Reduced graphene oxide electrodes meet lateral flow assays: A promising path to advanced point-of-care diagnostics.

Research in electrochemical detection in lateral flow assays (LFAs) has gained significant momentum in recent years. The primary impetus for this surge in interest is the pursuit of achieving lower limits of detection, especially given that LFAs are the most widely employed point-of-care biosensors. Conventionally, the strategy for merging electrochemistry and LFAs has centered on the superposition of screen-printed electrodes onto nitrocellulose substrates during LFA fabrication. Nevertheless, this approach poses substantial limitations regarding scalability. In response, we have developed a novel method for the complete integration of reduced graphene oxide (rGO) electrodes into LFA strips. We employed a CO2 laser to concurrently reduce graphene oxide and pattern nitrocellulose, exposing its backing to create connection sites impervious to sample leakage. Subsequently, rGO and nitrocellulose were juxtaposed and introduced into a roll-to-roll system using a wax printer. The exerted pressure facilitated the transfer of rGO onto the nitrocellulose. We systematically evaluated several electrochemical strategies to harness the synergy between rGO and LFAs. While certain challenges persist, our rGO transfer technology presents compelling potential for setting a new standard in electrochemical LFA fabrication.

Low-cost inkjet-printed nanostructured biosensor based on CRISPR/Cas12a system for pathogen detection.

The escalating global incidence of infectious diseases caused by pathogenic bacteria, especially in developing countries, emphasises the urgent need for rapid and portable pathogen detection devices. This study introduces a sensitive and specific electrochemical biosensing platform utilising cost-effective electrodes fabricated by inkjet-printing gold and silver nanoparticles on a plastic substrate. The biosensor exploits the CRISPR/Cas12a system for detecting a specific DNA sequence selected from the genome of the target pathogen. Upon detection, the trans-activity of Cas12a/gRNA is triggered, leading to the cleavage of rationally designed single-strand DNA reporters (linear and hairpin) labelled with methylene blue (ssDNA-MB) and bound to the electrode surface. In principle, this sensing mechanism can be adapted to any bacterium by choosing a proper guide RNA to target a specific sequence of its DNA. The biosensor's performance was assessed for two representative pathogens (a Gram-negative, Escherichia coli, and a Gram-positive, Staphylococcus aureus), and results obtained with inkjet-printed gold electrodes were compared with those obtained by commercial screen-printed gold electrodes. Our results show that the use of inkjet-printed nanostructured gold electrodes, which provide a large surface area, in combination with the use of hairpin reporters containing a poly-T loop can increase the sensitivity of the assay corresponding to a signal variation of 86%. DNA targets amplified from various clinically isolated bacteria, have been tested and demonstrate the potential of the proposed platform for point-of-need applications.

Harnessing Bioluminescent Bacteria to Develop an Enzymatic-free Enzyme-linked immunosorbent assay for the Detection of Clinically Relevant Biomarkers.

Enzyme-linked immunosorbent assay (ELISA) is the gold standard technique for measuring protein biomarkers due to its high sensitivity, specificity, and throughput. Despite its success, continuous advancements in ELISA and immunoassay formats are crucial to meet evolving global challenges and to address new analytical needs in diverse applications. To expand the capabilities and applications of immunoassays, we introduce a novel ELISA-like assay that we call Bioluminescent-bacteria-linked immunosorbent assay (BBLISA). BBLISA is an enzyme-free assay that utilizes the inner filter effect between the bioluminescent bacteriaAllivibrio fischeriand metallic nanoparticles (gold nanoparticles and gold iridium oxide nanoflowers) as molecular absorbers. Functionalizing these nanoparticles with antibodies induces their accumulation in wells upon binding to molecular targets, forming the classical immune-sandwich complex. Thanks to their ability to adsorb the light emitted by the bacteria, the nanoparticles can suppress the bioluminescence signal, allowing the rapid quantification of the target. To demonstrate the bioanalytical properties of the novel immunoassay platform, as a proof of principle, we detected two clinically relevant biomarkers (human immunoglobulin G and SARS-CoV-2 nucleoprotein) in human serum, achieving the same sensitivity and precision as the classic ELISA. We believe that BBLISA can be a promising alternative to the standard ELISA techniques, offering potential advancements in biomarker detection and analysis by combining nanomaterials with a low-cost, portable bioluminescent platform.

Rationally Designed DNA-Based Scaffolds and Switching Probes for Protein Sensing.

The detection of a protein analyte and use of this type of information for disease diagnosis and physiological monitoring requires methods with high sensitivity and specificity that have to be also easy to use, rapid and, ideally, single step. In the last 10 years, a number of DNA-based sensing methods and sensors have been developed in order to achieve quantitative readout of protein biomarkers. Inspired by the speed, specificity, and versatility of naturally occurring chemosensors based on structure-switching biomolecules, significant efforts have been done to reproduce these mechanisms into the fabrication of artificial biosensors for protein detection. As an alternative, in scaffold DNA biosensors, different recognition elements (e.g., peptides, proteins, small molecules, and antibodies) can be conjugated to the DNA scaffold with high accuracy and precision in order to specifically interact with the target protein with high affinity and specificity. They have several advantages and potential, especially because the transduction signal can be drastically enhanced. Our aim here is to provide an overview of the best examples of structure switching-based and scaffold DNA sensors, as well as to introduce the reader to the rational design of innovative sensing mechanisms and strategies based on programmable functional DNA systems for protein detection.

Unleashing inkjet-printed nanostructured electrodes and battery-free potentiostat for the DNA-based multiplexed detection of SARS-CoV-2 genes.

Following the global COVID-19 pandemic triggered by SARS-CoV-2, the need for rapid, specific and cost-effective point-of-care diagnostic solutions remains paramount. Even though COVID-19 is no longer a public health emergency, the disease still poses a global threat leading to deaths, and it continues to change with the risk of new variants emerging causing a new surge in cases and deaths. Here, we address the urgent need for rapid, cost-effective and point-of-care diagnostic solutions for SARS-CoV-2. We propose a multiplexed DNA-based sensing platform that utilizes inkjet-printed nanostructured gold electrodes and an inkjet-printed battery-free near-field communication (NFC) potentiostat for the simultaneous quantitative detection of two SARS-CoV-2 genes, the ORF1ab and the N gene. The detection strategy based on the formation of an RNA-DNA sandwich structure leads to a highly specific electrochemical output. The inkjet-printed nanostructured gold electrodes providing a large surface area enable efficient binding and increase the sensitivity. The inkjet-printed battery-free NFC potentiostat enables rapid measurements and real-time data analysis via a smartphone application, making the platform accessible and portable. With the advantages of speed (5 min), simplicity, sensitivity (low pM range, ∼450% signal gain) and cost-effectiveness, the proposed platform is a promising alternative for point-of-care diagnostics and high-throughput analysis that complements the COVID-19 diagnostic toolkit.

Supramolecular Nucleic Acid-Based Organosilica Nanoparticles Responsive to Physical and Biological Inputs.

Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol-gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson-Crick-Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics.

Responsive Nucleic Acid-Based Organosilica Nanoparticles.

The development of smart nanoparticles (NPs) that encode responsive features in the structural framework promises to extend the applications of NP-based drugs, vaccines, and diagnostic tools. New nanocarriers would ideally consist of a minimal number of biocompatible components and exhibit multiresponsive behavior to specific biomolecules, but progress is limited by the difficulty of synthesizing suitable building blocks. Through a nature-inspired approach that combines the programmability of nucleic acid interactions and sol-gel chemistry, we report the incorporation of synthetic nucleic acids and analogs, as constitutive components, into organosilica NPs. We prepared different nanomaterials containing single-stranded nucleic acids that are covalently embedded in the silica network. Through the incorporation of functional nucleic acids into the organosilica framework, the particles respond to various biological, physical, and chemical inputs, resulting in detectable physicochemical changes. The one-step bottom-up approach used to prepare organosilica NPs provides multifunctional systems that combine the tunability of oligonucleotides with the stiffness, low cost, and biocompatibility of silica for different applications ranging from drug delivery to sensing.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

DNA-Based Nanoswitches: Insights into Electrochemiluminescence Signal Enhancement.

Electrochemiluminescence (ECL) is a powerful transduction technique that has rapidly gained importance as a powerful analytical technique. Since ECL is a surface-confined process, a comprehensive understanding of the generation of ECL signal at a nanometric distance from the electrode could lead to several highly promising applications. In this work, we explored the mechanism underlying ECL signal generation on the nanoscale using luminophore-reporter-modified DNA-based nanoswitches (i.e., molecular beacon) with different stem stabilities. ECL is generated according to the "oxidative-reduction" strategy using tri-n-propylamine (TPrA) as a coreactant and Ru(bpy)32+ as a luminophore. Our findings suggest that by tuning the stem stability of DNA nanoswitches we can activate different ECL mechanisms (direct and remote) and, under specific conditions, a "digital-like" association curve, i.e., with an extremely steep transition after the addition of increasing concentrations of DNA target, a large signal variation, and low preliminary analytical performance (LOD 22 nM for 1GC DNA-nanoswtich and 16 nM for 5GC DNA-nanoswitch). In particular, we were able to achieve higher signal gain (i.e., 10 times) with respect to the standard "signal-off" electrochemical readout. We demonstrated the copresence of two different ECL generation mechanisms on the nanoscale that open the way for the design of customized DNA devices for highly efficient dual-signal-output ratiometric-like ECL systems.

Rapid, Cost-Effective Peptide/Nucleic Acid-Based Platform for Therapeutic Antibody Monitoring in Clinical Samples.

We demonstrate here a homogeneous assay, named NanoHybrid, for monoclonal antibody quantification directly in serum samples in a single-step format. NanoHybrid is composed of both synthetic peptide nucleic acids (PNAs) and nucleic acid strands conjugated to recognition elements and optical labels and is designed to allow fast fluorescence quantification of a therapeutic antibody. More specifically, we have characterized our analytical assay for the detection of trastuzumab (Herceptin), a monoclonal antibody (mAb) drug used for breast cancer treatment and for tumors overexpressing the HER2/neu protein. We show here that NanoHybrid is capable of performing fast drug quantification directly in blood serum. The results obtained with a pool of samples from breast cancer patients under trastuzumab treatment are compared with CE-IVD ELISA (enzyme-linked immunosorbent assay) showing a good agreement (Cohen's K = 0.729). Due to the modular nature of the NanoHybrid platform, this technology can be programmed to potentially detect and quantify any antibody for which a high-affinity recognition element has been characterized. We envision the application of NanoHybrid in a point-of-care (POC) drug monitoring system based on disposable kits for therapeutic drug management.

Programming DNA-Based Systems through Effective Molarity Enforced by Biomolecular Confinement.

The fundamental concept of effective molarity is observed in a variety of biological processes, such as protein compartmentalization within organelles, membrane localization and signaling paths. To control molecular encountering and promote effective interactions, nature places biomolecules in specific sites inside the cell in order to generate a high, localized concentration different from the bulk concentration. Inspired by this mechanism, scientists have artificially recreated in the lab the same strategy to actuate and control artificial DNA-based functional systems. Here, it is discussed how harnessing effective molarity has led to the development of a number of proximity-induced strategies, with applications ranging from DNA-templated organic chemistry and catalysis, to biosensing and protein-supported DNA assembly.

Harnessing Effective Molarity to Design an Electrochemical DNA-based Platform for Clinically Relevant Antibody Detection.

Easy-to-use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi-step, time-consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity-driven electrochemical DNA-based detection of target antibodies. We show a highly selective, signal-on DNA-based sensor that takes advantage of antibody-binding-induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.

Programmable RNA-based systems for sensing and diagnostic applications.

The emerging field of RNA nanotechnology harnesses the versatility of RNA molecules to generate nature-inspired systems with programmable structure and functionality. Such methodology has therefore gained appeal in the fields of biosensing and diagnostics, where specific molecular recognition and advanced input/output processing are demanded. The use of RNA modules and components allows for achieving diversity in structure and function, for processing information with molecular precision, and for programming dynamic operations on the grounds of predictable non-covalent interactions. When RNA nanotechnology meets bioanalytical chemistry, sensing of target molecules can be performed by harnessing programmable interactions of RNA modules, advanced field-ready biosensors can be manufactured by interfacing RNA-based devices with supporting portable platforms, and RNA sensors can be engineered to be genetically encoded allowing for real-time imaging of biomolecules in living cells. In this article, we report recent advances in RNA-based sensing technologies and discuss current trends in RNA nanotechnology-enabled biomedical diagnostics. In particular, we describe programmable sensors that leverage modular designs comprising dynamic aptamer-based units, synthetic RNA nanodevices able to perform target-responsive regulation of gene expression, and paper-based sensors incorporating artificial RNA networks. Graphical Abstract ᅟ.

Antibody-Mediated Small Molecule Detection Using Programmable DNA-Switches.

The development of rapid, cost-effective, and single-step methods for the detection of small molecules is crucial for improving the quality and efficiency of many applications ranging from life science to environmental analysis. Unfortunately, current methodologies still require multiple complex, time-consuming washing and incubation steps, which limit their applicability. In this work we present a competitive DNA-based platform that makes use of both programmable DNA-switches and antibodies to detect small target molecules. The strategy exploits both the advantages of proximity-based methods and structure-switching DNA-probes. The platform is modular and versatile and it can potentially be applied for the detection of any small target molecule that can be conjugated to a nucleic acid sequence. Here the rational design of programmable DNA-switches is discussed, and the sensitive, rapid, and single-step detection of different environmentally relevant small target molecules is demonstrated.

Allosterically regulated DNA-based switches: From design to bioanalytical applications.

DNA-based switches are structure-switching biomolecules widely employed in different bioanalytical applications. Of particular interest are DNA-based switches whose activity is regulated through the use of allostery. Allostery is a naturally occurring mechanism in which ligand binding induces the modulation and fine control of a connected biomolecule function as a consequence of changes in concentration of the effector. Through this general mechanism, many different allosteric DNA-based switches able to respond in a highly controlled way at the presence of a specific molecular effector have been engineered. Here, we discuss how to design allosterically regulated DNA-based switches and their applications in the field of molecular sensing, diagnostic and drug release.

Allosteric DNA nanoswitches for controlled release of a molecular cargo triggered by biological inputs.

Here we demonstrate the rational design of a new class of DNA-based nanoswitches which are allosterically regulated by specific biological targets, antibodies and transcription factors, and are able to load and release a molecular cargo (i.e. doxorubicin) in a controlled fashion. In our first model system we rationally designed a stem-loop DNA-nanoswitch that adopts two mutually exclusive conformations: a "Load" conformation containing a doxorubicin-intercalating domain and a "Release" conformation containing a duplex portion recognized by a specific transcription-factor (here Tata Binding Protein). The binding of the transcription factor pushes this conformational equilibrium towards the "Release" state thus leading to doxorubicin release from the nanoswitch. In our second model system we designed a similar stem-loop DNA-nanoswitch for which conformational change and subsequent doxorubicin release can be triggered by a specific antibody. Our approach augments the current tool kit of smart drug release mechanisms regulated by different biological inputs.

Electronic Activation of a DNA Nanodevice Using a Multilayer Nanofilm.

A method to control activation of a DNA nanodevice by supplying a complementary DNA (cDNA) strand from an electro-responsive nanoplatform is reported. To develop functional nanoplatform, hexalayer nanofilm is precisely designed by layer-by-layer assembly technique based on electrostatic interaction with four kinds of materials: Hydrolyzed poly(ß-amino ester) can help cDNA release from the film. A cDNA is used as a key building block to activate DNA nanodevice. Reduced graphene oxides (rGOs) and the conductive polymer provide conductivity. In particular, rGOs efficiently incorporate a cDNA in the film via several interactions and act as a barrier. Depending on the types of applied electronic stimuli (reductive and oxidative potentials), a cDNA released from the electrode can quantitatively control the activation of DNA nanodevice. From this report, a new system is successfully demonstrated to precisely control DNA release on demand. By applying more advanced form of DNA-based nanodevices into multilayer system, the electro-responsive nanoplatform will expand the availability of DNA nanotechnology allowing its improved application in areas such as diagnosis, biosensing, bioimaging, and drug delivery.

A Modular, DNA-Based Beacon for Single-Step Fluorescence Detection of Antibodies and Other Proteins.

A versatile platform for the one-step fluorescence detection of both monovalent and multivalent proteins has been developed. This system is based on a conformation-switching stem-loop DNA scaffold that presents a small-molecule, polypeptide, or nucleic-acid recognition element on each of its two stem strands. The steric strain associated with the binding of one (multivalent) or two (monovalent) target molecules to these elements opens the stem, enhancing the emission of an attached fluorophore/quencher pair. The sensors respond rapidly (<10 min) and selectively, enabling the facile detection of specific proteins even in complex samples, such as blood serum. The versatility of the platform was demonstrated by detecting five bivalent proteins (four antibodies and the chemokine platelet-derived growth factor) and two monovalent proteins (a Fab fragment and the transcription factor TBP) with low nanomolar detection limits and no detectable cross-reactivity.

Social support and long-term mortality in the elderly: role of comorbidity.

Several studies have demonstrated a global increase in morbidity and mortality in elderly subjects with low social support or high comorbidity. However, the relationship between social support and comorbidity on long-term mortality in elderly people is not yet known. Thus, the present study was performed to evaluate the relationship between social support and comorbidity on 12-year mortality of elderly people. A random sample of 1288 subjects aged 65-95 years interviewed in 1992 was studied. Comorbidity by Charlson Comorbidity Index (CCI) score and Social Support by a scale in which total score ranges from 0 to 17, assigning to lowest social support the highest score, were evaluated. At 12-year follow-up, mortality progressively increase with low social support and comorbidity increasing (from 41.5% to 66.7% and from 41.2% to 68.3%, respectively; p<0.001). Moreover, low social support progressively increases with comorbidity increasing (and 12.4±2.5 to 14.3±2.6; p<0.001). Accordingly, multivariate analysis shows an increased mortality risk of 23% for each increase of tertile of social support scale (Hazard ratio=HR=1.23; 95% CI=1.01-1.51; p=0.045). Moreover, when the analysis was performed considering different degrees of comorbidity we found that social support level was predictive of mortality only in subjects with the highest comorbidity (HR=1.39; 95% CI=1.082-1.78; p=0.01). Thus, low social support is predictive of long-term mortality in the elderly. Moreover, the effect of social support on mortality increases in subjects with the highest comorbidity.

Intrapetrous internal carotid artery dissection and essential thrombocythemia: what relationship? A case report.

Internal carotid artery (ICA) dissection is responsible for 10-20% of strokes in young and middle-aged patients. Isolated ICA dissection involving the intrapetrous carotid canal is particularly rare, and no case has been reported to describe an association between intrapetrous ICA dissection and essential thrombocythemia. We report a case of ischemic stroke in the presence of intrapetrous right ICA dissection and essential thrombocythemia. The diagnosis of essential thrombocythemia was performed by bone marrow biopsy. The essential thrombocythemia may cause endothelial dysfunction and predispose to vascular damage such as carotid artery dissection.