal stability, and accurate quantification. The single-particle method was performed on biotin-streptavidin interactions and DNA-DNA interactions in which a single-base mismatch was specifically differentiated. Subsequently, SARS-CoV-2-related antibodies and nucleic acids were examined by a digital immunomagnetic assay derived from SiPMI. In addition, a magnetic separation process improved the detection sensitivity and dynamic range by more than 3 orders of magnitude and also the specificity. This digital magnetic platform is applicable to extensive biomolecular interaction studies and ultrasensitive biomedical assays.

Dumbbell-like upconversion nanoparticles synthesized by controlled epitaxial growth for light-heat-color tri-modal sensing of carcinoembryonic antigen

Accurate quantitative analysis of tumor markers in a wide linear range has important practical significance towards complex clinical samples in cancer identification and monitoring of tumor development stages, but remains challenging. Herein, three-layer dumbbell-like upconversion nanoparticles NaErF4:Tm@NaYF4@NaNdF4 (labeled as UCNPs) combined with G-quadruplex (G4) DNAzyme are reported for tri-modal sensing of carcinoembryonic antigen (CEA) in a wide range using upconversion luminescence (UCL), photothermal and catalysis signal readouts. Initially, dumbbell-like UCNPs were controlled synthesized by a three-dimensional epitaxial growth strategy through tuning the concentration of Nd precursors. After surface functionalization, G4zyme-UCNPs-cDNA/Apt-MB was subsequently fabricated by biotin-streptavidin interaction and DNA hybridization. Quantitative detection of CEA was achieved by competitive interaction and magnetic separation, and the intensities of tri-modal signals (light, heat and catalysis-based chrominance) of dissociative probes are linearly related to the concentration of CEA. The results showed that the tri-modal sensing method exhibited a wide linear range (0.005–2000 ng/mL) and low limit of detection (LOD) across three models: the luminescence model (0.005–50 ng/mL, LOD = 0.910 pg/mL), the catalysis model (10–1000 ng/mL, LOD = 0.387 ng/mL), and the temperature model (50–2000 ng/mL, LOD = 1.114 ng/mL). These findings suggest that the tri-modal sensing platform is suitable for use in the analysis of a wide range of complex and diverse clinical samples.

Tools to compare antibody gold nanoparticle conjugates for a small molecule immunoassay

Antibody gold nanoparticle conjugates as recognition elements are essential for the overall performance of lateral flow assays. When immobilizing antibodies on gold nanoparticles, the challenge is to prevent aggregation and to ensure that the antibodies are correctly oriented so that they remain functional and their paratopes remain accessible. There are many methods available, and it is difficult to decide which one to use. To help selecting the most appropriate conjugate production method, different synthetic routes of binding antibodies to gold nanoparticles are systematically investigated for the purpose of a quantitative lateral flow test for small molecules. The direct comparison of different conjugate syntheses shows how to select a suitable conjugate for a lateral flow assay. The syntheses examined are direct adsorption of antibody, direct adsorption of reduced antibody, covalent binding to polyethylene glycol linker, and binding via biotin-streptavidin interaction. The conjugates are characterized using UV-Vis spectroscopy and dynamic light scattering to determine their stability. Their performance on structured lateral flow test strips is examined using calibrations for different amitriptyline concentrations. It was shown that the best conjugate for quantification of amitriptyline was realized by direct adsorption of an UV-light irradiated antibody to gold nanoparticles. The methods employed can serve as a guide for selecting the most appropriate conjugate for an application and enhance the performance of lateral flow assays.Graphical abstract

A Lab-on-a-Chip Approach for the Detection of the Quarantine Potato Cyst Nematode Globodera pallida.

The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin−streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor’s electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor’s potential as a bioanalytical device for field applications and border phytosanitary inspections.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2fgmL-1 and the linear dynamic detection range was 0.015 ~ 158.5pgmL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A.

Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

Low cost and massively parallel force spectroscopy with fluid loading on a chip

Current approaches for single molecule force spectroscopy are typically constrained by low throughput and high instrumentation cost. Herein, a low-cost, high throughput technique is demonstrated using microfluidics for multiplexed mechanical manipulation of up to ~4000 individual molecules via molecular fluid loading on-a-chip (FLO-Chip). The FLO-Chip consists of serially connected microchannels with varying width, allowing for simultaneous testing at multiple loading rates. Molecular force measurements are demonstrated by dissociating Biotin-Streptavidin and Digoxigenin-AntiDigoxigenin interactions along with unzipping of double stranded DNA of varying sequence under different dynamic loading rates and solution conditions. Rupture force results under varying loading rates and solution conditions are in good agreement with prior studies, verifying a versatile approach for single molecule biophysics and molecular mechanobiology. FLO-Chip enables straightforward, rapid, low-cost, and portable mechanical testing of single molecules that can be implemented on a wide range of microscopes to broaden access and may enable new applications of molecular force spectroscopy.

Small-Molecule Modulated Affinity-Tunable Semisynthetic Protein Switches.

Engineered protein switches have been widely applied in cell-based protein sensors and point-of-care diagnosis for the rapid and simple analysis of a wide variety of proteins, metabolites, nucleic acids, and enzymatic activities. Currently, these protein switches are based on two main types of switching mechanisms to transduce the target binding event to a quantitative signal, through a change in the optical properties of fluorescent molecules and the activation of enzymatic activities. In this paper, we introduce a new affinity-tunable protein switch strategy in which the binding of a small-molecule target with the protein activates the streptavidin-biotin interaction to generate a readout signal. In the absence of a target, the biotinylated protein switch forms a closed conformation where the biotin is positioned in close proximity to the protein, imposing a large steric hindrance to prevent the effective binding with streptavidin. In the presence of the target molecule, this steric hindrance is removed, thereby exposing the biotin for streptavidin binding to produce strong fluorescent signals. With this modular sensing concept, various sulfonamide, methotrexate, and trimethoprim drugs can be selectively detected on the cell surface of native and genetically engineered cells using different fluorescent dyes and detection techniques.

Multimodal Ochratoxin A-Aptasensor Using 3′-FAM-Enhanced Exonuclease I Tool and Magnetic Microbead Carrier

Thanks to its preparatory ease, close affinity, and low cost, the aptasensor can serve as a promising substitute for antibody-dependent biosensors. However, the available aptasensors are mostly subject to a single-mode readout and the interference of unbound aptamers in solution and non-target-induced transition events. Herein, we proposed a multimodal aptasensor for multimode detection of ochratoxin A (OTA) with cross-validation using the 3'-6-carboxyfluorescein (FAM)-enhanced exonuclease I (Exo I) tool and magnetic microbead carrier. Specifically, the 3'-FAM-labeled aptamer/biotinylated-cDNA hybrids were immobilized onto streptavidin-magnetic microbeads via streptavidin-biotin interaction. With the presence of OTA, an antiparallel G-quadruplex conformation was formed, protecting the 3'-FAM labels from Exo I digestion, and then anti-FAM-horseradish peroxidase (HRP) was bound via specific antigen-antibody affinity; for the aptamers without the protection of OTA, the distal ssDNA was hydrolyzed from 3' → 5', releasing 3'-FAM labels to the solution. Therefore, the OTA was detected by analyzing the "signal-off" fluorescence of the supernatant and two "signal-on" signals in electrochemistry and colorimetry through the detection of the coating magnetic microbeads in HRP's substrate. The results showed that the 3'-FAM labels increased the activity of Exo I, producing a low background due to a more thorough digestion of unbound aptamers. The proposed multimodal aptasensor successfully detected the OTA in actual samples. This work first provides a novel strategy for the development of aptasensors with Exo I and 3'-FAM labels, broadening the application of aptamer in the multimode detection of small molecules.

MOPSS: Toward high-fidelity oligonucleotides for clinical applications.

MOPSS: Toward high-fidelity oligonucleotides for clinical applications.

Hydroxymethylation-Specific Ligation-Mediated Single Quantum Dot-Based Nanosensors for Sensitive Detection of 5-Hydroxymethylcytosine in Cancer Cells.

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 β-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

Development of nano Bio LC columns for the search of acetylcholinesterase molecular targets.

A novel acetylcholinesterase Nano liquid chromatography capillary column (75 μm i.d. × 50mm length) was developed for the fast screening of acetylcholinesterase inhibitors and the evaluation of their molecular recognition mechanism. Biotinylated acetylcholinesterase was immobilized on a streptavidin Nano liquid chromatography capillary column. Because of the very strong streptavidin-biotin interaction, the acetylcholinesterase immobilization step performed by frontal analysis is very fast (required less than 10min), and the amount of immobilized acetylcholinesterase was in the microgram range (1μg). The yellow anion obtained from the enzymatic reaction detected at 412nm was achieved within 60 s, and the immobilized acetylcholinesterase retained 96% of the initial activity beyond 90 days. This column was successfully applied for the discrimination of weak affinity ligands to acetylcholinesterase from nonbinders, which is the heart of fragment-based drug design. This column was used for the determination of the IC50 values of a series of inhibitor molecules. In addition, it was demonstrated that competitive experiments could be performed with our miniaturized system to confirm the existence and binding pocket of a ligand to acetylcholinesterase contained in a methanol plant extract. The results revealed that our acetylcholinesterase Nano liquid chromatography capillary column developed in this work represents a useful tool for the rapid screening of inhibitor candidates and evaluation of the action mechanism.

Highly sensitive electrochemical aptasensor for SARS-CoV-2 antigen detection based on aptamer-binding induced multiple hairpin assembly signal amplification

In this work, a brief electrochemical aptasensor was developed for highly sensitive detection of SARS-CoV-2 antigen utilizing an aptamer-binding induced multiple hairpin assembly strategy for signal amplification. In the presence of SARS-CoV-2, a pair of aptamers was brought in a close proximity according to the aptamer-protein antigen binding, which initiated strand displacement reaction thereby triggering a multiple hairpin assembly to obtain long linear DNA concatemers on the electrode surface. As the fabricated hairpin probes were labeled with biotin, massive streptavidin-alkaline phosphatases (ST-ALP) could be further introduced on the electrode interface via biotin-streptavidin interaction thus generating strong electrochemical signal in electrolyte solution containing 1-naphthol phosphate. Benefiting from the non-enzymatic multiple hairpin assembly signal amplification strategy, the designed aptasensor for SARS-CoV-2 spike protein detection exhibited the wide linear range from 50 fg·mL−1 to 50 ng·mL−1 and low detection limit of 9.79 fg·mL−1. Meaningfully, this proposed electrochemical assay provided a potential application for the point of care analysis of viral diseases under ambient temperature.

Profiling the ubiquitinated proteome in human cells

Ubiquitination is a post‐translational modification that determines the half‐life of many cellular proteins by conjugating a chain of the small protein ubiquitin to a target protein. The ubiquitin chain is then recognized by the 26S proteasome, and subsequently, the ubiquitinated protein is degraded. This ubiquitin‐proteosome system (UPS) contains various components that function cooperatively to determine the ubiquitination status of cellular proteins that play key roles in biological events. It has become clear that mis‐regulation of protein ubiquitination can lead to diverse human diseases, and multiple UPS components have been identified as promising drug targets. To facilitate studies that define roles of the UPS components in protein ubiquitination, a method that probes the ubiquitination status of the whole proteome in human cells is necessary. Tandem ubiquitin binding entities (TUBEs) is a synthetic protein that binds poly‐ubiquitin chains with a dissociation constant in the nanomolar range. Therefore, TUBEs can recognize the poly‐ubiquitinated protein and enrich them from human cell lysate. Combining the TUBE‐dependent affinity precipitation, stable isotope labeling by amino acids in cell culture (SILAC), and mass spectrometry, we are establishing a method for quantitatively accessing the ubiquitinated proteome in human cells. To preserve the poly‐ubiquitinated protein, HEK293 cells were treated with bortezomib, a proteosome inhibitor, to prevent the ubiquitinated proteins from degradation. To perform the affinity precipitation, the recombinant TUBE was conjugated to biotin and immobilized on resin via the Streptavidin‐biotin interaction. The lysate of the bortezomib‐treated cells was incubated with the immobilized BiotinTUBE to enrich poly‐ubiquitinated proteins, which were then digested by the trypsin protease. Following the trypsin digestion, ubiquitinated proteins generated peptides with the Lys‐ɛ‐Gly‐Gly (K‐ɛ‐GG) remnant, which was further immunoprecipitated by antibodies specifically recognizing the K‐ɛ‐GG remnant and identified by mass spectrometry. To probe proteins with different ubiquitination status under different conditions, SILAC was used to grow one cell sample in normal (light) media and the other cell sample in (heavy) media containing stable isotope labeled amino acids. By comparing the abundance of the light versus heavy peptides from the same ubiquitinated protein, changes in the ubiquitination status of each detected protein can be determined. We anticipate that this method will help profile changes in the ubiquitinated proteome when the UPS components are mutated or interfered by drugs.

High-avidity binding drives nucleation of amyloidogenic transthyretin monomer.

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursors. Transthyretin (TTR) naturally folds into a stable tetramer, whereas conditions and mutations that foster aberrant monomer formations facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the early assembly of oligomers by WT TTR compared with its V30M and V122I variants. We monitored time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20–40 TTR subunits via engineered biotin-streptavidin (SA) interactions. As expected, these multimer seeds rapidly nucleated TTR monomers into larger complexes, while having less effect on dimers and tetramers. In vivo, SA-induced multimers formed TTR-like deposits in the heart and the kidney following i.v. injection in mice. While all 3 variants prominently deposited glomerulus in the kidney, only V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized in the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR engaging in high-avidity interactions with tissue amyloids. Our multimeric induction approach provides a model for studying the initiation of TTR deposition in the heart.

Biorecognition Layer Based On Biotin-Containing [1]Benzothieno[3,2-b][1]benzothiophene Derivative for Biosensing by Electrolyte-Gated Organic Field-Effect Transistors.

Requirements of speed and simplicity in testing stimulate the development of modern biosensors. Electrolyte-gated organic field-effect transistors (EGOFETs) are a promising platform for ultrasensitive, fast, and reliable detection of biological molecules for low-cost, point-of-care bioelectronic sensing. Biosensitivity of the EGOFET devices can be achieved by modification with receptors of one of the electronic active interfaces of the transistor gate or organic semiconductor surface. Functionalization of the latter gives the advantage in the creation of a planar architecture and compact devices for lab-on-chip design. Herein, we propose a universal, fast, and simple technique based on doctor blading and Langmuir-Schaefer methods for functionalization of the semiconducting surface of C8-BTBT-C8, allowing the fabrication of a large-scale biorecognition layer based on the novel functional derivative of BTBT-containing biotin fragments as a foundation for further biomodification. The fabricated devices are very efficient and operate stably in phosphate-buffered saline solution with high reproducibility of electrical properties in the EGOFET regime. The development of biorecognition properties of the proposed biolayer is based on the streptavidin-biotin interactions between the consecutive layers and can be used for a wide variety of receptors. As a proof-of-concept, we demonstrate the specific response of the BTBT-based biorecognition layer in EGOFETs to influenza A virus (H7N1 strain). The elaborated approach to biorecognition layer formation is appropriate but not limited to aptamer-based receptor molecules and can be further applied for fabricating several biosensors for various analytes on one substrate and paves the way for "electronic tongue" creation.

Click reaction-assisted construction of antifouling immunosensors for electrochemical detection of cancer biomarkers in human serum

Herein, an electrochemical immunosensor capable of detecting targets in human serum with ultralow fouling and high sensitivity was successfully constructed through a click reaction. One designed biotinylated peptide was associated with the other peptide functionalized with biotin and azide groups at its two terminals, to form a branched antifouling peptide through the streptavidin-biotin affinity interaction, and it was attached to the electrode to construct an antifouling interface. Then, the exposed azide group on the branched peptide was used to link the antibody modified with 5′-dibenzocyclooctyne (DBCO), and the immunosensor was thus constructed with the aid of the click reaction between the azide group and the DBCO. The developed antifouling immunosensor demonstrated a wide linear response range for carcinoembryonic antigen (CEA) and a low limit of detection of 40 fg mL−1, which is much superior to the sensing performance of the immunosensor prepared through the normal EDC/NHS method. The immunosensor was capable of assaying CEA in undiluted serum samples with satisfying accuracy when compared with a clinically approved method. This work offered an effective strategy to develop various biosensors through the conjugation of antifouling peptides with different antibodies.