Robust Amide-Linked Fluorinated Covalent Organic Framework for Long-Term Oxygen Reduction Reaction Electrocatalysis.

The high energy demand of the evolving world opens the door to develop more sustainable and environmentally friendly energy sources. Oxygen reduction reaction (ORR) is a promising candidate, being the 2e- pathway of great interest for the green production of hydrogen peroxide. Metal-free covalent organic frameworks (COFs) electrocatalysts present a suitable alternative to substitute the noble-metals more commonly employed in this application. However, the lability of the linkages building up the framework raises an issue for their long-term use and application in aggressive media. Herein, a stable amide-linked COF is reported through post-synthetic modification of a previously reported imine-linked COF proven to be effective as an electrocatalyst, enhancing its chemical stability and electrochemical response. It is found that after the linkage transformation, the new electrocatalyst displays a higher selectivity toward the H2O2 production (98.5%) and an enhanced turnover frequency of 0.155 s-1, which is among the bests reported to date for metal-free and COF based electrocatalysts. The results represent a promising step forward for metal-free non pyrolyzed electrocatalysts, improving their properties through post-synthetic linkage modification for long-term operation.

Scalable Synthesis and Electrocatalytic Performance of Highly Fluorinated Covalent Organic Frameworks for Oxygen Reduction.

In this study, we present a novel approach for the synthesis of covalent organic frameworks (COFs) that overcomes the common limitations of non-scalable solvothermal procedures. Our method allows for the room-temperature and scalable synthesis of a highly fluorinated DFTAPB-TFTA-COF, which exhibits intrinsic hydrophobicity. We used DFT-based calculations to elucidate the role of the fluorine atoms in enhancing the crystallinity of the material through corrugation effects, resulting in maximized interlayer interactions, as disclosed both from PXRD structural resolution and theoretical simulations. We further investigated the electrocatalytic properties of this material towards the oxygen reduction reaction (ORR). Our results show that the fluorinated COF produces hydrogen peroxide selectively with low overpotential (0.062 V) and high turnover frequency (0.0757 s-1 ) without the addition of any conductive additives. These values are among the best reported for non-pyrolyzed and metal-free electrocatalysts. Finally, we employed DFT-based calculations to analyse the reaction mechanism, highlighting the crucial role of the fluorine atom in the active site assembly. Our findings shed light on the potential of fluorinated COFs as promising electrocatalysts for the ORR, as well as their potential applications in other fields.

Neutral Red-carbon nanodots for selective fluorescent DNA sensing.

Carbon nanodots modified with Neutral Red covalently inserted in the nanostructure (NR-CDs) have been prepared by a simple synthesis method based on microwave irradiation under controlled temperature and pressure. The synthetized NR-CDs have been characterized by different techniques, demonstrating the covalent bonding of Neutral Red molecules to the carbon dots nanostructure. Fluorescence activity of the prepare NR-CDs has been explored showing different interaction pathways with singled and doubled stranded DNA. These studies have been successfully applied to develop a new fluorescence DNA hybridization assay to the detection of a specific DNA sequence of Escherichia coli bacteria.

Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures for sensitive and selective SARS-CoV-2 sensing.

The development of DNA-sensing platforms based on new synthetized Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures (AuNs), as a new pathway to develop a selective and sensitive methodology for SARS-CoV-2 detection is presented. A mixture of gold nanoparticles and gold nanotriangles have been synthetized to modify disposable electrodes that act as an enhanced nanostructured electrochemical surface for DNA probe immobilization. On the other hand, modified carbon nanodots prepared a la carte to contain Methylene Blue (MB-CDs) are used as electrochemical indicators of the hybridization event. These MB-CDs, due to their structure, are able to interact differently with double and single-stranded DNA molecules. Based on this strategy, target sequences of the SARS-CoV-2 virus have been detected in a straightforward way and rapidly with a detection limit of 2.00 aM. Moreover, this platform allows the detection of the SARS-CoV-2 sequence in the presence of other viruses, and also a single nucleotide polymorphism (SNPs). The developed approach has been tested directly on RNA obtained from nasopharyngeal samples from COVID-19 patients, avoiding any amplification process. The results agree well with those obtained by RT-qPCR or reverse transcription quantitative polymerase chain reaction technique.

Amplification-free detection of SARS-CoV-2 using gold nanotriangles functionalized with oligonucleotides.

Gold nanotriangles (AuNTs) functionalized with dithiolated oligonucleotides have been employed to develop an amplification-free electrochemical biosensor for SARS-CoV-2 in patient samples. Gold nanotriangles, prepared through a seed-mediated growth method and exhaustively characterized by different techniques, serve as an improved electrochemical platform and for DNA probe immobilization. Azure A is used as an electrochemical indicator of the hybridization event. The biosensor detects either single stranded DNA or RNA sequences of SARS-CoV-2 of different lengths, with a low detection limit of 22.2 fM. In addition, it allows to detect point mutations in SARS-CoV-2 genome with the aim to detect more infective SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron. Results obtained with the biosensor in nasopharyngeal swab samples from COVID-19 patients show the possibility to clearly discriminate between non-infected and infected patient samples as well as patient samples with different viral load. Furthermore, the results correlate well with those obtained by the gold standard technique RT-qPCR, with the advantage of avoiding the amplification process and the need of sophisticated equipment.

Electrochemical (Bio)Sensors Based on Covalent Organic Frameworks (COFs).

Covalent organic frameworks (COFs) are defined as crystalline organic polymers with programmable topological architectures using properly predesigned building blocks precursors. Since the development of the first COF in 2005, many works are emerging using this kind of material for different applications, such as the development of electrochemical sensors and biosensors. COF shows superb characteristics, such as tuneable pore size and structure, permanent porosity, high surface area, thermal stability, and low density. Apart from these special properties, COF's electrochemical behaviour can be modulated using electroactive building blocks. Furthermore, the great variety of functional groups that can be inserted in their structures makes them interesting materials to be conjugated with biological recognition elements, such as antibodies, enzymes, DNA probe, aptamer, etc. Moreover, the possibility of linking them with other special nanomaterials opens a wide range of possibilities to develop new electrochemical sensors and biosensors.

Carbon nanodot-based electrogenerated chemiluminescence biosensor for miRNA-21 detection.

A simple carbon nanodot-based electrogenerated chemiluminescence biosensor is described for sensitive and selective detection of microRNA-21 (miRNA-21), a biomarker of several pathologies including cardiovascular diseases (CVDs). The photoluminescent carbon nanodots (CNDs) were obtained using a new synthesis method, simply by treating tiger nut milk in a microwave reactor. The synthesis is environmentally friendly, simple, and efficient. The optical properties and morphological characteristics of the CNDs were exhaustively investigated, confirming that they have oxygen and nitrogen functional groups on their surfaces and exhibit excitation-dependent fluorescence emission, as well as photostability. They act as co-reactant agents in the anodic electrochemiluminescence (ECL) of [Ru(bpy)3]2+, producing different signals for the probe (single-stranded DNA) and the hybridized target (double-stranded DNA). These results paved the way for the development of a sensitive ECL biosensor for the detection of miRNA-21. This was developed by immobilization of a thiolated oligonucleotide, fully complementary to the miRNA-21 sequence, on the disposable gold electrode. The target miRNA-21 was hybridized with the probe on the electrode surface, and the hybridization was detected by the enhancement of the [Ru(bpy)3]2+/DNA ECL signal using CNDs. The biosensor shows a linear response to miRNA-21 concentration up to 100.0 pM with a detection limit of 0.721 fM. The method does not require complex labeling steps, and has a rapid response. It was successfully used to detect miRNA-21 directly in serum samples from heart failure patients without previous RNA extraction neither amplification process.

Fluorescent C-NanoDots for rapid detection of BRCA1, CFTR and MRP3 gene mutations.

The authors report on a fluorometric method for the rapid detection of BRCA1, CFRT and MRP3 gene mutations. These are associated with breast cancer, cystic fibrosis and autoimmune hepatitis diseases, respectively. Carbon nanodots with blue fluorescence (with excitation/emission maxima at 340/440 nm) were synthesized and characterized, and their interactions with DNA were investigated. Changes in the fluorescence intensity following interaction with ssDNA and dsDNA were used for specific DNA sequence of BRCA1, CFRT and MRP3 genes detection. The response to DNAs is linear up to 200 nM and the detection limit is 270 pM. The assay selectivity allows the detection of single gene mutations. Under optimum conditions, the assay can rapidly discriminate between wild type and mutated samples. Graphical abstract Schematic representation of fluorescence assay for rapid detection of gene mutation based on fluorescent carbon nanodots.

Enhanced Performance of Reagent-Less Carbon Nanodots Based Enzyme Electrochemical Biosensors.

This work reports on the advantages of using carbon nanodots (CNDs) in the development of reagent-less oxidoreductase-based biosensors. Biosensor responses are based on the detection of H2O2, generated in the enzymatic reaction, at 0.4 V. A simple and fast method, consisting of direct adsorption of the bioconjugate, formed by mixing lactate oxidase, glucose oxidase, or uricase with CNDs, is employed to develop the nanostructured biosensors. Peripherical amide groups enriched CNDs are prepared from ethyleneglycol bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid and tris(hydroxymethyl)aminomethane, and used as precursors. The bioconjugate formed between lactate oxidase and CNDs was chosen as a case study to determine the analytical parameters of the resulting L-lactate biosensor. A linear concentration range of 3.0 to 500 µM, a sensitivity of 4.98 × 10-3 µA·µM-1, and a detection limit of 0.9 µM were obtained for the L-lactate biosensing platform. The reproducibility of the biosensor was found to be 8.6%. The biosensor was applied to the L-lactate quantification in a commercial human serum sample. The standard addition method was employed. L-lactate concentration in the serum extract of 0.9 ± 0.3 mM (n = 3) was calculated. The result agrees well with the one obtained in 0.9 ± 0.2 mM, using a commercial spectrophotometric enzymatic kit.

Metallacarboranes on the Road to Anticancer Therapies: Cellular Uptake, DNA Interaction, and Biological Evaluation of Cobaltabisdicarbollide [COSAN].

After uptake by U87 MG and A375 cancer cells, cobaltabisdicarbollide [COSAN]- distributes between membrane and nucleus and presents no relevant cytotoxicity against both cell lines even for long incubation times. The cytotoxicity of Na[COSAN] was also tested towards one normal cell line, the V79 fibroblasts, in order to ascertain the noncytotoxic profile of the compound. As the cell's nucleus contains DNA, the interaction between [COSAN]- and double-stranded calf thymus DNA (CT-dsDNA) has been investigated. There is a strong interaction between both molecules forming a nanohybrid CT-dsDNA-[COSAN] biomaterial, which was fully characterized. Moreover, Na[COSAN] shows characteristic redox peaks ascribed to the oxidation/reduction of Co3+/2+ at a formal potential of -1.444 V and it can be accumulated at a surface-immobilized DNA layer of glassy carbon electrodes. The equilibrium surface-binding constants (Kox /Kred ), which confirm that [COSAN]- interacts with DNA by an intercalative or electrostatic mode, depending on the ionic strength of the solution, were estimated. In addition, high binding affinity of Na[COSAN] to proteins was observed by 11 B{1 H} NMR and confirmed in vivo. Finally, biodistribution studies of [COSAN]- in normal mice were run. After administration, Na[COSAN] was distributed into many organs but mainly accumulated in the reticuloendothelial system (RES), including liver and spleen. After 1 h, the formation of aggregates by plasma protein interaction plays a role in the biodistribution profile; the aggregates accumulate mostly in the lungs. Na[COSAN], which displays low toxicity and high uptake by relevant cancer cells accumulating boron within the nucleus, could act as a suitable compound for further developments as boron neutron capture therapy (BNCT) agents.

Reagent-Less and Robust Biosensor for Direct Determination of Lactate in Food Samples.

Lactic acid is a relevant analyte in the food industry, since it affects the flavor, freshness, and storage quality of several products, such as milk and dairy products, juices, or wines. It is the product of lactose or malo-lactic fermentation. In this work, we developed a lactate biosensor based on the immobilization of lactate oxidase (LOx) onto N,N'-Bis(3,4-dihydroxybenzylidene) -1,2-diaminobenzene Schiff base tetradentate ligand-modified gold nanoparticles (3,4DHS-AuNPs) deposited onto screen-printed carbon electrodes, which exhibit a potent electrocatalytic effect towards hydrogen peroxide oxidation/reduction. 3,4DHS-AuNPs were synthesized within a unique reaction step, in which 3,4DHS acts as reducing/capping/modifier agent for the generation of stable colloidal suspensions of Schiff base ligand-AuNPs assemblies of controlled size. The ligand-in addition to its reduction action-provides a robust coating to gold nanoparticles and a catalytic function. Lactate oxidase (LOx) catalyzes the conversion of l-lactate to pyruvate in the presence of oxygen, producing hydrogen peroxide, which is catalytically oxidized at 3,4DHS-AuNPs modified screen-printed carbon electrodes at +0.2 V. The measured electrocatalytic current is directly proportional to the concentration of peroxide, which is related to the amount of lactate present in the sample. The developed biosensor shows a detection limit of 2.6 µM lactate and a sensitivity of 5.1 ± 0.1 µA·mM-1. The utility of the device has been demonstrated by the determination of the lactate content in different matrixes (white wine, beer, and yogurt). The obtained results compare well to those obtained using a standard enzymatic-spectrophotometric assay kit.

Multi-tasking Schiff base ligand: a new concept of AuNPs synthesis.

Multi-tasking 3,4-dihydroxysalophen Schiff base tetradentate ligand (3,4-DHS) as reductant, stabilizer, and catalyst in a new concept of gold nanoparticles (AuNPs) synthesis is demonstrated. 3,4-DHS is able to reduce HAuCl4 in water, acting also as capping agent for the generation of stable colloidal suspensions of Schiff base ligand-AuNPs assemblies of controlled size by providing a robust coating to AuNPs, within a unique reaction step. Once deposited on carbon electrodes, 3,4-DHS-AuNPs assemblies show a potent electrocatalytic effect towards hydrazine oxidation and hydrogen peroxide oxidation/reduction.

Glutathione immunosensing platform based on total internal reflection ellipsometry enhanced by functionalized gold nanoparticles.

An immunosensor to detect small molecules, such as glutathione (GSH), has been developed by combination of ellipsometry and Kretschmann surface plasmon resonance (SPR). The Au thin film used for surface plasmon polariton (SPP) excitation is functionalized with anti-GSH to specifically bind GSH. At low concentrations, the small refractive index changes caused by the low molecular weight of GSH induced only negligible shifts in the plasmon resonant energy during GSH binding. To improve sensitivity, gold nanoparticles (AuNPs) are functionalized with glutathione acting as amplifiers of the antigen-antibody interaction. Changes induced by the AuNP adsorption are monitored using Ψ and Δ ellipsometric functions. After performing competitive assays using solutions containing different concentrations of free GSH and a constant amount of functionalized AuNPs, it was concluded that the resonant energy linearly shifts as the relative concentration of free GSH increases. A detection limit for free GSH in the nanomolar range is found, demonstrating the effectiveness of AuNPs to enhance the sensitivity to immunoreactions in total internal reflection ellipsometry.

Electrochemically generated nanoparticles of halogen-bridged mixed-valence binuclear metal complex chains.

Spherical nanoparticles composed of MMX chains can be made by a polymerization strategy driven by electrochemical processes. In particular, the [Pt2(MeCS2)4I2] (MMI2) dimetal subunit is employed as a monomer for the formation of [Pt2(MeCS2)4I]n spherical nanostructures on surfaces. We have paid particular attention to elucidating the general mechanism of the deposition process on the basis of in situ electrochemical measurements. The reduction of MMI2 to give the electrodeposition of nanostructures agrees well with formation of the reduced [MMI2](-) species followed by a disproportionation mechanism mediated by iodide anions. The chemical composition of the particles was determined by energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS) to reveal the MMI2 polymer.

A "signal off-on" fluorescence bioassay based on 2D-MoS2-tetrahedral DNA bioconjugate for rapid virus detection.

In this work we present the preparation of a 2D molybdenum disulphide nanosheets (2D-MoS2) and tetrahedral DNA nanostructures (TDNs) bioconjugate, and its application to the development of a bioassay for rapid and easy virus detection. The bioconjugate has been prepared by using TDNs carrying the capture probe labelled with 6-carboxyfluoresceine (6-FAM). As case of study to assess the utility of the assay developed, we have chosen the SARS-CoV-2 virus. Hence, as probe we have used a DNA sequence complementary to a region of the SARS-CoV-2 ORF1ab gene (TDN-ORF-FAM). This 6-FAM labelled capture probe is located on the top vertex of the tetrahedral DNA nanostructure, the three left vertices of TDNs have a thiol group. These TDNs are bounded to 2D-MoS2 surface through the three thiol groups, allowing the capture probe to be oriented to favour the biorecognition reaction with the analyte. This biorecognition resulting platform has finally been challenged to the detection of the SARS-CoV-2 ORF1ab gene sequence as the target model by measuring fluorescence before and after the hybridization event with a detection limit of 19.7fM. Furthermore, due to high sensitivity of the proposed methodology, it has been applied to directly detect the virus in nasopharyngeal samples of infected patients without the need of any amplification step. The developed bioassay has a wide range of applicability since it can be applied to the detection of any pathogen by changing the probe corresponding to the target sequence. Thus, a novel, hands-on strategy for rapid pathogen detection has proposed and has a high potential application value in the early diagnosis of infections causes by virus or bacteria.

Free PCR virus detection via few-layer bismuthene and tetrahedral DNA nanostructured assemblies.

In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of few-layer bismuthene (FLB) as a platform for the oriented immobilization of tetrahedral DNA nanostructures (TDNs) specifically designed and synthetized to detect a specific SARS-CoV-2 gene sequence. In one of its vertices, these TDNs contain a DNA capture probe of the open reading frame 1 ab (ORF1ab) of the virus, available for the biorecognition of the target DNA/RNA. At the other three vertices, there are thiol groups that enable the stable anchoring/binding to the FLB surface. This novel geometry/approach enables not only the binding of the TDNs to surfaces, but also the orientation of the capture probe in a direction normal to the bismuthine surface so that it is readily accessible for binding/recognition of the specific SARS-CoV-2 sequence. The analytical signal is based on the anodic electrochemiluminescence (ECL) intensity of luminol which, in turn, arises as a result of the reaction with H2O2, generated by the enzymatic reaction of glucose oxidation, catalyzed by the biocatalytic label avidin-glucose oxidase conjugate (Av-GOx), which acts as co-reactant in the electrochemiluminescent reaction. The method exhibits a limit of detection (LOD) of 4.31 aM and a wide linear range from 14.4 aM to 1.00 µM, and its applicability was confirmed by detecting SARS-CoV-2 in nasopharyngeal samples from COVID-19 patients without the need of any amplification process.

Rapid and simple viral protein detection by functionalized 2D MoS2/graphene electrochemiluminescence aptasensor.

In this work we present the development of an electrochemiluminescence aptasensor based on electrografting molybdenum disulphide nanosheets functionalized with diazonium salt (MoS2-N2+) upon screen-printed electrodes of graphene (SPEs GPH) for viral proteins detection. In brief, this aptasensor consists of SPEs GPH electrografted with MoS2-N2+ and modified with a thiolated aptamer, which can specifically recognize the target protein analyte. In this case, we have used SARS-CoV-2 spike protein as model protein. Electrochemiluminescence detection was performed by using the [Ru(bpy)3]2+/TPRA (tripropylamine) system, which allows the specific detection of the SARS-CoV-2 spike protein easily and rapidly with a detection limit of 9.74 fg/mL and a linear range from 32.5 fg/mL to 50.0 pg/mL. Moreover, the applicability of the aptasensor has been confirmed by the detection of the protein directly in human saliva samples. Comparing our device with a traditional saliva antigen test, our aptasensor can detect the spike protein even when the saliva antigen test gives a negative result.

Bismuthene - Tetrahedral DNA nanobioconjugate for virus detection.

In this work, we present an electrochemical sensor for fast, low-cost, and easy detection of the SARS-CoV-2 spike protein in infected patients. The sensor is based on a selected combination of nanomaterials with a specific purpose. A bioconjugate formed by Few-layer bismuthene nanosheets (FLB) and tetrahedral DNA nanostructures (TDNs) is immobilized on Carbon Screen-Printed Electrodes (CSPE). The TDNs contain on the top vertex an aptamer that specifically binds to the SARS-CoV-2 spike protein, and a thiol group at the three basal vertices to anchor to the FLB. The TDNs are also marked with a redox indicator, Azure A (AA), which allows the direct detection of SARS-CoV-2 spike protein through changes in the current intensity of its electrolysis before and after the biorecognition reaction. The developed sensor can detect SARS-CoV-2 spike protein with a detection limit of 1.74 fg mL-1 directly in nasopharyngeal swab human samples. Therefore, this study offers a new strategy for rapid virus detection since it is versatile enough for different viruses and pathogens.

MoS2-Carbon Nanodots as a New Electrochemiluminescence Platform for Breast Cancer Biomarker Detection.

In this work, we present the combination of two different types of nanomaterials, 2D molybdenum disulfide nanosheets (MoS2-NS) and zero-dimensional carbon nanodots (CDs), for the development of a new electrochemiluminescence (ECL) platform for the early detection and quantification of the biomarker human epidermal growth factor receptor 2 (HER2), whose overexpression is associated with breast cancer. MoS2-NS are used as an immobilization platform for the thiolated aptamer, which can recognize the HER2 epitope peptide with high affinity, and CDs act as coreactants of the anodic oxidation of the luminophore [Ru(bpy)3]2+. The HER2 biomarker is detected by changes in the ECL signal of the [Ru(bpy)3]2+/CD system, with a low detection limit of 1.84 fg/mL and a wide linear range. The proposed method has been successfully applied to detect the HER2 biomarker in human serum samples.

Bifunctional Au@Pt/Au nanoparticles as electrochemiluminescence signaling probes for SARS-CoV-2 detection.

A novel immunosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) for the sensitive determination of N protein of the SARS-CoV-2 coronavirus is described. For this purpose, bifunctional core@shell nanoparticles composed of a Pt-coated Au core and finally decorated with small Au inlays (Au@Pt/Au NPs) have been synthesized to act as ECL acceptor, using [Ru (bpy)3]2+ as ECL donor. These nanoparticles are efficient signaling probes in the immunosensor developed. The proposed ECL-RET immunosensor has a wide linear response to the concentration of N protein of the SARS-CoV-2 coronavirus with a detection limit of 1.27 pg/mL. Moreover, it has a high stability and shows no response to other proteins related to different virus. The immunosensor has achieved the quantification of N protein of the SARS-CoV-2 coronavirus in saliva samples. Results are consistent with those provided by a commercial colorimetric ELISA kit. Therefore, the developed immunosensor provides a feasible and reliable tool for early and effective detection of the virus to protect the population.