Multiplex protein profiling method for extracellular vesicle protein detection.

Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 µL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.

Functional Comparison of Bioactive Cellulose Materials Incorporating Engineered Binding Proteins.

Whatman No. 1 chromatography paper is widely used as a substrate for cellulose-based immunoassays. The immobilized proteins are used to capture target biomarkers for detection. However, alternative paper substrates may facilitate mass production of immunoassays as diagnostic tests. Here, we assessed the physical characteristics and protein immobilization capabilities of different commercial papers. Some substrates fulfilled our design criteria, including adequate flow rate and sufficient protein immobilization for efficient target capture. This study demonstrates that a variety of paper substrates can be bioactivated and used to capture target biomarkers, enabling development of affordable diagnostic tests from a range of starting materials.

A Surface Plasmon Resonance Biosensor Based on Directly Immobilized Hemoglobin and Myoglobin.

Immobilization of proteins on a surface plasmon resonance (SPR) transducer is a delicate procedure since loss of protein bioactivity can occur upon contact with the untreated metal surface. Solution to the problem is the use of an immobilization matrix having a complex structure. However, this is at the expense of biosensor selectivity and sensitivity. It has been shown that the matrix-assisted pulsed laser evaporation (MAPLE) method has been successfully applied for direct immobilization (without a built-in matrix) of proteins, preserving their bioactivity. So far, MAPLE deposition has not been performed on a gold surface as required for SPR biosensors. In this paper we study the impact of direct immobilization of heme proteins (hemoglobin (Hb) and myoglobin (Mb)) on their bioactivity. For the purpose, Hb and Mb were directly immobilized by MAPLE technique on a SPR transducer. The bioactivity of the ligands immobilized in the above-mentioned way was assessed by SPR registration of the molecular reactions of various Hb/Mb functional groups. By SPR we studied the reaction between the beta chain of the Hb molecule and glucose, which shows the structural integrity of the immobilized Hb. A supplementary study of films deposited by FTIR and AFM was provided. The experimental facts showed that direct immobilization of an intact molecule was achieved.

Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot.

The article presents a new method of immunoblotting for simple, rapid, and highly sensitive detection of proteins. Electrophoretic separation of sample is carried out under non-denaturing conditions in a thin conductive layer between cellulose membranes without polyacrylamide gel. The membrane surface is preliminarily modified with azidophenyl groups to photochemically immobilize proteins in situ. For visualization of protein bands, the membranes are treated with magnetic beads coated with specific antibodies, unbound particles are then removed with a magnet. The detection limit in the model system with biotinylated BSA and magnetic beads coated with streptavidin reaches 10 fg or about 105 molecules, while the total blotting time does not exceed 5 min. The method was applied for detection of IgA in a sample of human exhaled air. The method can be used for the analysis of various complex biological samples containing low amounts of the analyte.

Protein Assays on Organic Electronics: Rational Device and Material Designs for Organic Transistor-Based Sensors.

Artificial receptor-based protein assays have various attractive features such as a long-term stability, a low-cost production process, and the ease of tuning the target specificity. However, such protein sensors are still immature compared with conventional immunoassays. To enhance the application potential of synthetic sensing materials, organic field-effect transistors (OFETs) are some of the suitable platforms for protein assays because of their solution processability, durability, and compact integration. Importantly, OFETs enable the electrical readout of the protein recognition phenomena of artificial receptors on sensing electrodes. Thus, we believe that OFETs functionalized with artificial protein receptors will be a powerful tool for the on-site analyses of target proteins. In this Minireview, we summarize the recent progress of the OFET-based protein assays including the rational design strategies for devices and sensing materials.

Electrochemical selection of a DNA aptamer, and an impedimetric method for determination of the dedicator of cytokinesis 8 by self-assembly of a thiolated aptamer on a gold electrode.

The autosomal recessive-hyper immunoglobulin E syndromes (AR-HIES) are inherited inborn primary immunodeficiency disorders caused mainly by mutations in the dedicator of cytokinesis 8 (DOCK8) gene. A method is described for the selection of DNA aptamers against DOCK8 protein. The selection was performed by using a gold electrode as the solid matrix for immobilization of DOCK8. This enables voltammetric monitoring of the bound DNA after each selection cycle. After eight rounds of selection, high affinity DNA aptamers for DOCK8 were identified with dissociation constants (Kds) ranging from 3.3 to 66 nM. The aptamer which a Kd of 8.8 nM was used in an aptasensor. A gold electrode was modified by self-assembly of the thiolated aptamer, and the response to the DOCK8 protein was detected by monitoring the change in the electron transfer resistance using the ferro/ferricyanide system as a redox probe. The aptasensor works in the 100 pg.mL-1 to 100 ng.mL-1 DOCK8 concentration range, has a detection limit of 81 pg.mL-1 and good selectivity over other proteins in the serum. Graphical abstractSchematic representation of an electrochemical screening protocol for the selection of DNA aptamer against dedicator of cytokinesis 8 protein using electrode as solid support for target immobilization.

A surface plasmon resonance immunoassay for the rapid analysis of methamphetamine in forensic oral fluid.

BACKGROUND: Current chromatographic methods applied for the forensic analysis of methamphetamine are costly, time-consuming, and require complicated pretreatment procedures. Thus, the rapid detection of methamphetamine is a critical and unmet need. In this study, a surface plasmon resonance (SPR) system based on indirect inhibitive immunoassay was designed for the analysis of methamphetamine in forensic oral fluid samples. METHODS: For the inhibition immunoassay, the diluted oral fluid was mixed with methamphetamine antibody and then injected into the SPR sensor chip. The biosensor chip was constructed by covalently immobilizing of methamphetamine-bovine serum albumin conjugate onto a carboxymethyl dextran surface at an optimized pH. The concentration of antibody was also optimized. RESULTS: The SPR biosensor showed good sensitivity with a limit of detection of 0.44 ng/mL and was comparable or lower than the pre-existing methods. The method was finally tested using oral fluid samples from 20 suspected drug abusers in forensic cases, and it provided an acceptable recovery of 113.2%, indicating good anti-interference capability of the SPR sensor. CONCLUSION: The SPR biosensor was rapid, reproducible, and had a great potential approach for the forensic detection of methamphetamine.

Optimization of protein entrapment in affinity microcolumns using hydrazide-activated silica and glycogen as a capping agent.

Several approaches were compared for the entrapment of proteins within hydrazide-activated silica for use in affinity microcolumns and high performance affinity chromatography. Human serum albumin (HSA) and concanavalin A (Con A) were used as model proteins for this work. Items considered in this study included the role played by the solution volume, amount of added protein, and use of slurry vs. on-column entrapment on the levels of solute retention and extent of protein immobilization that could be obtained by means of entrapment. The levels of retention and protein immobilization were evaluated by injecting warfarin or 4-methylumbellipheryl α-D-mannopyranoside as solutes with known binding properties for HSA or Con A. Altering both the solution volume and amount of added protein led to an increase of up to 17-fold in the extent of protein immobilization for HSA in slurry-based entrapment; on-column entrapment provided an additional 3.6-fold increase in protein content vs. the optimized slurry method. Similar general trends were seen for Con A. The protein contents obtained by entrapment for HSA or Con A (i.e., up to ~87 and 46 mg/g silica, respectively) were comparable to or higher than levels reported for the covalent immobilization of these proteins onto silica. The retention of warfarin on the entrapped HSA was at least 1.7-fold higher than has been obtained under comparable support and mobile phase conditions when using covalent immobilization. These results indicated that entrapment can be an attractive alternative to covalent immobilization for proteins such as HSA and Con A, with this approach serving as a potential means for obtaining good solute binding and retention in work with affinity microcolumns or related microscale devices.

Indirect Microcontact Printing to Create Functional Patterns of Physisorbed Antibodies.

Microcontact printing (µCP) is a practical and versatile approach to create nanostructured patterns of biomolecular probes, but it involves conformational changes on the patterned bioreceptors that often lead to a loss on the biological activity of the resulting structures. Herein we introduce indirect µCP to create functional patterns of bioreceptors on solid substrates. This is a simple strategy that relies on physisorbing biomolecular probes of interest in the nanostructured gaps that result after patterning backfilling agents by standard µCP. This study presents the approach, assesses bovine serum albumin as backfilling agent for indirect µCP on different materials, reports the limitations of standard µCP on the functionality of patterned antibodies, and demonstrates the capabilities of indirect µCP to solve this issue. Bioreceptors were herein structured as diffractive gratings and used to measure biorecognition events in label-free conditions. Besides, as a preliminary approach towards sensing biomarkers, this work also reports the implementation of indirect µCP in an immunoassay to detect human immunoglobulin E.

Cytokeratins Biosensing Using Tilted Fiber Gratings.

Optical fiber gratings have widely proven their applicability in biosensing, especially when they are coupled with antibodies for specific antigen recognition. While this is customarily done with fibers coated by a thin metal film to benefit from plasmonic enhancement, in this paper, we propose to study their intrinsic properties, developing a label-free sensor for the detection of biomarkers in real-time without metal coatings for surface plasmon resonances. We focus on the inner properties of our modal sensor by immobilizing receptors directly on the silica surface, and reporting the sensitivity of bare tilted fiber Bragg gratings (TFBGs) used at near infrared wavelengths. We test different strategies to build our sensing surface against cytokeratins and show that the most reliable functionalization method is the electrostatic adsorption of antibodies on the fiber, allowing a limit of detection reaching 14 pM by following the guided cladding modes near the cut-off area. These results present the biodetection performance that TFBGs bring through their modal properties for different functionalizations and data processing strategies.

Stabilization of dry protein coatings with compatible solutes.

Exposure of protein modified surfaces to air may be necessary in several applications. For example, air contact may be inevitable during the implantation of biomedical devices, for analysis of protein modified surfaces, or for sensor applications. Protein coatings are very sensitive to dehydration and can undergo significant and irreversible alterations of their conformations upon exposure to air. With the use of two compatible solutes from extremophilic bacteria, ectoine and hydroxyectoine, the authors were able to preserve the activity of dried protein monolayers for up to >24 h. The protective effect can be explained by the preferred exclusion model; i.e., the solutes trap a thin water layer around the protein, retaining an aqueous environment and preventing unfolding of the protein. Horseradish peroxidase (HRP) immobilized on compact TiO2 was used as a model system. Structural differences between the compatible solute stabilized and unstabilized protein films, and between different solutes, were analyzed by static time-of-flight secondary ion mass spectrometry (ToF-SIMS). The biological activity difference observed in a colorimetric activity assay was correlated to changes in protein conformation by application of principal component analysis to the static ToF-SIMS data. Additionally, rehydration of the denatured HRP was observed in ToF-SIMS with an exposure of denatured protein coatings to ectoine and hydroxyectoine solutions.

Quantification of immobilized protein in pharmaceutical production by bio-assisted potentiometric multisensor system.

Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system. Surface-immobilized recombinant protein A from Staphylococcus aureus (SpA, expressed in Escherichia coli), which is commonly used as affinity ligand immobilized to stationary phase (сhromatography media) for monoclonal antibody purification was employed as the model object. Chromatography media samples containing different amounts of immobilized SpA were analyzed. Proteinase K from Tritirachium album was employed as a bio-transducer. We demonstrated that the suggested approach provides information about immobilized SpA concentration with 0.8mg/ml accuracy in the range 1-6.7mg/ml and within just 16min. Moreover, the proposed procedure requires no expensive materials and equipment and no bio-transducer immobilization. This method has potential of application for fast monitoring of other immobilized proteins in different tasks.

In situ surface protein conjugation of small molecules for SPR immunoassays.

Solution based protein conjugation of small molecules involves multiple steps of chemical syntheses, bio-conjugation and purifications which are labor intensive and time-consuming. Since many small molecules have limited water solubility, conjugation to a protein is also a relatively low efficiency process in aqueous solutions. In this study, a model in situ protein conjugation of small molecules was achieved onto SPR surfaces, using progesterone and ovalbumin as a model small-molecule-protein system. The in situ protein conjugation not only eliminated the requirement for wet chemistry, but also provided an easy way to optimize the assay performance and screen various molecular linkers.

Rapid signal enhancement method for nanoprobe-based biosensing.

The introduction of nanomaterials as detection reagents has enabled improved sensitivity and facilitated detection in a variety of bioanalytical assays. However, high nanoprobe densities are typically needed for colorimetric detection and to circumvent this limitation several enhancement protocols have been reported. Nevertheless, there is currently a lack of universal, enzyme-free and versatile methods that can be readily applied to existing as well as new biosensing strategies. The novel method presented here is shown to enhance the signal of gold nanoparticles enabling visual detection of a spot containing <10 nanoparticles. Detection of Protein G on paper arrays was improved by a 100-fold amplification factor in under five minutes of assay time, using IgG-labelled gold, silver, silica and iron oxide nanoprobes. Furthermore, we show that the presented protocol can be applied to a commercial allergen microarray assay, ImmunoCAP ISAC sIgE 112, attaining a good agreement with fluorescent detection when analysing human clinical samples.

Protein-Adsorbed Magnetic-Nanoparticle-Mediated Assay for Rapid Detection of Bacterial Antibiotic Resistance.

Antibiotic susceptibility tests have been used for years as a crucial diagnostic tool against antibiotic-resistant bacteria. However, due to a lack of biomarkers specific to resistant types, these approaches are often time-consuming, inaccurate, and inflexible in drug selections. Here, we present a novel susceptibility test method named protein-adsorbed nanoparticle-mediated matrix-assisted laser desorption-ionization mass spectrometry, or PANMS. Briefly, we adsorb five different proteins (ß-casein, α-lactalbumin, human serum albumin, fibrinogen, and avidin) onto the surface of Fe3O4. Upon interaction with bacteria surface, proteins were displaced from the nanoparticle surface, the amounts of which were quantified by matrix-assisted laser desorption ionization mass spectrometry. We find that the protein displacement profile was different distinctive among different bacteria strains and, in particular, between wild-type and drug-resistant strains. More excitingly, we observe bacteria resistant to drugs of the same mechanisms share similar displacement profiles on a linear discriminant analysis (LDA) map. This suggests the possibility of using PANMS to identify the type of mechanism behind antibiotic resistance, which was confirmed in a blind test. Given that PANMS is free of drug incubation and the whole procedure takes less than 50 min, it holds great potential as a high-throughput, low-cost, and accurate drug susceptibility test in the clinic.

Food protein-based phytosterol nanoparticles: fabrication and characterization.

The development of food-grade (nano)particles as a delivery system for poorly water soluble bioactives has recently attracted increasing attention. This work is an attempt to fabricate food protein-based nanoparticles as delivery systems for improving the water dispersion and bioaccessibility of phytosterols (PS) by an emulsification-evaporation method. The fabricated PS nanoparticles were characterized in terms of particle size, encapsulation efficiency (EE%) and loading amount (LA), and ξ-potential. Among all the test proteins, including soy protein isolate (SPI), whey protein concentrate (WPC) and sodium caseinate (SC), SC was confirmed to be the most suitable protein for the PS nano-formulation. Besides the type of protein, the particle size, EE% and LA of PS in the nanoparticles varied with the applied protein concentration in the aqueous phase and organic volume fraction. The freeze-dried PS nanoparticles with SC exhibited good water re-dispersion behavior and low crystallinity of PS. The LA of PS in the nanoparticles decreased upon storage, especially at high temperatures (e.g., >25 °C). The PS in the fabricated nanoparticles exhibited much better bioaccessibility than free PS. The findings would be of relevance for the fabrication of food-grade colloidal phytosterols, with great potential to be applied in functional food formulations.

Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample.

Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the 'footprint' of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

Development of quantitative radioactive methodologies on paper to determine important lateral-flow immunoassay parameters.

The lateral-flow immunoassay (LFA) is a well-established diagnostic technology that has recently seen significant advancements due in part to the rapidly expanding fields of paper diagnostics and paper-fluidics. As LFA-based diagnostics become more complex, it becomes increasingly important to quantitatively determine important parameters during the design and evaluation process. However, current experimental methods for determining these parameters have certain limitations when applied to LFA systems. In this work, we describe our novel methods of combining paper and radioactive measurements to determine nanoprobe molarity, the number of antibodies per nanoprobe, and the forward and reverse rate constants for nanoprobe binding to immobilized target on the LFA test line. Using a model LFA system that detects for the presence of the protein transferrin (Tf), we demonstrate the application of our methods, which involve quantitative experimentation and mathematical modeling. We also compare the results of our rate constant experiments with traditional experiments to demonstrate how our methods more appropriately capture the influence of the LFA environment on the binding interaction. Our novel experimental approaches can therefore more efficiently guide the research process for LFA design, leading to more rapid advancement of the field of paper-based diagnostics.

A Bioorthogonal Approach for the Preparation of a Titanium-Binding Insulin-like Growth-Factor-1 Derivative by Using Tyrosinase.

The generation of metal surfaces with biological properties, such as cell-growth-enhancing and differentiation-inducing abilities, could be potentially exciting for the development of functional materials for use in humans, including artificial dental implants and joint replacements. However, currently the immobilization of proteins on the surfaces of the metals are limited. In this study, we have used a mussel-inspired bioorthogonal approach to design a 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth-factor-1 using a combination of recombinant DNA technology and tyrosinase treatment for the surface modification of titanium. The modified growth factor prepared in this study exhibited strong binding affinity to titanium, and significantly enhanced the growth of NIH3T3 cells on the surface of titanium.

Competitive immunosensor based on gliadin immobilization on disposable carbon-nanogold screen-printed electrodes for rapid determination of celiotoxic prolamins.

The first competitive disposable amperometric immunosensor based on gliadin-functionalized carbon/nanogold screen-printed electrodes was developed for rapid determination of celiotoxic prolamins. To date, no competitive spectrophotometric or electrochemical immunoassays have yet been successfully applied to gluten detection in processed food samples, which require the use of complex prolamin extraction solutions containing additives with denaturing, reducing and disaggregating functions. Thus, in this work, great effort was put into the optimization and performance evaluation of the immunosensor in terms of suitability as a screening tool for analysis of cereal-based food samples. For this purpose, aqueous ethanol or complex extraction mixtures, as the patented Cocktail Solution®, were proved effective in the extraction of gliadin. Good sensitivity was achieved after optimization of the immunocompetitive assay, giving limit of detection and limit of quantitation of 8 and 22 ng/ml of gliadin, respectively, for ethanol extracts. The immunosensor was proved to be suitable also for samples extracted with Cocktail Solution® after a proper dilution. Analysis of real samples of different flours proved the suitability of the immunosensing device as a powerful tool for safety assessment of raw materials used for the formulation of dietary products for celiac disease patients. This immunosensor combines good analytical performance using a very simplified set-up protocol with suitability for rapid screening analysis performed using inexpensive and portable instrumentation. Graphical abstract Depiction of the development and working principle of the competitive immunosensor.