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.

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.

ZnO nanowire-based fluorometric enzymatic assays for lactate and cholesterol.

A rapid fluorometric method is described for the determination of lactate and cholesterol by using ZnO nanowires (ZnO NWs). The assay is based on the detection of the hydrogen peroxide generated during the enzymatic reactions of the oxidation of lactate or cholesterol. Taking advantage of the electrostatic interactions between the enzymes and the ZnO NWs, two bioconjugates were prepared by mixing the nanomaterial and the enzymes, viz. lactate oxidase (LOx) or cholesterol oxidase (ChOx). The enzymatically generated hydrogen peroxide quenches the fluorescence of the ZnO NWs, which have emission peaks at 384 nm and at 520 nm under 330 nm photoexcitation. H2O2 quenches the 520 nm band more strongly. Response is linear up to 1.9 µM lactate concentration, and up to 1.1 µM cholesterol concentration. Relative standard deviation was found to be 5%. The detection limits for lactate and cholesterol are 0.54 and 0.24 µM, respectively. Graphical abstractSchematic representation of fluorescence assay based on ZnO nanowires photoluminiscence for lactate and colesterol detection.

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.

Functionalized N95 Face Mask with a Chemical-Free Paper-Based Collector for Exhaled Breath Analysis: SARS-CoV-2 Detection with a Printed Immunosensor as a Case Study.

Exhaled breath electrochemical sensing is a promising biomedical technology owing to its portability, painlessness, cost-effectiveness, and user-friendliness. Here, we present a novel approach for target analysis in exhaled breath by integrating a comfortable paper-based collector into an N95 face mask, providing a universal solution for analyzing several biomarkers. As a model analyte, we detected SARS-CoV-2 spike protein from the exhaled breath by sampling the target analyte into the collector, followed by its detection out of the N95 face mask using a magnetic bead-based electrochemical immunosensor. This approach was designed to avoid any contact between humans and the chemicals. To simulate human exhaled breath, untreated saliva samples were nebulized on the paper collector, revealing a detection limit of 1 ng/mL and a wide linear range of 3.7-10,000 ng/mL. Additionally, the developed immunosensor exhibited high selectivity toward the SARS-CoV-2 spike protein, compared to other airborne microorganisms, and the SARS-CoV-2 nucleocapsid protein. Accuracy assessments were conducted by analyzing the simulated breath samples spiked with varying concentrations of SARS-CoV-2 spike protein, resulting in satisfactory recovery values (ranging from 97 ± 4 to 118 ± 1%). Finally, the paper-based hybrid immunosensor was successfully applied for the detection of SARS-CoV-2 in real human exhaled breath samples. The position of the collector in the N95 mask was evaluated as well as the ability of this paper-based analytical tool to identify the positive patient.

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.

Compelling DNA intercalation through 'anion-anion' anti-coulombic interactions: boron cluster self-vehicles as promising anticancer agents.

Anticancer drugs inhibit DNA replication by intercalating between DNA base pairs, forming covalent bonds with nucleotide bases, or binding to the DNA groove. To develop safer drugs, novel molecular structures with alternative binding mechanisms are essential. Stable boron hydrides offer a promising alternative for cancer therapy, opening up additional options like boron neutron capture therapy based on 10B and thermal neutron beams or proton boron fusion therapy using 11B and proton beams. These therapies are more efficient when the boron compound is ideally located inside cancer cells, particularly in the nucleus. Current cancer treatments often utilize small, polycyclic, aromatic, planar molecules that intercalate between ds-DNA base pairs, requiring only a spacing of approximately 0.34 nm. In this paper, we demonstrate another type of intercalation. Notably, [3,3'-Fe(1,2-C2B9H11)2]-, ([o-FESAN]-), a compact 3D molecule measuring 1.1 nm × 0.6 nm, can as well intercalate by strong non-bonding interactions preferentially with guanine. Unlike known intercalators, which are positive or neutral, [o-FESAN]- is a negative species and when an [o-FESAN]- molecule approaches the negatively charged DNA phosphate chain an anion-anion interaction consistently anti-electrostatic via Ccluster-H⋯O-P bonds occurs. Then, when more molecules approach, an elongated outstandingly self-assembled structure of [o-FESAN]--[o-FESAN]- forms moving anions towards the interthread region to interact with base pairs and form aggregates of four [o-FESAN]- anions per base pair. These aggregates, in this environment, are generated by Ccluster-H⋯O-C, N-H⋯H-B and Ccluster-H⋯H-B interactions. The ferrabis(dicarbollide) boron-rich small molecules not only effectively penetrate the nucleus but also intercalate with ds-DNA, making them promising for cancer treatment. This amphiphilic anionic molecule, used as a carrier-free drug, can enhance radiotherapy in a multimodal perspective, providing healthcare professionals with improved tools for cancer treatment. This work demonstrates these findings with a plethora of techniques.

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.

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.

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.

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.

Multiplex Portable Biosensor for Bacteria Detection.

An advanced, cost-effective, and portable DNA biosensor capable of detecting multiple bacteria simultaneously has been developed. The biosensor comprises a fast and inexpensive potentiostat that controls the applied potential to a screen-printed electrochemical array platform functionalized with MoS2 flakes and bacterial DNA probes. The current response obtained by à la carte thionine functionalized carbon nanodots (Ty-CDs) is monitored as an electrochemical indicator of the hybridization event. The design of the potentiostat prioritizes achieving an optimal signal-to-noise ratio and incorporates a user-friendly interface compatible with various devices, including computers, mobile phones, and tablets. The device is compact, lightweight, and manufactured at a low cost. The key components of the potentiostat include a data acquisition board capable of analyzing multiple samples simultaneously and a controller board. The results of this study confirm the ability of the multiplex portable biosensor to successfully detect specific bacterial DNA sequences, demonstrating its reliability and superior performance compared with a traditional, more complex, and laboratory-oriented potentiostat.

Potential application of metallacarboranes as an internal reference: an electrochemical comparative study to ferrocene.

Ferrocene and its derivatives have been extensively used as an internal reference in electrochemical processes. Yet, they possess limitations such as solvent restrictions that require chemical modifications. In this regard, we have studied the use of metallacarboranes [3,3'-M(1,2-C2B9H11)2]- (M = Co, Fe) as general internal reference systems and have proven their suitability by thoroughly investigating their electrochemical properties in both aqueous and organic electrolytes without any derivatization.

Electrochemiluminescent nanostructured DNA biosensor for SARS-CoV-2 detection.

This work focuses on the development of an electrochemiluminescent nanostructured DNA biosensor for SARS-CoV-2 detection. Gold nanomaterials (AuNMs), specifically, a mixture of gold nanotriangles (AuNTs) and gold nanoparticles (AuNPs), are used to modified disposable electrodes that serve as an improved nanostructured electrochemiluminescent platform for DNA detection. Carbon nanodots (CDs), prepared by green chemistry, are used as coreactants agents in the [Ru(bpy)3]2+ anodic electrochemiluminescence (ECL) and the hybridization is detected by changes in the ECL signal of [Ru(bpy)3]2+/CDs in combination with AuNMs nanostructures. The biosensor is shown to detect a DNA sequence corresponding to SARS-CoV-2 with a detection limit of 514 aM.

Paving the way to point of care (POC) devices for SARS-CoV-2 detection.

In this work we present a powerful, affordable, and portable biosensor to develop Point of care (POC) SARS-CoV-2 virus detection. It is constructed from a fast, low cost, portable and electronically automatized potentiostat that controls the potential applied to a disposable screen-printed electrochemical platform and the current response. The potentiostat was designed to get the best signal-to-noise ratio, a very simple user interface offering the possibility to be used by any device (computer, mobile phone or tablet), to have a small and portable size, and a cheap manufacturing cost. Furthermore, the device includes as main components, a data acquisition board, a controller board and a hybridization chamber with a final size of 10 × 8 × 4 cm. The device has been tested by detecting specific SARS-CoV-2 virus sequences, reaching a detection limit of 22.1 fM. Results agree well with those obtained using a conventional potentiostat, which validate the device and pave the way to the development of POC biosensors. In this sense, the device has finally applied to directly detect the presence of the virus in nasopharyngeal samples of COVID-19 patients and results confirm its utility for the rapid detection infected samples avoiding any amplification process.

A MoS2 platform and thionine-carbon nanodots for sensitive and selective detection of pathogens.

This work focuses on the combination of molybdenum disulfide (MoS2) and à la carte functionalized carbon nanodots (CNDs) for the development of DNA biosensors for selective and sensitive detection of pathogens. MoS2 flakes prepared through liquid-phase exfoliation, serves as platform for thiolated DNA probe immobilization, while thionine functionalized carbon nanodots (Thi-CNDs) are used as electrochemical indicator of the hybridization event. Spectroscopic and electrochemical studies confirmed the interaction of Thi-CNDs with DNA. As an illustration of the pathogen biosensor functioning, DNA sequences from InIA gen of Listeria monocytogenes bacteria and open reading frame sequence (ORF1ab) of SARS-CoV-2 virus were detected and quantified with a detection limit of 67.0 fM and 1.01 pM, respectively. Given the paradigmatic selectivity of the DNA hybridization, this approach allows pathogen detection in the presence of other pathogens, demonstrated by the detection of Listeria monocytogenes in presence of Escherichia coli. We note that this design is in principle amenable to any pathogen for which the DNA has been sequenced, including other viruses and bacteria. As example of the application of the method in real samples it has been used to directly detect Listeria monocytogenes in cultures without any DNA Polymerase Chain Reaction (PCR) amplification process.