Electrochemiluminescent immunosensor for detection of carcinoembryonic antigen using luminol-coated silver nanoparticles.

Recently, electrochemiluminescent (ECL) immunosensors have received much attention in the field of biomarker detection. Here, a highly enhanced ECL immunosensing platform was designed for ultrasensitive detection of carcinoembryonic antigen (CEA). The surface of the glassy carbon electrode was enhanced by applying functional nanostructures such as thiolated graphene oxide (S-GO) and streptavidin-coated gold nanoparticles (SA-AuNPs). The selectivity and sensitivity of the designed immunosensor were improved by entrapping CEA biomolecules using a sandwich approach. Luminol/silver nanoparticles (Lu-SNPs) were applied as the main core of the signaling probe, which were then coated with streptavidin to provide overloading of the secondary antibody. The highly ECL signal enhancement was obtained due to the presence of horseradish peroxidase (HRP) in the signaling probe, in which the presence of H2O2 further amplified the intensity of the signals. The engineered immunosensor presented excellent sensitivity for CEA detection, with limit of detection (LOD) and linear detection range (LDR) values of 58 fg mL-1 and 0.1 pg mL-1 to 5 pg mL-1 (R2 = 0.9944), respectively. Besides its sensitivity, the fabricated ECL immunosensor presented outstanding selectivity for the detection of CEA in the presence of various similar agents. Additionally, the developed immunosensor showed an appropriate repeatability (RSD 3.8%) and proper stability (2 weeks). Having indicated a robust performance in the real human serum with stated LOD and LDR, the engineered immunosensor can be considered for the detection and monitoring of CEA in the clinic.

Iron oxide nanoparticles/nanocomposites derived from steel and iron wastes for water treatment: A review.

Iron oxide nanoparticles (IONPs) are characterized by superior magnetic properties, high surface area to volume ratio, and active surface functional groups. These properties aid in removal of pollutants from water, through adsorption and/or photocatalysis, justifying the choice of IONPs in water treatment systems. IONPs are usually developed from commercial chemicals of ferric and ferrous salts alongside other reagents, a procedure that is costly, environmentally unfriendly and limits their mass production. On the other hand, steel and iron industries produce both solid and liquid wastes which in most cases are piled, discharged into water streams or landfilled as strategies to dispose them off. Such practices are detrimental to environmental ecosystems. Given the high content of iron present in these wastes, they can be used to generate IONPs. This work reviewed published literature through selected key words on the deployment of steel and/or iron-based wastes as IONPs precursors for water treatment. The findings reveal that steel waste-derived IONPs have properties such as specific surface area, particle sizes, saturation magnetization, and surface functional groups that are comparable or sometimes better than those synthesized from commercial salts. Furthermore, the steel waste-derived IONPs have high removal efficacy for heavy metals and dyes from water with possibilities of being regenerated. The performance of steel waste-derived IONPs can be enhanced by functionalization with different reagents such as chitosan, graphene, and biomass based activated carbons. Nonetheless, there is need to explore the potential of steel waste-based IONPs in removing contaminants of emerging concern, modifying pollutant detection sensors, their techno-economic feasibility in large treatment plants, toxicity of these nanoparticles when ingested into the human body, among other areas.

Electrochemical Assay for Extremely Selective Recognition of Fructose Based on 4-Ferrocene-Phenylboronic Acid Probe and ß-Cyclodextrins Supramolecular Complex.

The aim of the present paper is to highlight a novel electrochemical assay for an extremely-selective detection of fructose thanks to the use of a supramolecular complex between ß-cyclodextrins (ß-CDs) and a chemically modified ferrocene with boronic acid named 4-Fc-PB/natural-ß-CDs. Another kind of ß-CDs, the 4-Fc-PB/3-phenylboronic-ß-CDs, is proposed for the detection of glucose. The novel electrochemical probe is fully characterized by 1 H nuclear magnetic resonance, mass spectroscopy, and elemental analysis, while the superior electrochemical performance is assessed in terms of sensitivity and detection limit. The novelty of the present work consists in the role of CDs that for the first time are employed in electrochemistry with a unique detection mechanism based on specific chemical interactions with the target molecule by the introduction of proper binding groups. A highly selective detection of fructose is obtained and it is believed that the proposed mechanism of detection represents a new way to electrochemically sense other molecules by varying the combination of specific groups of the supramolecular complex. The findings are of impactful importance since a quick, easy, cheap, and extremely selective detection of fructose is not yet available in the market, here achieved by using electrochemical methods which are a very growing field.

Microfluidics by Additive Manufacturing for Wearable Biosensors: A Review.

Wearable devices are nowadays at the edge-front in both academic research as well as in industry, and several wearable devices have been already introduced in the market. One of the most recent advancements in wearable technologies for biosensing is in the area of the remote monitoring of human health by detection on-the-skin. However, almost all the wearable devices present in the market nowadays are still providing information not related to human 'metabolites and/or disease' biomarkers, excluding the well-known case of the continuous monitoring of glucose in diabetic patients. Moreover, even in this last case, the glycaemic level is acquired under-the-skin and not on-the-skin. On the other hand, it has been proven that human sweat is very rich in molecules and other biomarkers (e.g., ions), which makes sweat a quite interesting human liquid with regards to gathering medical information at the molecular level in a totally non-invasive manner. Of course, a proper collection of sweat as it is emerging on top of the skin is required to correctly convey such liquid to the molecular biosensors on board of the wearable system. Microfluidic systems have efficiently come to the aid of wearable sensors, in this case. These devices were originally built using methods such as photolithographic and chemical etching techniques with rigid materials. Nowadays, fabrication methods of microfluidic systems are moving towards three-dimensional (3D) printing methods. These methods overcome some of the limitations of the previous method, including expensiveness and non-flexibility. The 3D printing methods have a high speed and according to the application, can control the textures and mechanical properties of an object by using multiple materials in a cheaper way. Therefore, the aim of this paper is to review all the most recent advancements in the methods for 3D printing to fabricate wearable fluidics and provide a critical frame for the future developments of a wearable device for the remote monitoring of the human metabolism directly on-the-skin.

Fast Procedures for the Electrodeposition of Platinum Nanostructures on Miniaturized Electrodes for Improved Ion Sensing.

Nanostructured materials have attracted considerable interest over the last few decades to enhance sensing capabilities thanks to their unique properties and large surface area. In particular, noble metal nanostructures offer several advantages including high stability, non-toxicity and excellent electrochemical behaviour. However, in recent years the great expansion of point-of-care (POC) and wearable systems and the attempt to perform measurements in tiny spaces have also risen the need of increasing sensors miniaturization. Fast constant potential electrodeposition techniques have been proven to be an efficient way to obtain conformal platinum and gold nanostructured layers on macro-electrodes. However, this technique is not effective on micro-electrodes. In this paper, we investigate an alternative one-step deposition technique of platinum nanoflowers on micro-electrodes by linear sweep voltammetry (LSV). The effective deposition of platinum nanoflowers with similar properties to the ones deposited on macro-electrodes is confirmed by morphological analysis and by the similar roughness factor (~200) and capacitance (~18 µ F/mm 2 ). The electrochemical behaviour of the nanostructured layer is then tested in an solid-contact (SC) L i + -selective micro-electrode and compared to the case of macro-electrodes. The sensor offers Nernstian calibration with same response time (~15 s) and a one-order of magnitude smaller limit of detection (LOD) ( 2.6 × 10 - 6 ) with respect to the macro-ion-selective sensors (ISE). Finally, sensor reversibility and stability in both wet and dry conditions is proven.

A bimetallic nanocoral Au decorated with Pt nanoflowers (bio)sensor for H2O2 detection at low potential.

In this work, we have developed for the first time a method to make novel gold and platinum hybrid bimetallic nanostructures differing in shape and size. Au-Pt nanostructures were prepared by electrodeposition in two simple steps. The first step consists of the electrodeposition of nanocoral Au onto a gold substrate using hydrogen as a dynamic template in an ammonium chloride solution. After that, the Pt nanostructures were deposited onto the nanocoral Au organized in pores. Using Pt (II) and Pt (IV), we realized nanocoral Au decorated with Pt nanospheres and nanocoral Au decorated with Pt nanoflowers, respectively. The bimetallic nanostructures showed better capability to electrochemically oxidize hydrogen peroxide compared with nanocoral Au. Moreover, Au-Pt nanostructures were able to lower the potential of detection and a higher performance was obtained at a low applied potential. Then, glucose oxidase was immobilized onto the bimetallic Au-Pt nanostructure using cross-linking with glutaraldehyde. The biosensor was characterized by chronoamperometry at +0.15V vs. Ag pseudo-reference electrode (PRE) and showed good analytical performances with a linear range from 0.01 to 2.00mM and a sensitivity of 33.66µA/mMcm2. The good value of Kmapp (2.28mM) demonstrates that the hybrid nanostructure is a favorable environment for the enzyme. Moreover, the low working potential can minimize the interference from ascorbic acid and uric acid as well as reducing power consumption to effect sensing. The simple procedure to realize this nanostructure and to immobilize enzymes, as well as the analytical performances of the resulting devices, encourage the use of this technology for the development of biosensors for clinical analysis.

Label-Free Ultrasensitive Memristive Aptasensor.

We present the very first worldwide ever-reported electrochemical biosensor based on a memristive effect and DNA aptamers. This novel device is developed to propose a completely new approach in cancer diagnostics. In this study, an affinity-based technique is presented for the detection of the prostate specific antigen (PSA) using DNA aptamers. The hysteretic properties of memristive silicon nanowires functionalized with these DNA aptamers provide a label-free and ultrasensitive biodetection technique. The ultrasensitive detection is hereby demonstrated for PSA with a limit of detection down to 23 aM, best ever published value for electrochemical biosensors in PSA detection. The effect of polyelectrolytes on our memristive devices is also reported to further show how positive or negative charges affect the memristive hysteresis. With such an approach, combining memristive nanowires and aptamers, memristive aptamer-based biosensors can be proposed to detect a wide range of cancer markers with unprecedent ultrasensitivities to also address the issue of an early detection of cancer.

Enzymatic and Nonenzymatic Electrochemical Interaction of Abiraterone (Antiprostate Cancer Drug) with Multiwalled Carbon Nanotube Bioelectrodes.

Unexplored electrochemical behavior of abiraterone, a recent and widely used prostate cancer drug, in interaction with cytochrome P450 3A4 (CYP3A4) enzyme and multiwalled carbon nanotubes (MWCNTs) is investigated in this work. The results reported in this work are significant for personalized medicine and point-of-care chemical treatment, especially to improve the life expectancy and quality of life of patients with prostate-cancer. To this purpose, enzymatic and nonenzymatic electrochemical biosensors were developed and characterized with different concentrations of abiraterone. Nonenzymatic biosensors were functionalized with MWCNTs as a catalyst for signal enhancement, while enzymatic biosensors have been obtained with CYP3A4 protein immobilized on MWCNTs as recognition biomolecule. Enzymatic electrochemical experiments demonstrated an inhibition effect on the CYP3A4, clearly observed as a diminished electrocatalytic activity of the enzyme. Electrochemical responses of nonenzymatic biosensors clearly demonstrated the direct electroactivity of abiraterone when reacting with MWCNT as well as an electrode-fouling effect.

High-performance multipanel biosensors based on a selective integration of nanographite petals.

We report the first selective growth of nanographite petals and various carbon nanomaterials onto a multipanel electrochemical platform. Different types of nanomaterials can be obtained by fine-tuning the growth parameters of the chemical vapor deposition (CVD) process. First, absolute novelty is the catalytic CVD selective growth of different carbon nanomaterials only on the working electrodes of the platform. A second novelty is the growth obtained at complementary metal-oxide-semiconductor compatible temperatures. These novel electrodes have been incorporated in sensors in which performance characteristics improve with the content of nanostructures. Unprecedented sensing parameters with respect to both direct and enzyme-mediated electrochemical biodetection have been obtained.

Electrochemical sensors modified with iron oxide nanoparticles/nanocomposites for voltammetric detection of Pb (II) in water: A review.

Permissible limits of Pb2+ in drinking water are being reduced from 10 µgL-1 to 5 µgL-1, which calls for rapid, and highly reliable detection techniques. Electrochemical sensors have garnered attention in detection of heavy metal ions in environmental samples due to their ease of operation, low cost, and rapid detection responses. Selectivity, sensitivity and detection capabilities of these sensors, can be enhanced by modifying their working electrodes (WEs) with iron oxide nanoparticles (IONPs) and/or their composites. Therefore, this review is an in-depth analysis of the deployment of IONPs/nanocomposites in modification of electrochemical sensors for detection of Pb2+ in drinking water over the past decade. From the analyzed studies (n = 23), the optimal solution pH, deposition potential, and deposition time ranged between 3 and 5.6, -0.7 to -1.4 V vs Ag/AgCl, and 100-400 s, respectively. Majority of the studies employed square wave anodic stripping voltammetry (n = 16), in 0.1 M acetate buffer solution (n = 19) for detection of Pb2+. Limits of detection obtained (2.5 x 10-9 - 4.5 µg/L) were below the permissible levels which indicated good sensitivities of the modified electrodes. Despite the great performance of these modified electrodes, the primary source of IONPs has always been commercial iron-based salts in addition to the use of so many materials as modifying agents of these IONPs. This may limit reproducibility and sustainability of the WEs due to lengthy and costly preparation protocols. Steel and/or iron industrial wastes can be alternatively employed in generation of IONPs for modification of electrochemical sensors. Additionally, biomass-based activated carbons enriched with surface functional groups are also used in modification of bare IONPs, and subsequently bare electrodes. However, these two areas still need to be fully explored.

In-Memory Sensing and Computing for Cancer Diagnostics: A Perspective Paper.

During the past two decades, a number of two-terminal switching devices have been demonstrated in the literature. They typically exhibit hysteric behavior in the current-to-voltage characteristics. These devices have often been also referred to as memristive devices. Their capacity to switch and exhibit electrical hysteresis has made them well-suited for applications such as data storage, in-memory computing, and in-sensor computing or in-memory sensing. The aim of this perspective paper is to is twofold. Firstly, it seeks to provide a comprehensive examination of the existing research findings in the field and engage in a critical discussion regarding the potential for the development of new non-Von-Neumann computing machines that can seamlessly integrate sensing and computing within memory units. Secondly, this paper aims to demonstrate the practical application of such an innovative approach in the realm of cancer medicine. Specifically, it explores the modern concept of employing multiple cancer markers simultaneously to enhance the efficiency of diagnostic processes in cancer medicine.

A Frequency-Switching Inductive Power Transfer System for Wireless, Miniaturised and Large-Scale Neural Interfaces.

Three-coil inductive power transfer is the state-of-the-art solution to power multiple miniaturised neural implants. However, the maximum delivered power is limited by the efficiency of the powering link and safety constrains. Here we propose a frequency-switching inductive link, where the passive resonator normally used in a three-coil link is replaced by an active resonator. It receives power from the external transmitter via a two-coil inductive link at the low frequency of 13.56 MHz. Then, it switches the operating frequency to the higher frequency of 433.92 MHz through a dedicated circuitry. Last, it transmits power to 1024 miniaturised implants via a three-coil inductive link using an array of 37 focusing resonators for a brain coverage of 163.84 mm 2. Our simulations reported a power transfer efficiency of 0.013 % and a maximum power delivered to the load of 1970 µW under safety-constrains, which are respectively two orders of magnitude and more than six decades higher compared to an equivalent passive three-coil link. The frequency-switching inductive system is a scalable and highly versatile solution for wireless, miniaturised and large-scale neural interfaces.

A Wearable Real-Time System for Simultaneous Wireless Power and Data Transmission to Cortical Visual Prosthesis.

Wireless, miniaturised and distributed neural interfaces are emerging neurotechnologies. Although extensive research efforts contribute to their technological advancement, the need for real-time systems enabling simultaneous wireless information and power transfer toward distributed neural implants remains crucial. Here we present a complete wearable system including a software for real-time image capturing, processing and digital data transfer; an hardware for high radiofrequency generation and modulation via amplitude shift keying; and a 3-coil inductive link adapt to operate with multiple miniaturised receivers. The system operates in real-time with a maximum frame rate of 20 Hz, reconstructing each frame with a matrix of 32 × 32 pixels. The device generates a carrier frequency of 433.92 MHz. It transmits the highest power of 32 dBm with a data rate of 6 Mbps and a variable modulation index as low as 8 %, thus potentially enabling wireless communication with 1024 miniaturised and distributed intracortical microstimulators. The system is primarily conceived as an external wearable device for distributed cortical visual prosthesis covering a visual field of 20 °. At the same time, it is modular and versatile, being suitable for multiple applications requiring simultaneous wireless information and power transfer to large-scale neural interfaces.

Image thresholding techniques for localization of sub-resolution fluorescent biomarkers.

In this article, we explore adaptive global and local segmentation techniques for a lab-on-chip nutrition monitoring system (NutriChip). The experimental setup consists of Caco-2 intestinal cells that can be artificially stimulated to trigger an immune response. The eventual response is optically monitored using immunofluoresence techniques targeting toll-like receptor 2 (TLR2). Two problems of interest need to be addressed by means of image processing. First, a new cell sample must be properly classified as stimulated or not. Second, the location of the stained TLR2 must be recovered in case the sample has been stimulated. The algorithmic approach to solving these problems is based on the ability of a segmentation technique to properly segment fluorescent spots. The sample classification is based on the amount and intensity of the segmented pixels, while the various segmenting blobs provide an approximate localization of TLR2. A novel local thresholding algorithm and three well-known spot segmentation techniques are compared in this study. Quantitative assessment of these techniques based on real and synthesized data demonstrates the improved segmentation capabilities of the proposed algorithm.

Portable impedance-sensing device for microorganism characterization in the field.

A variety of biosensors have been proposed to quickly detect and measure the properties of individual microorganisms among heterogeneous populations, but challenges related to cost, portability, stability, sensitivity, and power consumption limit their applicability. This study proposes a portable microfluidic device based on impedance flow-cytometry and electrical impedance spectroscopy that can detect and quantify the size of microparticles larger than 45 µm, such as algae and microplastics. The system is low cost ($300), portable (5 cm [Formula: see text] 5 cm), low-power (1.2 W), and easily fabricated utilizing a 3D-printer and industrial printed circuit board technology. The main novelty we demonstrate is the use of square wave excitation signal for impedance measurements with quadrature phase-sensitive detectors. A linked algorithm removes the errors associated to higher order harmonics. After validating the performance of the device for complex impedance models, we used it to detect and differentiate between polyethylene microbeads of sizes between 63 and 83 µm, and buccal cells between 45 and 70 µm. A precision of 3% is reported for the measured impedance and a minimum size of 45 µm is reported for the particle characterization.

Soft Epidermal Paperfluidics for Sweat Analysis by Ratiometric Raman Spectroscopy.

The expanding interest in digital biomarker analysis focused on non-invasive human bodily fluids, such as sweat, highlights the pressing need for easily manufactured and highly efficient soft lab-on-skin solutions. Here, we report, for the first time, the integration of microfluidic paper-based devices (µPAD) and non-enhanced Raman-scattering-enabled optical biochemical sensing (Raman biosensing). Their integration merges the enormous benefits of µPAD, with high potential for commercialization and use in resource-limited settings, with biorecognition-element-free (but highly selective) optical Raman biosensing. The introduced thin (0.36 mm), ultra-lightweight (0.19 g), and compact footprint (3 cm2) opto-paperfluidic sweat patch is flexible, stretchable, and conforms, irritation-free, to hairless or minimally haired body regions to enable swift sweat collection. As a great advantage, this new bio-chemical sensory system excels through its absence of onboard biorecognition elements (bioreceptor-free) and omission of plasmonic nanomaterials. The proposed easy fabrication process is adaptable to mass production by following a fully sustainable and cost-effective process utilizing only basic tools by avoiding typically employed printing or laser patterning. Furthermore, efficient collection and transportation of precise sweat volumes, driven exclusively by the wicking properties of porous materials, shows high efficiency in liquid transportation and reduces biosensing latency by a factor of 5 compared to state-of-the-art epidermal microfluidics. The proposed unit enables electronic chip-free and imaging-less visual sweat loss quantification as well as optical biochemical analysis when coupled with Raman spectroscopy. We investigated the multimodal quantification of sweat urea and lactate levels ex vivo (with syntactic sweat including +30 sweat analytes on porcine skin) and achieved a linear dynamic range from 0 to 100 mmol/L during fully dynamic continuous flow characterization.

Biocompatible and Biodegradable 3D Printing from Bioplastics: A Review.

There has been a lot of interest in developing and producing biodegradable polymers to address the current environmental problem caused by the continued usage of synthetic polymers derived from petroleum products. Bioplastics have been identified as a possible alternative to the use of conventional plastics since they are biodegradable and/or derived from renewable resources. Additive manufacturing, also referred to as 3D printing, is a field of growing interest and can contribute towards a sustainable and circular economy. The manufacturing technology also provides a wide material selection with design flexibility increasing its usage in the manufacture of parts from bioplastics. With this material flexibility, efforts have been directed towards developing 3D printing filaments from bioplastics such as Poly (lactic acid) to substitute the common fossil- based conventional plastic filaments such as Acrylonitrile butadiene styrene. Plant biomass is now utilized in the development of biocomposite materials. A lot of literature presents work done toward improving the biodegradability of printing filaments. However, additive manufacture of biocomposites from plant biomass is faced with printing challenges such as warping, low agglomeration between layers and poor mechanical properties of the printed parts. The aim of this paper is to review the technology of 3D printing using bioplastics, study the materials that have been utilized in this technology and how challenges of working with biocomposites in additive manufacture have been addressed.

Changes Over Time in the Electrode/Brain Interface Impedance: An Ex-Vivo Study.

Closed-loop neural implants based on continuous brain activity recording and intracortical microstimulation are extremely effective and promising devices to monitor and address many neurodegenerative diseases. The efficiency of these devices depends on the robustness of the designed circuits which rely on precise electrical equivalent models of the electrode/brain interface. This is true in the case of amplifiers for differential recording, voltage or current drivers for neurostimulation, and potentiostats for electrochemical bio-sensing. This is of paramount importance, especially for the next generation of wireless and ultra-miniaturised CMOS neural implants. Circuits are usually designed and optimized considering the electrode/brain impedance with a simple electrical equivalent model whose parameters are stationary over time. However, the electrode/brain interfacial impedance varies simultaneously in frequency and in time after implantation. The aim of this study is to monitor the impedance changes occurring on microelectrodes inserted in ex-vivo porcine brains to derive an opportune electrode/brain model describing the system and its evolution in time. In particular, impedance spectroscopy measurements have been performed for 144 hours to characterise the evolution of the electrochemical behaviour in two different setups analysing both the neural recording and the chronic stimulation scenarios. Then, different equivalent electrical circuit models have been proposed to describe the system. Results showed a decrease in the resistance to charge transfer, attributed to the interaction between biological material and the electrode surface. These findings are crucial to support circuit designers in the field of neural implants.

Precision Oncology by Point-of-Care Therapeutic Drug Monitoring and Dosage Adjustment of Conventional Cytotoxic Chemotherapies: A Perspective.

Therapeutic drug monitoring (TDM) of conventional cytotoxic chemotherapies is strongly supported yet poorly implemented in daily practice in hospitals. Analytical methods for the quantification of cytotoxic drugs are instead widely presented in the scientific literature, while the use of these therapeutics is expected to keep going for longer. There are two main issues hindering the implementation of TDM: turnaround time, which is incompatible with the dosage profiles of these drugs, and exposure surrogate marker, namely total area under the curve (AUC). Therefore, this perspective article aims to define the adjustment needed from current to efficient TDM practice for cytotoxics, namely point-of-care (POC) TDM. For real-time dose adjustment, which is required for chemotherapies, such POC TDM is only achievable with analytical methods that match the sensitivity and selectivity of current methods, such as chromatography, as well as model-informed precision dosing platforms to assist the oncologist with dose fine-tuning based on quantification results and targeted intervals.

Nanosized optoelectronic devices based on photoactivated proteins.

Molecular nanoelectronics is attracting much attention, because of the possibility to add functionalities to silicon-based electronics by means of intrinsically nanoscale biological or organic materials. The contact point between active molecules and electrodes must present, besides nanoscale size, a very low resistance. To realize Metal-Molecule-Metal junctions it is, thus, mandatory to be able to control the formation of useful nanometric contacts. The distance between the electrodes has to be of the same size of the molecule being put in between. Nanogaps technology is a perfect fit to fulfill this requirement. In this work, nanogaps between gold electrodes have been used to develop optoelectronic devices based on photoactive proteins. Reaction Centers (RC) and Bacteriorhodopsin (BR) have been inserted in nanogaps by drop casting. Electrical characterizations of the obtained structures were performed. It has been demonstrated that these nanodevices working principle is based on charge separation and photovoltage response. The former is induced by the application of a proper voltage on the RC, while the latter comes from the activation of BR by light of appropriate wavelengths.