Investigation of Peptides for Molecular Recognition of C-Reactive Protein-Theoretical and Experimental Studies.

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 µg mL-1) with a detection limit (LOD = 3σ/S) of 0.34 µg mL-1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 µg mL-1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.

Controlling plasmon propagation and enhancement via reducing agent in wet chemistry synthesized silver nanowires.

Silver nanowires with varying diameters and submillimeter lengths were obtained by changing a reducing agent used during hydrothermal synthesis. The control over the nanowire diameter turns out to play a critical role in determining their plasmonic properties, including fluorescence enhancement and surface plasmon polariton propagation. Advanced fluorescence imaging of hybrid nanostructures assembled of silver nanowires and photoactive proteins indicates longer propagation lengths for nanowires featuring larger diameters. At the same time, with increasing diameter of the nanowires, we measure a substantial reduction of fluorescence enhancement. The results point at possible ways to control the influence of plasmon excitations in silver nanowires by tuning their morphology.

In Situ Interactions of Eu(TTA)3(H2O)2 with Latent Fingermark Components-A Time-Gated Visualization of Latent Fingermarks on Paper.

We introduce a new latent fingermark (LFM) development method, where compounds showing long lifetime luminescence are generated in situ by the reactions of Eu(TTA)3(H2O)2 with LFM components. Until now, time-gated imaging could not be used to develop LFM on porous surfaces due to the difficulties with selective binding of the developing agents to the fingermark ridges. The nature of the interactions of Eu(TTA)3(H2O)2 with the LFM material has been investigated for three model compounds commonly found in the LFM composition-oleic acid, l-serine, and squalene. The LFMs developed with the europium ß-diketonate complex have been successfully photographed using a time-gated imaging scheme. The presented new approach has been demonstrated to give similar or better results than developing agents commonly used for paper samples (ninhydrin and 1,2-indanedione). Moreover, contrary to the methods mentioned above, the new approach allows for the development of amino acid-poor LFM on paper.

Immunosensor Based on Long-Period Fiber Gratings for Detection of Viruses Causing Gastroenteritis.

Since the norovirus is the main cause of acute gastroenteritis all over the world, its fast detection is crucial in medical diagnostics. In this work, a rapid, sensitive, and selective optical fiber biosensor for the detection of norovirus virus-like particles (VLPs) is reported. The sensor is based on highly sensitive long-period fiber gratings (LPFGs) coated with antibodies against the main coat protein of the norovirus. Several modification methods were verified to obtain reliable immobilization of protein receptors on the LPFG surface. We were able to detect 1 ng/mL norovirus VLPs in a 40-min assay in a label-free manner. Thanks to the application of an optical fiber as the sensor, there is a possibility to increase the user's safety by separating the measurement point from the signal processing setup. Moreover, our sensor is small and light, and the proposed assay is straightforward. The designed LPFG-based biosensor could be applied in both fast norovirus detection and in vaccine testing.

Photochemical Printing of Plasmonically Active Silver Nanostructures.

In this paper, we demonstrate plasmonic substrates prepared on demand, using a straightforward technique, based on laser-induced photochemical reduction of silver compounds on a glass substrate. Importantly, the presented technique does not impose any restrictions regarding the shape and length of the metallic pattern. Plasmonic interactions have been probed using both Stokes and anti-Stokes types of emitters that served as photoluminescence probes. For both cases, we observed a pronounced increase of the photoluminescence intensity for emitters deposited on silver patterns. By studying the absorption and emission dynamics, we identified the mechanisms responsible for emission enhancement and the position of the plasmonic resonance.

Silver Island Film for Enhancing Light Harvesting in Natural Photosynthetic Proteins.

The effects of combining naturally evolved photosynthetic pigment-protein complexes with inorganic functional materials, especially plasmonically active metallic nanostructures, have been a widely studied topic in the last few decades. Besides other applications, it seems to be reasonable using such hybrid systems for designing future biomimetic solar cells. In this paper, we describe selected results that point out to various aspects of the interactions between photosynthetic complexes and plasmonic excitations in Silver Island Films (SIFs). In addition to simple light-harvesting complexes, like peridinin-chlorophyll-protein (PCP) or the Fenna-Matthews-Olson (FMO) complex, we also discuss the properties of large, photosynthetic reaction centers (RCs) and Photosystem I (PSI)-both prokaryotic PSI core complexes and eukaryotic PSI supercomplexes with attached antenna clusters (PSI-LHCI)-deposited on SIF substrates.

Spectrally selective fluorescence imaging of Chlorobaculum tepidum reaction centers conjugated to chelator-modified silver nanowires.

A polyhistidine tag (His-tag) present on Chlorobaculum tepidum reaction centers (RCs) was used to immobilize photosynthetic complexes on a silver nanowire (AgNW) modified with nickel-chelating nitrilo-triacetic acid (Ni-NTA). The optical properties of conjugated nanostructures were studied using wide-field and confocal fluorescence microscopy. Plasmonic enhancement of RCs conjugated to AgNWs was observed as their fluorescence intensity dependence on the excitation wavelength does not follow the excitation spectrum of RC complexes in solution. The strongest effect of plasmonic interactions on the emission intensity of RCs coincides with the absorption spectrum of AgNWs and is observed for excitation into the carotenoid absorption. From the absence of fluorescence decay shortening, we attribute the emission enhancement to increase of absorption in RC complexes.

Wide-Field Fluorescence Microscopy of Real-Time Bioconjugation Sensing.

We apply wide-field fluorescence microscopy to measure real-time attachment of photosynthetic proteins to plasmonically active silver nanowires. The observation of this effect is enabled, on the one hand, by sensitive detection of fluorescence and, on the other hand, by plasmonic enhancement of protein fluorescence. We examined two sample configurations with substrates being a bare glass coverslip and a coverslip functionalized with a monolayer of streptavidin. The different preparation of the substrate changes the observed behavior as far as attachment of the protein is concerned as well as its subsequent photobleaching. For the latter substrate the conjugation process is measurably slower. The described method can be universally applied in studying protein-nanostructure interactions for real-time fluorescence-based sensing.

Bacteriophage-Based Bioconjugates as a Flow Cytometry Probe for Fast Bacteria Detection.

Robust detection of bacteria can significantly reduce risks of nosocomial infections, which are a serious problem even in developed countries (4.1 million cases each year in Europe). Here we demonstrate utilization of novel multifunctional bioconjugates as specific probes for bacteria detection. Bifunctional magnetic-fluorescent microparticles are coupled with bacteriophages. The T4 bacteriophage, due to its natural affinity to bacterial receptors, namely, OmpC and LPS, enables specific and efficient detection of Escherichia coli bacteria. Prepared probes are cheap, accessible (even in nonbiological laboratories), as well as versatile and easily tunable for different bacteria species. The magnetic properties of the bioconjugates facilitate the separation of captured target bacteria from other components of complex samples and other bacteria strains. Fluorescence enables simple analysis. We chose flow cytometry as the detection method as it is fast and widely used for biotests. The capture efficiency of the prepared bioconjugates is close to 100% in the range of bacteria concentrations from tens to around 105 CFU/mL. The limit of detection is restricted by flow cytometry capabilities and in our case was around 104 CFU/mL.

Highly active 3-dimensional cobalt oxide nanostructures on the flexible carbon substrates for enzymeless glucose sensing.

The demand for electrochemical sensors with high sensitivity and reliability, fast response, and excellent selectivity has stimulated intensive research on developing highly active nanomaterials. In this work, freestanding 3D/Co3O4 thorn-like and wire-like (nanowires) nanostructures are directly grown on a flexible carbon fiber paper (CFP) substrate by a single-step hydrothermal process without using surfactants or templates. The 3D/Co3O4 thorn-like nanostructures show higher electrochemical activity than wire-like because of their high conductivity, large specific surface areas, and mesopores on their surface. The characterization of 3D/Co3O4 nanostructures is performed by using high resolution transmission electron microscopy (HRTEM), field emission scanning electron microscopy (FESEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction analysis (XRD), and electrochemical methods. The 3D/Co3O4 thorn-like nanostructures displayed non-enzymatic higher catalytic activity towards the electrochemical detection of glucose, compared to the 3D/Co3O4 wire-like morphology. The 3D/Co3O4 thorn-like nanostructures show a wide linear range response of glucose concentration ranging from 1 to 1000 µM with a detection limit of 0.046 µM (S/N = 3). The 3D/Co3O4 thorn-like nanostructure-modified CFP electrode selectively detects glucose in the presence of 100-fold excess of interfering compounds. The 3D/Co3O4 thorn-like nanostructure-modified CFP electrode is tested with human blood serum samples and validated with commercial glucose sensors. The newly developed sensor material shows potential for glucose monitoring in clinical and food samples.

Surface-enhanced Raman spectroscopy introduced into the International Standard Organization (ISO) regulations as an alternative method for detection and identification of pathogens in the food industry.

We show that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast, reliable, and easy method for detection and identification of food-borne bacteria, namely Salmonella spp., Listeria monocytogenes, and Cronobacter spp., in different types of food matrices (salmon, eggs, powdered infant formula milk, mixed herbs, respectively). The main aim of this work was to introduce the SERS technique into three ISO (6579:2002; 11290-1:1996/A1:2004; 22964:2006) standard procedures required for detection of these bacteria in food. Our study demonstrates that the SERS technique is effective in distinguishing very closely related bacteria within a genus grown on solid and liquid media. The advantages of the proposed ISO-SERS method for bacteria identification include simplicity and reduced time of analysis, from almost 144 h required by standard methods to 48 h for the SERS-based approach. Additionally, PCA allows one to perform statistical classification of studied bacteria and to identify the spectrum of an unknown sample. Calculated first and second principal components (PC-1, PC-2) account for 96, 98, and 90% of total variance in the spectra and enable one to identify the Salmonella spp., L. monocytogenes, and Cronobacter spp., respectively. Moreover, the presented study demonstrates the excellent possibility for simultaneous detection of analyzed food-borne bacteria in one sample test (98% of PC-1 and PC-2) with a goal of splitting the data set into three separated clusters corresponding to the three studied bacteria species. The studies described in this paper suggest that SERS represents an alternative to standard microorganism diagnostic procedures. Graphical Abstract New approach of the SERS strategy for detection and identification of food-borne bacteria, namely S. enterica, L. monocytogenes, and C. sakazakii in selected food matrices.

Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber.

With the advent of nanotechnology, carbon nanomaterials such as carbon nanofibers (CNF) have aroused substantial interest in various research fields, including energy storage and sensing. Further improvement of their properties might be achieved via the application of viral particles such as bacteriophages. In this report, we present a filamentous M13 bacteriophage with a point mutation in gene VII (pVII-mutant-M13) that selectively binds to the carbon nanofibers to form 3D structures. The phage-display technique was utilized for the selection of the pVII-mutant-M13 phage from the phage display peptide library. The properties of this phage make it a prospective candidate for a scaffold material for CNFs. The results for binding of CNF by mutant phage were compared with those for maternal bacteriophage (pVII-M13). The efficiency of binding between pVII-mutant-M13 and CNF is about 2 orders of magnitude higher compared to that of the pVII-M13. Binding affinity between pVII-mutant-M13 and CNF was also characterized using atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, which confirmed the specificity of the interaction of the phage pVII-mutant-M13 and the CNF; the binding occurs via the phage's ending, where the mutated pVII protein is located. No similar behavior has been observed for other carbon nanomaterials such as graphite, reduced graphene oxide, single-walled carbon nanotubes, and multiwalled carbon nanotubes. Infrared spectra confirmed differences in the interaction with CNF between the pVII-mutant-M13 and the pVII-M13. Basing on conducted research, we hypothesize that the interactions are noncovalent in nature, with π-π interactions playing the dominant role. Herein, the new bioconjugate material is introduced.

Tungsten carbide nanotubes supported platinum nanoparticles as a potential sensing platform for oxalic acid.

Supported tungsten carbide is an efficient and vital nanomaterial for the development of high-performance, sensitive, and selective electrochemical sensors. In this work, tungsten carbide with tube-like nanostructures (WC NTs) supported platinum nanoparticles (PtNPs) are synthesized and explored as an efficient catalyst toward electrochemical oxidation of oxalic acid for the first the time. The WC NTs supported PtNPs modified glassy carbon (GC) electrode is highly sensitive toward the electrochemical oxidation of oxalic acid. A large decrease in the oxidation overpotential (220 mV) and significant enhancement in the peak current compared to unmodified and Pt/C modified GC electrodes have been observed without using any redox mediator. Moreover, WC NTs supported PtNPs modified electrode possessed wide linear concentration ranges from 0 to 125 nM and a higher sensitivity toward the oxidation of oxalic acid (80 nA/nM) achieved by the amperometry method. The present modified electrode showed an experimentally determined lowest detection limit (LOD) of 12 nM (S/N = 3). Further, WC NTs supported PtNPs electrode can be demonstrated to have an excellent selectivity toward the detection of oxalic acid in the presence of a 200-fold excess of major important interferents. The practical application of WC NTs supported PtNPs has also been demonstrated in the detection of oxalic acid in tomato fruit sample, by differential pulse voltammetry under optimized conditions.

Antibody modified gold nanoparticles for fast and selective, colorimetric T7 bacteriophage detection.

Herein, we report a colorimetric immunosensor for T7 bacteriophage based on gold nanoparticles modified with covalently bonded anti-T7 antibodies. The new immunosensor allows for a fast, simple, and selective detection of T7 virus. T7 virions form immunological complexes with the antibody modified gold nanoparticles which causes them to aggregate. The aggregation can be observed with the naked eye as a color change from red to purple, as well as with a UV-vis spectrophotometer. The aggregate formation was confirmed with SEM imaging. Sensor selectivity against the M13 bacteriophage was demonstrated. The limit of detection (LOD) is 1.08 × 10(10) PFU/mL (18 pM) T7. The new method was compared with a traditional plaque test. In contrast to biological tests the colorimetric method allows for detection of all T7 phages, not only those biologically active. This includes phage ghosts and fragments of virions. T7 virus has been chosen as a model organism for adenoviruses. The described method has several advantages over the traditional ones. It is much faster than a standard plaque test. It is more robust since no bacteria-virus interactions are utilized in the detection process. Since antibodies are available for a large variety of pathogenic viruses, the described concept is very flexible and can be adapted to detect many different viruses, not only bacteriophages. Contrary to the classical immunoassays, it is a one-step detection method, and no additional amplification, e.g., enzymatic, is needed to read the result.

T7 bacteriophage induced changes of gold nanoparticle morphology: biopolymer capped gold nanoparticles as versatile probes for sensitive plasmonic biosensors.

The morphological changes of gold nanoparticles induced by T7 virus (bacteriophage) and the determination of its femtomolar concentration by a plasmonic method are presented. Carboxymethyl chitosan capped gold nanoparticles (CMC-AuNPs) are used as plasmonic probes and are synthesized by a simple one pot wet chemical method. HR-TEM images show that the spherical structure of the CMC-AuNPs is changed into chain-like nanostructures after the addition of T7 virus due to the strong coordination of CMC-AuNPs with T7. Since T7 capsids comprise a repeating motif of capsomers built from proteins that bind to the acid groups of chitosan, the conjugation of carboxymethyl chitosan-linked AuNPs with T7 virions enables colorimetric biosensing detection. The absorbance intensity (∼610 nm) increases in the concentration range of T7 from 2 × 10(-15) M to 2 × 10(-13) M and the detection limit is found to be 2 × 10(-15) M (2 fM). The present work demonstrates eco-friendly biopolymer stabilized AuNPs as potential nanomaterials for biosensing of viruses. Our method is very simple, low cost, selective and highly sensitive, and provides new insight into virus induced chain-like morphology of AuNPs.

Selective electrochemical detection of dopamine in a microfluidic channel on carbon nanoparticulate electrodes.

There is a continuous need for the construction of detection systems in microfluidic devices. In particular, electrochemical detection allows the separation of signals from the analyte and interfering substances in the potential domain. Here, a simple microfluidic device for the sensitive and selective determination of dopamine in the presence of interfering substances was constructed and tested. It employs a carbon nanoparticulate electrode allowing the separation of voltammetric signals of dopamine and common interfering substances (ascorbic acid and acetaminophen) both in quiescent conditions and in flow due to the electrocatalytic effect. These voltammograms were also successfully simulated. The limit of detection of dopamine detected by square wave voltammetry in 1 mM solutions of interfering substances in phosphate buffered saline is about 100 nM. In human serum a clear voltammetric signal could be seen for a 200 nM solution, sufficient to detect dopamine in the cerebral fluid. Flow injection analysis allows a decrease in the limit of detection down to 3.5 nM.

An impedimetric immunosensor based on diamond nanowires decorated with nickel nanoparticles.

Nanostructured boron-doped diamond has been investigated as a sensitive impedimetric electrode for the detection of immunoglobulin G (IgG). The immunosensor was constructed in a three-step process: (i) reactive ion etching of flat boron-doped diamond (BDD) interfaces to synthesize BDD nanowires (BDD NWs), (ii) electrochemical deposition of nickel nanoparticles (Ni NPs) on the BDD NWs, and (iii) immobilization of biotin-tagged anti-IgG onto the Ni NPs. Electrochemical impedance spectroscopy (EIS) was used to follow the binding of IgG at different concentrations without the use of any additional label. A detection limit of 0.3 ng mL(-1) (2 nM) with a dynamic range up to 300 ng mL(-1) (2 µM) was obtained with the interface. Moreover, the study demonstrated that this immunosensor exhibits good stability over time and allows regeneration by incubation in ethylenediaminetetraacetic acid (EDTA) aqueous solution.

NFC Smartphone-Based Electrochemical Microfluidic Device Integrated with Nanobody Recognition for C-Reactive Protein.

Point-of-care testing (POCT) devices play a crucial role as tools for disease diagnostics, and the integration of biorecognition elements with electronic components into these devices widens their functionalities and facilitates the development of complex quantitative assays. Unfortunately, biosensors that exploit large conventional IgG antibodies to capture relevant biomarkers are often limited in terms of sensitivity, selectivity, and storage stability, considerably restricting the use of POCT in real-world applications. Therefore, we used nanobodies as they are more suitable for fabricating electrochemical biosensors with near-field communication (NFC) technology. Moreover, a flow-through microfluidic device was implemented in this system for the detection of C-reactive protein (CRP), an inflammation biomarker, and a model analyte. The resulting sensors not only have high sensitivity and portability but also retain automated sequential flow properties through capillary transport without the need for an external pump. We also compared the accuracy of CRP quantitative analyses between commercial PalmSens4 and NFC-based potentiostats. Furthermore, the sensor reliability was evaluated using three biological samples (artificial serum, plasma, and whole blood without any pretreatment). This platform will streamline the development of POCT devices by combining operational simplicity, low cost, fast analysis, and portability.

Thiol-yne click reactions on alkynyl-dopamine-modified reduced graphene oxide.

The large-scale preparation of graphene is of great importance due to its potential applications in various fields. We report herein a simple method for the simultaneous exfoliation and reduction of graphene oxide (GO) to reduced GO (rGO) by using alkynyl-terminated dopamine as the reducing agent. The reaction was performed under mild conditions to yield rGO functionalized with the dopamine derivative. The chemical reactivity of the alkynyl function was demonstrated by post-functionalization with two thiolated precursors, namely 6-(ferrocenyl)hexanethiol and 1H,1H,2H,2H-perfluorodecanethiol. X-ray photoelectron spectroscopy, UV/Vis spectrophotometry, Raman spectroscopy, conductivity measurements, and cyclic voltammetry were used to characterize the resulting surfaces.

Aligned silver nanowires for plasmonically-enhanced fluorescence detection of photoactive proteins in wet and dry environment.

We developed a method of aligning silver nanowires in a microchannel and fixing them to glass substrates via appropriate functionalization. The attachment of nanowires to the substrate is robust with no variation of their angles over minutes. Specific conjugation with photoactive proteins is observed using wide-field fluorescence imaging in real-time for highly concentrated protein solution, both in a microchannel and in a chip geometry. In the latter case we can detect the presence of the proteins in the dropcasted solution down to single proteins. The results point towards possible implementation of aligned silver nanowires as geometrically defined plasmonic fluorescence sensing platforms.