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.

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.

Determination of the Dissociation Constant for Polyvalent Receptors Using ELISA: A Case of M13 Phages Displaying Troponin T-Specific Peptides.

Phage-derived affinity peptides have become widespread thanks to their easy selection via phage display. Interactions between a target protein and its specific peptide are similar to those between antibodies and antigens. The strength of these non-covalent complexes may be described by the dissociation constant (Kd). In this paper, protein-specific peptides are exposed on the pIII protein present in the M13 bacteriophage virion with up to five copies. Therefore, one phage particle can bind from one to five ligands. Here, we discuss the dependences between phage-displayed peptides and their ligands in solution using a model system based on troponin T (TnT) binding phages. Moreover, a method of calculating Kd values from ELISA experiments was developed and is presented. The determined Kd values are in the picomolar range.

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.

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection.

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 µg/mL.

Fingermark detection using upconverting nanoparticles and comparison with cyanoacrylate fuming.

This paper reports the synthesis of high-quality upconverting nanoparticles (UCNPs) - sodium yttrium tetrafluoride doped with ytterbium and erbium (NaYF4:Yb,Er) with a silica shell and capped with phenyl functional groups. The main goal of this research was to design tailor-made UCNPs for fingermark detection, to test and validate a nanoparticle-based detection technique and to compare their performance against a benchmark method to assess potential implementation in routine practice by law enforcement agencies. The water-based UCNPs solution was applied to natural fingermarks on a number of substrates. This is the first ever systematic comparative study between UCNPs and a benchmark fingermark detection technique - cyanoacrylate fuming (CAF) followed by luminescent dye staining. Fingermark detection effectiveness was studied by treating 300 latent fingermark specimens on aluminium foil, polyethylene, polypropylene and glass slides. It was concluded that, on average, CAF performed better across the substrates tested. Nevertheless, UCNPs can be advantageous for fingermark detection on multicoloured, patterned or luminescent substrates due to their unique optical properties. There are, however, shortfalls associated with their synthesis and use that need to be addressed before they can be considered for operational purposes.

Adsorption of bacteriophages on polypropylene labware affects the reproducibility of phage research.

Hydrophobicity is one of the most critical factors governing the adsorption of molecules and objects, such as virions, on surfaces. Even moderate change of wetting angle of plastic surfaces causes a drastic decrease ranging from 2 to 5 logs of the viruses (e.g., T4 phage) in the suspension due to adsorption on polymer vials' walls. The effect varies immensely in seemingly identical containers but purchased from different vendors. Comparison of glass, polyethylene, polypropylene, and polystyrene containers revealed a threshold in the wetting angle of around 95°: virions adsorb on the surface of more hydrophobic containers, while in more hydrophilic vials, phage suspensions are stable. The polypropylene surface of the Eppendorf-type and Falcon-type can accommodate from around 108 PFU/ml to around 1010 PFU/ml from the suspension. The adsorption onto the container's wall might result in complete scavenging of virions from the bulk. We developed two methods to overcome this issue. The addition of surfactant Tween20 and/or plasma treatment provides a remedy by modulating surface wettability and inhibiting virions' adsorption. Plastic containers are essential consumables in the daily use of many bio-laboratories. Thus, this is important not only for phage-related research (e.g., the use of phage therapies as an alternative for antibiotics) but also for data comparison and reproducibility in the field of biochemistry and virology.

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.

Real-time fluorescence sensing of single photoactive proteins using silver nanowires.

We demonstrate that single functionalized silver nanowires form a geometric platform suitable for efficient real-time detection of single photoactive proteins. By collecting series of images using wide-field fluorescence microscopy, events of single protein attachment can be distinguished with the signal to noise ratio further improved by fluorescence enhancement due to plasmon excitations in the nanowires. The enhancement is evidenced by strong shortening of the fluorescence decay of single photoactive proteins conjugated to the silver nanowires.

CRP-binding bacteriophage as a new element of layer-by-layer assembly carbon nanofiber modified electrodes.

Recently, bacteriophage particles have started to be applied as a new biomaterial for developing sensing platforms. They can be used as both a recognition element or/and as building blocks, template/scaffold. In this paper, we studied a bacteriophage selected through phage-display technology. The chosen bacteriophage acted as a building block for creating a carbon nanofiber-based electrode and as a new receptor/binding element that recognizes C-reactive protein (CRP) - one of the markers of inflammatory processes in the human body. The binding efficiency of the selected phage towards CRP is two orders of magnitude higher than in the wild type. We demonstrate that the phage-based sensor is selective against other proteins. Finally, we show that layer-by-layer methods are suitable for deposition of negatively charged phages (wild or CRP-binding) with positively charged carbon nanofibers for electrode surface modification. A three-layered electrode was successfully used for molecular recognition of CRP, and the molecular interactions were studied using electrochemical, biological, and optical methods, including microscopic and spectroscopic analyses.

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.

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.

Simultaneous optical and electrochemical label-free biosensing with ITO-coated lossy-mode resonance sensor.

In this work we discuss a new label-free biosensing device based on indium tin oxide (ITO) overlaid section of a multimode optical fiber fused silica core. The sensor has been used to optical measurements also simultaneously interrogated electrochemically (EC). Due to optimized thickness and optical properties of ITO film, a lossy-mode resonance (LMR) could be observed in the optical domain, where electrical properties of the film allowed for application of the sensor as a working electrode in an EC setup. It has been confirmed that the LMR response depends on optical properties of the external medium, as well as potential applied to the electrode during cyclic voltammetry. After the ITO surface functionalization with amine groups and covalently attached biotin, the device has been applied for label-free biosensing of avidin in both the domains simultaneously. On the example of biotin-avidin detection system it was demonstrated that when avidin concentration increases a decrease in current and increase in LMR wavelength shift were recorded in EC and optical domain, respectively. Both optical and EC responses follow the protein interaction process, and thus can be used as cross-verification of the readouts. Moreover, an extended information has been achieved comparing to solely EC interrogation, i.e., the grafting process of biotin and avidin was directly monitored optically displaying individual steps of an incubation procedure.

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.

Zinc oxide quantum dots embedded in hydrophobic silica particles for latent fingermarks visualization based on time-gated luminescence measurements.

In this work we demonstrate a composite material based on silica particles. The particles have been doped with zinc oxide quantum dots which possess long living luminescence. The surface of the particles has been functionalized with phenyl groups using sol-gel process. The new material has been successfully applied for visualization of natural latent fingermarks on several surfaces, in particular, those showing their own luminescence and intensive background staining while using powder dusting, what is of the vital interest of forensic science. The time-gated imaging allows to overcome the background luminescence problem and surface functionalization increases the affinity of the particles to the fingermarks, what improves the selectivity of a new developing agent. The main novelty of the presented approach is the use of composite material that combines two main features-long lifetime luminescence and the ability to preferentially attach to the fingermark, due to hydrophobic interactions. Moreover,the utilization of deposition from the suspension instead of simple powder dusting allows for development of latent fingermarks on the surfaces that are difficult to work with powders (e.g. sticky side of the adhesive tape).

Plasmonics with Metallic Nanowires.

The purpose of this review is to introduce and present the concept of metallic nanowires as building-blocks of plasmonically active structures. In addition to concise description of both the basic physical properties associated with the electron oscillations as well as energy propagation in metallic nanostructures, and methods of fabrication of metallic nanowires, we will demonstrate several key ideas that involve interactions between plasmon excitations and electronic states in surrounding molecules or other emitters. Particular emphasis will be placed on the effects that involve not only plasmonic enhancement or quenching of fluorescence, but also propagation of energy on lengths that exceed the wavelength of light.

Ultrasensitive tantalum oxide nano-coated long-period gratings for detection of various biological targets.

In this work we discussed a label-free biosensing application of long-period gratings (LPGs) optimized in refractive index (RI) sensitivity by deposition of thin tantalum oxide (TaOx) overlays. Comparing to other thin film and materials already applied for maximizing the RI sensitivity, TaOx offers good chemical and mechanical stability during its surface functionalization and other biosensing experiments. It was shown theoretically and experimentally that when RI of the overlay is as high as 2 in IR spectral range, for obtaining LPGs ultrasensitive to RI, the overlay's thickness must be determined with subnanometer precision. In this experiment the TaOx overlays were deposited using Atomic Layer Deposition method that allowed for achieving overlays with exceptionally well-defined thickness and optical properties. The TaOx nano-coated LPGs show RI sensitivity determined for a single resonance exceeding 11,500 nm/RIU in RI range nD= 1.335-1.345 RIU, as expected for label-free biosensing applications. Capability for detection of various in size biological targets, i.e., proteins (avidin) and bacteria (Escherichia coli), with TaOx-coated LPGs was verified using biotin and bacteriophage adhesin as recognition elements, respectively. It has been shown that functionalization process, as well as type of recognition elements and target analyte must be taken into consideration when the LPG sensitivity is optimized. In this work optimized approach made possible detection of small in size biological targets such as proteins with sensitivity reaching 10.21 nm/log(ng/ml).

Optical Properties of Submillimeter Silver Nanowires Synthesized Using the Hydrothermal Method.

We report on the synthesis of long silver nanowires using the hydrothermal method, with H2O2 as the reducing agent. Our approach yields nanowires with an average diameter and length of about 100 nm and 160 µm, respectively, reaching the maximum length of 800 µm. Scanning electron microscopy (SEM) measurements revealed the presence of a thick, inhomogeneous poly(vinylpyrrolidone) (PVP) layer covering the nanowires, which with time becomes much more uniform, leading to well-defined extinction peaks in the ultraviolet-visible (UV-Vis) spectra. This change in morphology is evidenced also by the fluorescence enhancement behavior probed using protein complexes. Wide-field and confocal fluorescence microscopy measurements demonstrate strong, 10-fold enhancement of the protein emission intensity, accompanied by a reduction of the fluorescence decay time. In addition, for the aged, one-month-old nanowires, the uniformity of the intensity profile along them was substantially improved as compared with the as-synthesized ones. The results point towards the importance of the morphology of plasmonically active silver nanowires when considering their application in enhancing optical properties or achieving energy propagation over submillimeter distances.