Advances and New Perspectives in Micro-Nanofabricated Sensors for Bioanalysis.

"Micro-Nanofabricated Sensors for Bioanalysis" represents a cutting-edge field in biosensing technology which leverages the integration of micro- and nanoscale fabrication techniques [...].

Integration of Ultrathin Bubble Walls and Electrochemistry: Innovation in Microsensing for Forensic Nitrite Detection and Microscale Metallic Film Deposition.

We present a strategy for electrochemical measurements using a durable minute bubble wall with a thickness of 27 µm (D = 1.8 cm) as an innovative electrochemical medium. The composition, thickness, and volume of the tiny bubble film were investigated and estimated using the spectroscopic method and the Beer-Lambert law. A carbon microelectrode (D = 10 µm) was then employed as the working electrode, inserted through the bubble wall to function as the solution interface. First, the potential of this method for microelectrodeposition of metallic Ag and Pd films in a tiny bubble was investigated. Interestingly, microscopic images of the deposited film clearly demonstrated that the bubble thickness determines and confines the electrochemical deposition zone. In other words, innovative template-free microelectrodeposition was achieved. In the second phase of our work, microelectroanalysis of trace levels of nitrite ions was performed within the bubble wall and on a foam-covered hand, between the fingers directly, with a low limit of detection of 28 µM. This technique holds significance in criminal investigations, as the presence of NO2- ions on the hand indicates the potential presence of gunshot residue and aids in identifying suspects. In comparison to current methods, this approach is rapid, simple, cost-effective, and amenable to on-site applications, eliminating the need for sample treatment. Ultimately, the utilization of a bubble wall as a novel electrochemical microreactor can open new ways in microelectrochemical analysis, presenting novel opportunities and applications in the field of electrochemical sensors.

Electrochemical Wearable Biosensors and Bioelectronic Devices Based on Hydrogels: Mechanical Properties and Electrochemical Behavior.

Hydrogel-based wearable electrochemical biosensors (HWEBs) are emerging biomedical devices that have recently received immense interest. The exceptional properties of HWEBs include excellent biocompatibility with hydrophilic nature, high porosity, tailorable permeability, the capability of reliable and accurate detection of disease biomarkers, suitable device-human interface, facile adjustability, and stimuli responsive to the nanofiller materials. Although the biomimetic three-dimensional hydrogels can immobilize bioreceptors, such as enzymes and aptamers, without any loss in their activities. However, most HWEBs suffer from low mechanical strength and electrical conductivity. Many studies have been performed on emerging electroactive nanofillers, including biomacromolecules, carbon-based materials, and inorganic and organic nanomaterials, to tackle these issues. Non-conductive hydrogels and even conductive hydrogels may be modified by nanofillers, as well as redox species. All these modifications have led to the design and development of efficient nanocomposites as electrochemical biosensors. In this review, both conductive-based and non-conductive-based hydrogels derived from natural and synthetic polymers are systematically reviewed. The main synthesis methods and characterization techniques are addressed. The mechanical properties and electrochemical behavior of HWEBs are discussed in detail. Finally, the prospects and potential applications of HWEBs in biosensing, healthcare monitoring, and clinical diagnostics are highlighted.

Advances in nano/microscale electrochemical sensors and biosensors for analysis of single vesicles, a key nanoscale organelle in cellular communication.

The study of subcellular targets and biochemical processes within a living cell is valuable for biological and medical research. Secretory vesicles, one such important intracellular target, are nanoscale lipid structures that are capable of storage, transport, and secretion of, for example, neurotransmitters, hormones, proteins or waste products. Vesicles play an essential role in intercellular communication systems, as they facilitate the release of chemical messaging agents. If deregulated, these communication processes can be a central part in the pathogenesis of some neurodegenerative diseases or diabetes. Generally, due to their nanometer size and intracellular location, the analysis of single vesicles and their content is a great challenge. It requires sensitive techniques, micro/nanoscale tools and sensitive instruments with extreme spatio-temporal resolution. This review focuses on electrochemical sensors to study the biochemistry and quantification of messenger molecules and other species (e.g., reactive oxygen and nitrogen species) stored in organelles, providing new trends and developments in this field. Furthermore, we review the effect of the chemical environment of single cells (e.g., treatment with chemicals, drugs, lipids, and ions) on regulation of the physical and chemical properties of vesicles. Finally, unsolved challenges of and perspectives on vesicle electroanalysis are discussed.

Direct Acquisition of the Gap Height of Biological Tissue-Electronic Chemical Sensor Interfaces.

Interfacing biological tissues with electronic sensors offers the exciting opportunity to accurately investigate multiple biological processes. Accurate signal collection and application are the foundation of these measurements, but a long-term issue is the signal distortion resulting from the interface gap. The height of the gap is the key characteristic needed to evaluate or model the distortion, but it is difficult to measure. By integrating a pair of nanopores at the electronic sensor plane and measuring the ion conductance between them, we developed a versatile and straightforward strategy to realize the direct cooperative evaluation of the gap height during exocytotic release from adrenal gland tissues. The signaling distortion of this gap has been theoretically evaluated and shows almost no influence on the amperometric recording of exocytosis in a classic "semi-artificial synapse" configuration. This strategy should benefit research concerning various bio/chemical/machine interfaces.

Quantifying Intracellular Single Vesicular Catecholamine Concentration with Open Carbon Nanopipettes to Unveil the Effect of L-DOPA on Vesicular Structure.

Understanding the regulatory mechanisms of exocytosis is essential for uncovering the pathologies of neuronal disorders and developing related pharmaceuticals. In this work intracellular vesicle impact electrochemical cytometry (IVIEC) measurements with different-sized (50-500 nm radius) open carbon nanopipettes (CNPs) were performed to quantify the vesicular content and release kinetics of specific vesicle populations grouped by orifice sizes. Intracellular vesicles with radius below 100 nm were captured and narrowed between 50 and 100 nm. On the basis of this, single vesicular catecholamine concentrations in the intracellular environment were quantified as 0.23-1.1 M. Our results with L-3,4-dihydroxyphenylalanine (L-DOPA)-exposure indicate that L-DOPA regulates exocytosis by increasing the dense core size and vesicular content while catecholamine concentrations did not show obvious alterations. These were all achieved simultaneously and relatively noninvasively with open CNPs.

Vesicle Impact Electrochemical Cytometry to Determine Carbon Nanotube-Induced Fusion of Intracellular Vesicles.

Carbon nanotube (CNT)-modified electrodes are used to obtain new measurements of vesicle content via amperometry. We have investigated the interaction between CNTs and isolated adrenal chromaffin vesicles (as a model) by vesicle impact electrochemical cytometry. Our data show that the presence of CNTs not only significantly increased the vesicular catecholamine number from 2,250,000 ± 112,766 molecules on a bare electrode to 3,880,000 ± 686,573 molecules on CNT/carbon fiber electrodes but also caused an enhancement in the maximum intensity of the current, which implies the existence of strong interactions between vesicle biolayers and CNTs and an altered electroporation process. We suggest that CNTs might perturb and destabilize the membrane structure of intracellular vesicles and cause the aggregation or fusion of vesicles into new vesicles with larger size and higher content. Our findings are consistent with previous computational and experimental results and support the hypothesis that CNTs as a mediator can rearrange the phospholipid bilayer membrane and trigger homotypic fusion of intracellular vesicles.

Nanoscale Amperometry Reveals that Only a Fraction of Vesicular Serotonin Content is Released During Exocytosis from Beta Cells.

Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all-or-none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all-or-none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317±1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310±1127) for each stimulated exocytosis event. Thus, beta cells release only one-third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type-2 diabetes.

Correlating Molecule Count and Release Kinetics with Vesicular Size Using Open Carbon Nanopipettes.

In this work, open carbon nanopipettes (CNPs) with radius between 50 and 600 nm were used to control translocation of different-sized vesicles through the pipette orifice followed by nanoelectrochemical analysis. Vesicle impact electrochemical cytometry (VIEC) was used to determine the number of catecholamine molecules expelled from single vesicles onto an inner-wall carbon surface, where the duration of transmitter release was quantified and correlated to the vesicle size all in the same nanotip. This in turn allowed us to both size and count molecules for vesicles in a living cell. Here, small and sharp open CNPs were employed to carry out intracellular VIEC with minimal invasion and high sensitivity. Our findings with VIEC reveal that the vesicular content increases with vesicle size. The release kinetics of vesicular transmitters and dense core size have the same relation with the vesicle size, implying that the vesicular dense core size determines the speed of each release event. This direct correlation unravels one of the complexities of exocytosis.

Simultaneous Quantification of Vesicle Size and Catecholamine Content by Resistive Pulses in Nanopores and Vesicle Impact Electrochemical Cytometry.

We have developed the means to simultaneously measure the physical size and count catecholamine molecules in individual nanometer transmitter vesicles. This is done by combining resistive pulse (RP) measurements in a nanopore pipet and vesicle impact electrochemical cytometry (VIEC) at an electrode as the vesicle exits the nanopore. Analysis of freshly isolated bovine adrenal vesicles shows that the size and internal catecholamine concentration of vesicles varies with the occurrence of a dense core inside the vesicles. These results might benefit the understanding about the vesicles maturation, especially involving the "sorting by retention" process and concentration increase of intravesicular catecholamine. The methodology is applicable to understanding soft nanoparticle collisions on electrodes, vesicles in exocytosis and phagocytosis, intracellular vesicle transport, and analysis of electroactive drugs in exosomes.

Nanomaterial-Modified Conducting Paper: Fabrication, Properties, and Emerging Biomedical Applications.

The emerging demand for wearable, lightweight portable devices has led to the development of new materials for flexible electronics using non-rigid substrates. In this context, nanomaterial-modified conducting paper (CP) represents a new concept that utilizes paper as a functional part in various devices. Paper has drawn significant interest among the research community because it is ubiquitous, cheap, and environmentally friendly. This review provides information on the basic characteristics of paper and its functionalization with nanomaterials, methodology for device fabrication, and their various applications. It also highlights some of the exciting applications of CP in point-of-care diagnostics for biomedical applications. Furthermore, recent challenges and opportunities in paper-based devices are summarized.

Mechanochemical Green Synthesis of Exfoliated Edge-Functionalized Boron Nitride Quantum Dots: Application to Vitamin C Sensing through Hybridization with Gold Electrodes.

Two-dimensional boron nitride quantum dots (2D BNQDs) with excellent chemical stability, high photoluminescence efficiency, and low toxicity are a new class of advanced materials for biosensing and bioimaging applications. To overcome the current challenge about the lack of facile, scalable, and reproducible synthesis approach of BNQDs, we introduce a green and facile approach based on mechanochemical exfoliation of bulk h-BN particles in ethanol. Few-layered hydroxylated-functionalized QDs with a thickness of 1-2 nm and a lateral dimension of 2-6 nm have been prepared. The synthesized nanocrystals exhibit a strong fluorescence emission at 407 and 425 nm with a quantum efficiency of ∼6.2%. Spectroscopic analyses determine that interactions between oxygen groups of the solvent with boron sites occur, which along with the mechanical forces, lead to efficient exfoliation of the hexagonal structure and surface functionalization with -OH groups. We also demonstrate that the orbital interaction between BNQDs and the gold surface results in a profound electrochemical catalytic activity toward oxidation of vitamin C. It is shown that the BNQD-modified screen-printed gold electrode exhibits a decreased onset oxidation potential for about 0.37 V/AgCl. In addition to high catalytic activity, electrochemical studies also reveal that this electrode allows selective and sensitive detection of vitamin C with a good response over a wide range from 0.80 µM to 5.0 mM with a detection limit of 0.45 µM (S/N = 3) and a sensitivity of 1.3 µA µM-1 cm-2. Finally, the potential application of the hybrid sensor for detecting vitamin C in commercial drinks is demonstrated.

Graphitic carbon nitride nanosheets as a fluorescent probe for chromium speciation.

A fluorometric method was developed for simultaneous determination of Cr(VI) and Cr(III) ions using graphitic carbon nitride nanosheets (g-C3N4 NS) as a nanosized fluorescent indicator probe. The g-C3N4 NS were prepared using high-temperature carbonization of melamine followed by ultrasonication-assisted liquid exfoliation. The g-C3N4 NS display fluorescence with excitation/emission peaks located at 320 and 450 nm. The chromium speciation is based on the quenching of g-C3N4 NS fluorescence. The total concentration of chromium is determined after oxidation of Cr(III) to Cr(VI). The Cr(III) content was then calculated by subtracting the concentration of Cr(VI) from that of total chromium. The effects of pH value, probe amount, and contact time are optimized. Under optimum conditions, calibration plots are linear in the range in the 0.01 to 100 µM chromium concentration range. The limit of detection is 3 nM for for Cr(VI). The intra- and inter-day relative standard deviations (RSD) of the assay are 3.6-7.5% and 4.1-8.5%, respectively. The indicator probe was applied to the determination of chromium species in spiked water and food samples, and recoveries were satisfactory (93.9-107.0%). Graphical abstract Graphitic carbon nitride nanosheets are synthesized by melamine carbonization and employed for Cr speciation in water and food real samples. Total Cr(VI) and Cr(VI) are assessed based on the quenching of the fluorescence of nanosheets by Cr(VI).

Trace analysis of nitrite ions in environmental samples by using in-situ synthesized Zein biopolymeric nanoparticles as the novel green solid phase extractor.

For the first time, a novel green method using Zein biopolymeric nanoparticles as a green dispersive solid-phase extractor is reported for the separation and preconcentration of trace amount of nitrite (NO2-) ions in ppb levels. The Zein protein is a biodegradable hydrophobic plant protein that is obtained from corn and is composed of a number of hydrophobic amino acids. Zein bionanoparticles were synthesized in an anti-solvent process and used as a new biosorbent in the extraction technique. In the proposed technique, by using a standard method at first, a mixture of 1-naphthylamine and sulphanilic acid as selective regents was added to the samples, and in the presence of the nitrite ion, a red azo product was formed. After that, the ethanolic Zein solution (equal to 15mg) was injected rapidly into the sample, based on the anti-solvent process. Zein bionanoparticles (BNPs) were produced, the adsorbed colour product was separated by centrifugation, and finally samples were analysed with the spectrophotometric method. The influence of different variables such as pH, buffer and amount of buffer, amount of adsorbent and effect of time on extraction were investigated and Zein BNPs were characterized by TEM, SEM, and FT-IR techniques. The main advantages of Zein as a new solid-phase extractor are that this biopolymer is non-toxic, stable, widely available, biodegradable, very hydrophobic, and can be fabricated easily. Under optimal experimental conditions, the linear correlation coefficient (r2) was found to be 0.9972 at the concentration range of 5.0-1000ngmL-1. The limit of detection was 2.3ngmL-1 (0.05µM). This method was applied successfully for the analysis of sea and river waters as well as industrial wastewater samples. Finally, this method follows the US EPA (US Environmental Protection Agency) and WHO (World Health Organization) international standards for nitrite analysis. In addition, it has several advantages to warrant its applicability in the near future in separation science as a green biosorbent in both dispersive and normal solid-phase extraction.

Green synthesis of graphitic carbon nitride nanosheet (g-C3N4) and using it as a label-free fluorosensor for detection of metronidazole via quenching of the fluorescence.

In this research, g-C3N4nanosheets were facilely fabricated by thermal polymerization and then exfoliated into ultrathin nanosheets through ultrasonication in water media. Low-cost C-N nanosheets prepared by melamine possessed a highly π-conjugated structure and fluorescence property. In the present study, the g-C3N4nanosheet was used as a switch-off fluorescence sensor for rapid and sensitive sensing of metronidazole in biological fluids. These nanosheets were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. The fluorescence of the solution of the g-C3N4nanosheets was quenched effectively by metronidazole through two mechanisms: fluorescence resonance energy transfer and the formation of a donor-acceptor charge-transfer complex between π-electron rich donors. Under optimal conditions, the detection linear range for metronidazole was found to be from 0.01 to 0.10µgml-1, with a limit of detection (LOD) of 0.008µgml-1 which can cover standard range of metronidazole in real samples. Moreover, the proposed method has offered a green, rapid, and sensitive probe for quantitative determination of metronidazole in drug and biological fluids.

Impact of Pharmaceutical Impurities in Ecstasy Tablets: Gas Chromatography-Mass Spectrometry Study.

In this study, a simple and reliable method by gas chromatograph-mass spectrometry (GC-MS) was developed for the fast and regular identification of 3, 4-MDMA impurities in ecstasy tablets. In so doing, 8 samples of impurities were extracted by diethyl ether under alkaline condition and then analyzed by GC-MS. The results revealed high MDMA levels ranging from 37.6% to 57.7%. The GC-MS method showed that unambiguous identification can be achieved for MDMA from 3, 4-methylenedioxyamphetamine (MDA), Amphetamine (AM), methamphetamine (MA) and ketamine (Keta) compounds, respectively. The experimental results indicated the acceptable time window without interfering peaks. It is found that GC-MS was provided a suitable and rapid identification approach for MDMA (Ecstacy) tablets, particularly in the Forensic labs. Consequently, the intense MDMA levels would support the police to develop a simple quantification of impurity in Ecstasy tablets.

Hollow Fiber Liquid Based Microextraction of Nalidixic Acid in Urine Samples Using Aliquat 336 as a Carrier Combined with High-Performance Liquid Chromatography.

A new and simple hollow fiber liquid-phase microextraction was used in conjunction with HPLC for the extraction and quantitative determination of nalidixic acid (NA) in human urine samples. This study considers this technique an alternative to common methods of hollow fiber microextraction based on pH gradient and electromembrane extraction of NA in human urine samples. In this research, the drug was extracted from the source phase (donor phase) into a modified organic phase with Aliquat 336 (hydrophobic ion-pair reagent) as a carrier able to impregnate pores of the hollow fiber. In this study, the effects of several factors were tested on the extraction efficiency of the drug. Under optimum conditions, the linearity of the method was observed over the range 0.05-2.0 µg mL(-1) with a correlation coefficient of r = 0.9983. The results of tests on the method determined a good sensitivity with a limit of detection of 0.008 µg mL(-1). The intra-day relative standard deviation (n = 9) for 0.8 µg mL(-1) was 6.2%, and the inter-day relative standard deviation (n = 5) for 0.8 µg mL(-1) was 5.6%. This new strategy was successfully applied to analyze a real urine sample with satisfactory results.

Zinc oxide nanostructure-modified textile and its application to biosensing, photocatalysis, and as antibacterial material.

Recently, one-dimensional nanostructures with different morphologies (such as nanowires, nanorods (NRs), and nanotubes) have become the focus of intensive research, because of their unique properties with potential applications. Among them, zinc oxide (ZnO) nanomaterials has been found to be highly attractive, because of the remarkable potential for applications in many different areas such as solar cells, sensors, piezoelectric devices, photodiode devices, sun screens, antireflection coatings, and photocatalysis. Here, we present an innovative approach to create a new modified textile by direct in situ growth of vertically aligned one-dimensional (1D) ZnO NRs onto textile surfaces, which can serve with potential for biosensing, photocatalysis, and antibacterial applications. ZnO NRs were grown by using a simple aqueous chemical growth method. Results from analyses such as X-ray diffraction (XRD) and scanning electron microscopy (SEM) revealed that the ZnO NRs were dispersed over the entire surface of the textile. We have demonstrated the following applications of these multifunctional textiles: (1) as a flexible working electrode for the detection of aldicarb (ALD) pesticide, (2) as a photocatalyst for the degradation of organic molecules (i.e., Methylene Blue and Congo Red), and (3) as antibacterial agents against Escherichia coli. The ZnO-based textile exhibited excellent photocatalytic and antibacterial activities, and it showed a promising sensing response. The combination of sensing, photocatalysis, and antibacterial properties provided by the ZnO NRs brings us closer to the concept of smart textiles for wearable sensing without a deodorant and antibacterial control. Perhaps the best known of the products that is available in markets for such purposes are textiles with silver nanoparticles. Our modified textile is thus providing acceptable antibacterial properties, compared to available commercial modified textiles.

Colorimetric disposable paper coated with ZnO@ZnS core-shell nanoparticles for detection of copper ions in aqueous solutions.

In this study, we have proposed a new nanoparticle-containing test paper sensor that could be used as an inexpensive, easy-to-use, portable, and highly selective sensor to detect Cu(2+) ions in aqueous solutions. This disposable paper sensor is based on ZnO@ZnS core-shell nanoparticles. The core-shell nanoparticles were synthesized using a chemical method and then they were used for coating the paper. The synthesis of the ZnO@ZnS core-shell nanoparticles was performed at a temperature as low as 60 °C, and so far this is the lowest temperature for the synthesis of such core-shell nanoparticles. The sensitivity of the paper sensor was investigated for different Cu(2+) ion concentrations in aqueous solutions and the results show a direct linear relation between the Cu(2+) ions concentration and the color intensity of the paper sensor with a visual detection limit as low as 15 µM (∼0.96 ppm). Testing the present paper sensor on real river turbulent water shows a maximum 5% relative error for determining the Cu(2+) ions concentration, which confirms that the presented paper sensor can successfully be used efficiently for detection in complex solutions with high selectivity. Photographs of the paper sensor taken using a regular digital camera were transferred to a computer and analyzed by ImageJ Photoshop software. This finding demonstrates the potential of the present disposable paper sensor for the development of a portable, accurate, and selective heavy metal detection technology.