RESUMEN
Pre-treatment of diamond surface in low-temperature plasma for oxygenation and in acids for carboxylation was hypothesized to promote the branching density of the hyperbranched glycidol polymer. This was expected to increase the homogeneity of the branching level and suppress interactions with proteins. As a result, composite nanodiamonds with reduced hydrodynamic diameters that are maintained in physiological environments were anticipated. Surfaces of 140-nm-sized nanodiamonds were functionalized with oxygen and carboxyl groups for grafting of hyperbranched dendritic polyglycerol via anionic ring-opening polymerization of glycidol. The modification was verified with Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. Dynamic light scattering investigated colloidal stability in pH-diverse (2-12) solutions, concentrated phosphate-buffered saline, and cell culture media. Thermogravimetric analysis of nanodiamonds-protein incubations examined non-specific binding. Fluorescence emission was tested across pH conditions. Molecular dynamics simulations modeled interparticle interactions in ionic solutions. The hyperbranched polyglycerol grafting increased colloidal stability of nanodiamonds across diverse pH, high ionic media like 10 × concentrated phosphate-buffered saline, and physiological media like serum and cell culture medium. The hyperbranched polyglycerol suppressed non-specific protein adsorption while maintaining intensive fluorescence of nanodiamonds regardless of pH. Molecular modelling indicated reduced interparticle interactions in ionic solutions correlating with the improved colloidal stability.
RESUMEN
Maintaining both high sensitivity and large figure of merit (FoM) is crucial in regard to the performance of optical devices, particularly when they are intended for use as biosensors with extremely low limit of detection (LoD). Here, a stack of nano-assembled layers in the form of 1D photonic crystal, deposited on D-shaped single-mode fibers, is created to meet these criteria, resulting in the generation of Bloch surface wave resonances. The increase in the contrast between high and low refractive index (RI) nano-layers, along with the reduction of losses, enables not only to achieve high sensitivity, but also a narrowed resonance bandwidth, leading to a significant enhancement in the FoM. Preliminary testing for bulk RI sensitivity is carried out, and the effect of an additional nano-layer that mimics a biological layer where binding interactions occur is also considered. Finally, the biosensing capability is assessed by detecting immunoglobulin G in serum at very low concentrations, and a record LoD of 70 aM is achieved. An optical fiber biosensor that is capable of attaining extraordinarily low LoD in the attomolar range is not only a remarkable technical outcome, but can also be envisaged as a powerful tool for early diagnosis of diseases.
RESUMEN
This paper focuses on the development of a novel electrode based on boron-doped diamond nanosheet full-volume-enriched screen-printed carbon electrodes (BDDPE) for use as an impedimetric biosensor. Impedimetric biosensors offer high sensitivity and selectivity for virus detection, but their use as point-of-care devices is limited by the complexity of nanomaterials' architecture and the receptor immobilisation procedures. The study presents a two-step modification process involving the electroreduction of diazonium salt at the BDDPE and the immobilisation of antibodies using zero-length cross-linkers for a selective impedimetric biosensor of Haemophilus influenzae (Hi). The incorporation of diamond nanosheets into BDDPE leads to enhanced charge transfer and electrochemical behaviour, demonstrating greatly improved electrochemically active surface area compared with unmodified screen-printed electrodes (by 44% and 10% on average for [Ru(NH3)6]Cl2 and K3[Fe(CN)6], respectively). The presented sensing system shows high specificity towards protein D in Hi bacteria, as confirmed by negative controls against potential interference from other pathogens, with an estimated tolerance limit for interference under 12%. The Hi limit of detection by electrochemical impedance spectroscopy was 1 CFU/mL (measured at - 0.13 V vs BDDPE pseudo-reference), which was achieved in under 10 min, including 5 min sample incubation in the presence of the analyte.
Asunto(s)
Carbono , Diamante , Boro , Anticuerpos , Electrodos , Haemophilus influenzaeRESUMEN
Spectroelectrochemical (SEC) measurements play a crucial role in analytical chemistry, utilizing transparent or semitransparent electrodes for optical analysis of electrochemical (EC) processes. The EC readout provides information about the electrode's state, while changes in the transmitted optical spectrum help identify the products of EC reactions. To enhance SEC measurements, this study proposes the addition of optical monitoring of the electrode. The setup involves using a polymer-clad silica multimode fiber core coated with indium tin oxide (ITO), which serves as both the electrode and an optical fiber sensor. The ITO film is specifically tailored to exhibit the lossy-mode resonance (LMR) phenomenon, allowing for simultaneous optical monitoring alongside EC readouts. The LMR response depends on the properties of the ITO and the surrounding medium's optical properties. As a result, the setup offers three types of interrogation readouts: EC measurements, optical spectrum analysis corresponding to the volume of the analyte (similar to standard SEC), and LMR spectrum analysis reflecting the state of the sensor/electrode surface. In each interrogation path, cyclic voltammetry (CV) experiments were conducted individually with two oxidation-reduction reaction (redox) probes: potassium ferricyanide and methylene blue. Subsequently, simultaneous measurements were performed during chronoamperometry (CA) with the sensor, and the cross-correlation between the readouts was examined. Overall, this study presents a novel and enhanced SEC measurement approach that incorporates optical monitoring of the electrode. It provides a comprehensive understanding of EC processes and enables greater insights into the characteristics of the analyte.
RESUMEN
In this work, we study the electrodes with a periodic matrix of gold particles pattered by titanium dimples and modified by 3-mercaptopropionic acid (MPA) followed by CD147 receptor grafting for specific impedimetric detection of SARS-CoV-2 viral spike proteins. The synergistic DFT and MM/MD modeling revealed that MPA adsorption geometries on the Au-Ti surface have preferential and stronger binding patterns through the carboxyl bond inducing an enhanced surface coverage with CD147. Control of bonding at the surface is essential for oriented receptor assembling and boosted sensitivity. The complex Au-Ti electrode texture along with optimized MPA concentration is a crucial parameter, enabling to reach the detection limit of ca. 3 ng mL-1. Scanning electrochemical microscopy imaging and quantum molecular modeling were performed to understand the electrochemical performance and specific assembly of MPA displaying a free stereo orientation and not disturbed by direct interactions with closely adjacent receptors. This significantly limits nonspecific interceptor reactions, strongly decreasing the detection of receptor-binding domain proteins by saturation of binding groups. This method has been demonstrated for detecting the SARS virus but can generally be applied to a variety of protein-antigen systems. Moreover, the raster of the pattern can be tuned using various anodizing processes at the titania surfaces.
RESUMEN
Accurate and fast detection of viruses is crucial for controlling outbreaks of many diseases; therefore, to date, numerous sensing systems for their detection have been studied. On top of the performance of these sensing systems, the availability of biorecognition elements specific to especially the new etiological agents is an additional fundamental challenge. Therefore, besides high sensitivity and selectivity, such advantages as the size of the sensor and possibly low volume of analyzed samples are also important, especially at the stage of evaluating the receptor-target interactions in the case of new etiological agents when typically, only tiny amounts of the receptor are available for testing. This work introduces a real-time, highly miniaturized sensing solution based on microcavity in-line Mach-Zehnder interferometer (µIMZI) induced in optical fiber for SARS-CoV-2 virus-like particles detection. The assay is designed to detect conserved regions of the SARS-CoV-2 viral particles in a sample with a volume as small as hundreds of picoliters, reaching the detection limit at the single ng per mL level.
Asunto(s)
Técnicas Biosensibles , COVID-19 , Humanos , Fibras Ópticas , SARS-CoV-2 , Interferometría , COVID-19/diagnósticoRESUMEN
Monitoring cell adhesion and growth are crucial for various applications involving drug screening, cytotoxicity, and cytocompatibility studies. However, acquiring accurate information about the growing state and responsiveness to a treatment of a cell system in a real-time and label-free manner is still a challenge. This work presents the first research on direct, real-time, and label-free adherent cell culture monitoring using a microcavity in-line Mach-Zehnder interferometer (µIMZI) fabricated in an optical fiber. The sensing solution based on µIMZI offers a great advantage over many other monitoring concepts tracking the changes taking place on the microcavity's bottom surface and within its volume, thus offering a greater penetration depth. In this study, we verified performance of the approach using a non-cancer bone marrow stromal cell line HS-5. The results demonstrate that the changes of the acquired signal are closely related to the different states of cells' adhesion, proliferation, morphology, and variation of mass. Thus, this label-free, real-time µIMZI-based monitoring technique gives a great promise to the analysis or monitoring of relevant new treatments in future scientific, as well as clinical applications.
Asunto(s)
Técnicas Biosensibles , Fibras Ópticas , Técnicas de Cultivo de Célula , Interferometría/métodosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cells through the binding of spike protein to the host cell surface-expressing angiotensin-converting enzyme 2 (ACE2) or by endocytosis mediated by extracellular matrix metalloproteinase inducer (CD147). We present extended statistical studies of the multisine dynamic electrochemical impedance spectroscopy (DEIS) revealing interactions between Spike RBD and cellular receptors ACE2 and CD147, and a reference anti-RBD antibody (IgG2B) based on a functionalised boron-doped diamond (BDD) electrode. The DEIS was supported by a multivariate data analysis of a SARS-CoV-2 Spike RBD assay and cross-correlated with the atomic-level information revealed by molecular dynamics simulations. This approach allowed us to study and detect subtle changes in the electrical properties responsible for the susceptibility of cellular receptors to SARS-CoV-2, revealing their interactions. Changes in electrical homogeneity in the function of the RBD concentration led to the conclusion that the ACE2 receptor delivers the most homogeneous surface, delivered by the high electrostatic potential of the relevant docking regions. For higher RBD concentrations, the differences in electrical homogeneity between electrodes with different receptors vanish. Collectively, this study reveals interdependent virus entry pathways involving separately ACE2, CD147, and spike protein, as assessed using a biosensing platform for the rapid screening of cellular interactions (i.e. testing various mutations of SARS-CoV-2 or screening of therapeutic drugs).
RESUMEN
In this work, we demonstrate a novel method for multi-domain analysis of properties of analytes in volumes as small as picoliters, combining electrochemistry and optical measurements. A microcavity in-line Mach-Zehnder interferometer (µIMZI) obtained in a standard single-mode optical fiber using femtosecond laser micromachining was able to accommodate a microelectrode and optically monitor electrochemical processes inside the fiber. The interferometer shows exceptional sensitivity to changes in the optical properties of analytes in the microcavity. We show that the optical readout follows the electrochemical reactions. Here, the redox probe (ferrocenedimethanol) undergoing reactions of oxidation and reduction changes the optical properties of the analyte (refractive index and absorbance) that are monitored using the µIMZI. Measurements have been supported by numerical analysis of both optical and electrochemical phenomena. On top of the capability of the approach to perform analysis on a microscale, the difference between oxidized and reduced forms in the near-infrared region can be measured using the µIMZI, which is hardly possible using other optical techniques. The proposed multi-domain concept is a promising approach for highly reliable and ultrasensitive chemo- and biosensing.
Asunto(s)
Interferometría , Fibras Ópticas , Electroquímica , Microtecnología , RefractometríaRESUMEN
Rolling circle amplification (RCA) of DNA can be considered as a great alternative to the gold standard polymerase chain reaction (PCR), especially during this pandemic period, where rapid, sensitive, and reliable test results for hundreds of thousands of samples are required daily. This work presents the first research to date on direct, real-time and label-free isothermal DNA amplification monitoring using a microcavity in-line Mach-Zehnder interferometer (µIMZI) fabricated in an optical fiber. The solution based on µIMZI offers a great advantage over many other sensing concepts - making possible optical analysis in just picoliter sample volumes. The selectivity of the biosensor is determined by DNA primers immobilized on the microcavity's surface that act as selective biorecognition elements and trigger initiation of the DNA amplification process. In this study, we verified the sensing concept using circular DNA designed to target the H5N1 influenza virus. The developed biosensor exhibits an ultrahigh refractive index sensitivity reaching 14 000 nm per refractive index unit and a linear detection range between 9.4 aM and 94 pM of the target DNA sequence. Within a 30 min period, the amplification of as little as 9.4 aM DNA can be effectively detected, with a calculated limit of detection of as low as 0.2 aM DNA, suggesting that this methodology holds great promise in practical disease diagnosis applications in the future.
Asunto(s)
Técnicas Biosensibles , Subtipo H5N1 del Virus de la Influenza A , ADN/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Técnicas de Amplificación de Ácido Nucleico , Fibras ÓpticasRESUMEN
Selected optical fiber sensors offer extraordinary sensitivity to changes in external refractive (RI), which make them promising for label-free biosensing. In this work the most sensitive ones, namely long-period gratings working at (DTP-LPG) and micro-cavity in-line Mach-Zehnder interferometers (µIMZI) are discussed for application in bacteria sensing. We describe their working principles and RI sensitivity when operating in water environments, which is as high as 20,000 nm/RIU (Refractive index unit) for DTP-LPGs and 27,000 nm/RIU for µIMZIs. Special attention is paid to the methods to enhance the sensitivity by etching and nano-coatings. While the DTP-LPGs offer a greater interaction length and sensitivity to changes taking place at their surface, the µIMZIs are best suited for investigations of sub-nanoliter and picoliter volumes. The capabilities of both the platforms for bacteria sensing are presented and compared for strains of Escherichia coli, lipopolysaccharide E. coli, outer membrane proteins of E. coli, and Staphylococcus aureus. While DTP-LPGs have been more explored for bacteria detection in 102-106 Colony Forming Unit (CFU)/mL for S. aureus and 103-109 CFU/mL for E. coli, the µIMZIs reached 102-108 CFU/mL for E. coli and have a potential for becoming picoliter bacteria sensors.
Asunto(s)
Técnicas Biosensibles , Escherichia coli/aislamiento & purificación , Fibras Ópticas , Staphylococcus aureus/aislamiento & purificación , RefractometríaRESUMEN
In this work, we present a direct electrochemical biofunctionalization of an indium-tin-oxide-coated lossy-mode resonance optical fiber sensor. The functionalization using a biotin derivative was performed by cyclic voltammetry in a 10 mM biotin hydrazide solution. All stages of the experiment were simultaneously verified with optical and electrochemical techniques. Performed measurements indicate the presence of a poly-biotin layer on the sensor's surface. Furthermore, dual-domain detection of 0.01 and 0.1 mg/mL of avidin confirms the sensor's viability for label-free detection.
RESUMEN
This work discusses sensing properties of a long-period grating (LPG) and microcavity in-line Mach-Zehnder interferometer (µIMZI) when both are induced in the same single-mode optical fiber. LPGs were either etched or nanocoated with aluminum oxide (Al2O3) to increase its refractive index (RI) sensitivity up to ≈2000 and 9000 nm/RIU, respectively. The µIMZI was machined using a femtosecond laser as a cylindrical cavity (d = 60 µm) in the center of the LPG. In transmission measurements for various RI in the cavity and around the LPG we observed two effects coming from the two independently working sensors. This dual operation had no significant impact on either of the devices in terms of their functional properties, especially in a lower RI range. Moreover, due to the properties of combined sensors two major effects can be distinguished-sensitivity to the RI of the volume and sensitivity to the RI at the surface. Considering also the negligible temperature sensitivity of the µIMZI, it makes the combination of LPG and µIMZI sensors a promising approach to limit cross-sensitivity or tackle simultaneous measurements of multiple effects with high efficiency and reliability.
RESUMEN
In this Letter, we combined a promising bioreceptor, a cocaine aptamer MN6, with an ultrasensitive optical platform long-period fiber grating (LPFG) to create a new cocaine biosensor. The cocaine induces a conformational rearrangement of the aptamer which changes the refractive index around the LPFG producing a measurable shift of the transmission spectrum. We were able to track subtle interaction between the receptor and cocaine molecules over a concentration range of 25 to 100 µM. The presented biosensor does not require labeling or signal enhancement, resulting in a simple user-friendly device.
RESUMEN
The paper presents the first study to date on selective label-free biosensing with a microcavity in-line Mach-Zehnder interferometer induced in an optical fiber. The sensing structures were fabricated in a single-mode fiber by femtosecond laser micromachining. In contrast to other studies of this sensing scheme, where only the sensitivity to refractive index changes in the cavity was investigated, this research used chemical surface treatment of the sensor to ensure detection specificity. Immobilized MS2 bacteriophages were applied as recognition elements specifically targeting live E. coli C3000 bacteria. It is shown that the sensor allows for real-time monitoring of biological phenomena taking place on the surface of the microcavity. The developed biosensor exhibits ultrahigh refractive index sensitivity of 15,000 nm/RIU and is capable of detecting live E. coli bacteria concentrations as low as 100 colony forming units (CFU)/mL in liquid volume as low as picoliters.
Asunto(s)
Técnicas Biosensibles/métodos , Escherichia coli/aislamiento & purificación , Interferometría/métodos , Levivirus/fisiología , Sensibilidad y Especificidad , Acoplamiento ViralRESUMEN
In this paper, the enzymatic activity, substrate specificity and antibiofilm feature of bacteriophage dual-function tail proteins are presented. So far, tail tubular proteins A-TTPAgp31 and TTPAgp44-have been considered as structural proteins of Klebsiella pneumoniae bacteriophages KP32 and KP34, respectively. Our results show that TTPAgp31 is able to hydrolyze maltose as well as Red-starch. The activity of 1 µM of the protein was calculated as 47.6 milli-Units/assay relating to the α-amylase activity. It degrades capsular polysaccharides (cPS), slime polysaccharides (sPS) and lipopolysaccharide (LPS) of K. pneumoniae PCM 2713 and shows antibiofilm reactivity towards S. aureus PCM 519 and E. faecalis PCM 2673. TTPAgp44 hydrolyses trehalose and cPS of E. faecium PCM 1859. TTPAgp44's activity was also observed in the antibiofilm test against P. aeruginosa PCM 2710 and B. subtilis PCM 2021. TTPAgp31 has been identified as α-1,4-glucosidase whereas, TTPAgp44 exhibits trehalase-like activity. Both proteins contain aspartate and glutamate residues in the ß-stranded region which are essential for catalytic activity of glycoside hydrolases. The significant novelty of our results is that for the first time the bacteriophage tubular proteins are described as the unique enzymes displaying no similarity to any known phage hydrolases. They can be used as antibacterial agents directed against bacterial strains producing exopolysaccharides and forming a biofilm.
Asunto(s)
Bacteriófagos/metabolismo , Klebsiella pneumoniae/virología , Maltosa/metabolismo , Polisacáridos/metabolismo , Proteínas de la Cola de los Virus/metabolismo , Biopelículas , HidrólisisRESUMEN
In this work we discuss the refractive index (RI) sensitivity of a micro-cavity in-line Mach-Zehnder interferometer in the form of a cylindrical hole (40-50 µm in diameter) fabricated in a standard single-mode optical fiber using a femtosecond laser. The surface of the micro-cavity was coated with up to 400 nm aluminum oxide thin film using the atomic layer deposition method. Next, the film was progressively chemically etched and the influence on changes in the RI of liquid in the micro-cavity was determined at different stages of the experiment, i.e., at different thicknesses of the film. An effect of transition between sensitivity to the film thickness (surface) and the RI of liquid in the cavity (bulk) is demonstrated for the first time. We have found that depending on the interferometer working conditions determined by thin film properties, the device can be used for investigation of phenomena taking place at the surface, such as in case of specific label-free biosensing applications, or for small-volume RI analysis as required in analytical chemistry.
RESUMEN
This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria.