Single-stranded Nucleic Acids Research Articles

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3′→5′ exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5′-bond of phosphodiester from 3′ to 5′ end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3′-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA.

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3'→5' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5'-bond of phosphodiester from 3' to 5' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

Histamine-Responsive Hydrogel Biosensors Based on Aptamer Recognition and DNA-Driven Swelling Hydrogels.

Detection of chemical substances is essential for living a healthy and cultural life in the modern world. One type of chemical sensing technology, biosensing, uses biological components with molecular recognition abilities, enabling a broad spectrum of sensing targets. Short single-stranded nucleic acids called aptamers are one of the biological molecules used in biosensing, and sensing methods combining aptamers and hydrogels have been researched for simple sensing applications. In this research, we propose a hydrogel-based biosensor that uses aptamer recognition and DNA-driven swelling hydrogels for the rapid detection of histamine. Aptamer recognition and DNA-driven swelling hydrogels are directly linked via DNA molecular reactions, enabling rapid sensing. We selected histamine, a major food poisoning toxin, as our sensing target and detected the existence of histamine within 10 min with significance. Because this sensing foundation uses aptamers, which have a vast library of targets, we believe this system can be expanded to various targets, broadening the application of hydrogel-based biosensors.

In vitro selection of DNA aptamers against staphylococcal enterotoxin A

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Single-Stranded Nucleic Acid Transmembrane Molecular Carriers Based on Positively Charged Helical Foldamers.

Transmembrane delivery of biologically active nucleic acids is an important process in cells and has inspired one to develop advanced drug delivery techniques. In this contribution, molecular-level single-stranded nucleic acid transmembrane carriers are reported based on 3.2nm long Huc's foldamers (AOrnQ3Q3)8 and (mQ3Q2)8 with linearly and helically aligned positive charges, respectively. These two foldamers not only show very strong DNA affinity via electrostatic interactions but also discriminatively bind single-stranded DNA (ss-DNA) and double-stranded DNA (ds-DNA), corroborating the importance of precise charge arrangement in the electrostatic interactions. More importantly, these two foldamers are capable of efficiently transporting ss-DNA across the lipid membranes, and the ss-DNA transport activity of (AOrnQ3Q3)8 with linearly aligned charges is higher than that of (mQ3Q2)8 with helically aligned charges. Thus a type of novel single-stranded nucleic acid transmembrane molecular carriers based on positively charged helical foldamers are introduced. Further, effective and enhanced expression in EGFP-mRNA transfection experiments strongly demonstrates the potential of positively charged foldamers for RNA transmembrane transport and therapy.

Folding molecular origami from ribosomal RNA

Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg2+ concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired.Graphical

Cost-effective evaluation of Aptamer candidates in SELEX-based Aptamer isolation

Aptamers are short, single-stranded nucleic acids with high affinity and specificity for various targets, making them valuable in diagnostics and therapeutics. Their isolation traditionally involves a time-consuming and costly process called SELEX. While SELEX methods have evolved to improve binding and amplification, the crucial step of aptamer identification from sequencing data remains expensive and often overlooked. Common identification methods require modification of aptamer candidates with labels like biotin or fluorescent dyes, which becomes costly and cumbersome for high-throughput sequencing data. This paper presents an efficient and cost-effective approach to streamline aptamer identification. It employs asymmetric polymerase chain reaction (PCR) to generate modified single-stranded DNA copies of aptamer candidates, simplifying the modification process. By using excess modified forward primers and limited reverse primers, this method reduces costs since only unmodified candidates need to be synthesized initially. The approach was demonstrated with an IgE protein aptamer and successfully applied to identify aptamers from a pool of 12 candidates against a monoclonal antibody. The validity of the results was further confirmed through the direct synthesis of fluorophore-conjugated aptamer candidates, yielding consistent outcomes while reducing the cost by threefold. This approach addresses a critical bottleneck in aptamer discovery by significantly reducing the time and cost associated with aptamer identification, facilitating aptamer-based research and making aptamers more accessible for various applications in diagnostics and therapeutics.

Abstract LB036: Back and forth from structure to functional target: The interplay between Vimentin and non-canonical nucleic acid arrangements

Abstract Vimentin, a member of type III intermediate filaments (IFs), is a cytoskeletal protein that covers multiple architectural and functional roles, including the maintenance of organelle positioning in the cytoplasm, the regulation of cytoskeletal rearrangements, while more recently its potential involvement as a regulatory factor in gene expression emerged. Additionally, it is involved in cell migration and epithelial-to-mesenchymal transition (EMT). In previous works, we identified Vimentin as the first known protein that selectively binds G-quadruplexes repeats (G4-repeats). This DNA folding consists of repeated units of nucleic acid sequences arranged into G-quadruplex (G4s). Their three-dimensional features largely differ from the most common single-stranded or double-helix nucleic acids as well as from the isolated G4 modules. Noteworthy, they occur at a small subset of genomic sites, associated with cell proliferation and migration, where they can recruit the soluble nuclear pool of Vimentin. Altogether, this information led us to consider the Vimentin-DNA complex as a promising target for developing small molecule binders capable of inhibiting cell migration and, eventually, EMT. To properly set up a rational drug-design approach, we integrated different advanced biophysical tools (Hydrogen-Deuterium Exchange, Cryo-Electron Microscopy) to derive a comprehensive picture of the protein-DNA complex at the molecular level. This approach allowed us to map the interaction surface between the soluble tetrameric form of Vimentin and a G4-repeat. Based on this data, we initiated a screening campaign using focused libraries of small molecules and peptides, which were rationally designed according to the structural features of the Vimentin-G4-repeats complex. The capability of the identified hits to prevent the recognition of the nucleic acid by Vimentin has been investigated This approach allowed us to identify attractive novel entities that prevent the Vimentin-G4-repeat complex formation according to different molecular mechanisms. These systems are now under further refinement moving from the test tube to the cellular environment, where the dynamic behavior of these nucleic acid domains as well as the recruitment of different proteins by Vimentin might alter the architectural features of the target.

DNA-dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single-stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid-liquid phase separation (LLPS) propensity of Escherichia coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2's C-terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB-driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single-stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB-mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

In vivo interference of pea aphid endosymbiont Buchnera groEL gene by synthetic peptide nucleic acids

The unculturable nature of intracellular obligate symbionts presents a significant challenge for elucidating gene functionality, necessitating the development of gene manipulation techniques. One of the best-studied obligate symbioses is that between aphids and the bacterial endosymbiont Buchnera aphidicola. Given the extensive genome reduction observed in Buchnera, the remaining genes are crucial for understanding the host-symbiont relationship, but a lack of tools for manipulating gene function in the endosymbiont has significantly impeded the exploration of the molecular mechanisms underlying this mutualism. In this study, we introduced a novel gene manipulation technique employing synthetic single-stranded peptide nucleic acids (PNAs). We targeted the critical Buchnera groEL using specially designed antisense PNAs conjugated to an arginine-rich cell-penetrating peptide (CPP). Within 24 h of PNA administration via microinjection, we observed a significant reduction in groEL expression and Buchnera cell count. Notably, the interference of groEL led to profound morphological malformations in Buchnera, indicative of impaired cellular integrity. The gene knockdown technique developed in this study, involving the microinjection of CPP-conjugated antisense PNAs, provides a potent approach for in vivo gene manipulation of unculturable intracellular symbionts, offering valuable insights into their biology and interactions with hosts.

Aptamer Technologies in Neuroscience, Neuro-Diagnostics and Neuro-Medicine Development.

Aptamers developed using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology are single-stranded nucleic acids 10-100 nucleotides in length. Their targets, often with specificity and high affinity, range from ions and small molecules to proteins and other biological molecules as well as larger systems, including cells, tissues, and animals. Aptamers often rival conventional antibodies with improved performance, due to aptamers' unique biophysical and biochemical properties, including small size, synthetic accessibility, facile modification, low production cost, and low immunogenicity. Therefore, there is sustained interest in engineering and adapting aptamers for many applications, including diagnostics and therapeutics. Recently, aptamers have shown promise as early diagnostic biomarkers and in precision medicine for neurodegenerative and neurological diseases. Here, we critically review neuro-targeting aptamers and their potential applications in neuroscience research, neuro-diagnostics, and neuro-medicine. We also discuss challenges that must be overcome, including delivery across the blood-brain barrier, increased affinity, and improved in vivo stability and in vivo pharmacokinetic properties.

Sensitive Methods to Detect Single-Stranded Nucleic Acids of Food Pathogens Based on Cell-Free Protein Synthesis and Retroreflection Signal Detection.

Cell-free protein synthesis (CFPS) has recently gained considerable attention as a new platform for developing methods to detect various molecules, ranging from small chemicals to biological macromolecules. Retroreflection has been used as an alternative signal to develop analytical methods because it can be detected by using a simple instrument comprising a white light source and a camera. Here, we report a novel reporter protein that couples the capability of CFPS and the simplicity of retroreflection signal detection. The design of the reporter was based on two pairs of protein-peptide interactions, SpyCatcher003-SpyTag003 and MDM2-PMI(N8A). MDM2-MDM2-SpyCatcher003 was decided as the reporter protein, and the two peptides, SpyTag003 and PMI(N8A), were immobilized on the surfaces of retroreflective Janus particles and microfluidic chips, respectively. The developed retroreflection signal detection system was combined with a previously reported CFPS reaction that can transduce the presence of a single-stranded nucleic acid into protein synthesis. The resulting methods were applied to detect 16S rRNAs of several foodborne pathogens. Concentration-dependent relationships were observed over a range of 10° fM to 102 pM, with the limits of detection being single-digit femtomolar concentrations. Considering the designability of the CFPS system for other targets, the retroreflection signal detection method will enable the development of novel methods to detect various molecules.

Abstract B044: Intracellular sclerostin plays a vital role in tumor progression and metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited treatment options. It is essential to continue advancing well-defined molecular targets in TNBC. Sclerostin (SOST) is mostly concerned as an osteocyte secreted protein and a drug target for osteoporosis. Recently, sclerostin was reported to be expressed in a majority of clinical TNBC tissues. Upregulated SOST expression was reported to be associated with worse prognosis in breast cancer patients. This study aimed to investigate the role of sclerostin in TNBC. Methods: Cell proliferative ability was evaluated by colony formation assay. Cell migratory and invasive abilities were evaluated by transwell assay. The tumor growth and metastasis were evaluated in 4T1 orthotopic and metastatic mouse models. The cellular uptake of antibody and aptamer was evaluated by fluorescence microscopy. Results: It was found that sclerostin knockout demonstrated inhibitory effects on proliferation and migration/invasion in both human (MDA-MB-231) and murine (4T1) TNBC cell lines in vitro. Surprisingly, no tumor growth and metastasis were found in 4T1-sost KO inoculated orthotopic and metastatic mouse models compared to their wildtype counterparts. It suggested the vital role of sclerostin in TNBC. Anti-sclerostin antibody (Scl-Ab) could not be internalized by TNBC cells and demonstrated no effects on proliferation and migration/invasion in vitro. On the other hand, no difference in proliferation and migration/invasion was also observed between TNBC cells with and without supplementing sclerostin in vitro. Genetically, there was no difference in tumor growth between 4T1 inoculated WT mice and SOST -/− mice. Pharmacologically, no difference was also observed in tumor growth between Scl-Ab treatment and IgG control in 4T1 inoculated mice. It suggested that the role of extracellular and systemic sclerostin could be excluded in TNBC. Aptamers are single-strand nucleic acids which bind to specific targets. It was found that sclerostin-specific aptamer (Scl-Ap) could be internalized by TNBC cells and demonstrated inhibitory effects on proliferation and migration in vitro. Consistently, Scl-Ap demonstrated inhibitory effects on tumor growth in 4T1 inoculated mice. It suggested that intracellular sclerostin might play a vital role in TNBC. Conclusions: Intracellular sclerostin plays a vital role in TNBC progression and metastasis. Citation Format: Meiheng SUN, Shuangying Qiao, Hang Luo, Xin Yang, Liu Yang, Huarui Zhang, Zefeng Chen, Luyao Wang, Xiaohui Tao, Yuanyuan Yu, Yuan Ma, Fenglai Yuan, Daqing Ma, Qianjun Chen, Aiping Lu, Bao-ting Zhang, Ge Zhang, Fangfei Li. Intracellular sclerostin plays a vital role in tumor progression and metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B044.

Molecular dynamics simulations on the interactions between nucleic acids and a phospholipid bilayer

Recently, lipid nanoparticles (LNPs) have been extensively investigated as non-viral carriers of nucleic acid vaccines due to their high transport efficiency, safety, and straightforward production and scalability. However, the molecular mechanism underlying the interactions between nucleic acids and phospholipid bilayers within LNPs remains elusive. In this study, we employed the all-atom molecular dynamics simulation to investigate the interactions between single-stranded nucleic acids and a phospholipid bilayer. Our findings revealed that hydrophilic bases, specifically G in single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA), displayed a higher propensity to form hydrogen bonds with phospholipid head groups. Notably, ssRNA exhibited stronger binding energy than ssDNA. Furthermore, divalent ions, particularly Ca2+, facilitated the binding of ssRNA to phospholipids due to their higher binding energy and lower dissociation rate from phospholipids. Overall, our study provides valuable insights into the molecular mechanisms underlying nucleic acid-phospholipid interactions, with potential implications for the nucleic acids in biotherapies, particularly in the context of lipid carriers.

Differential expression of zinc finger CCHC-type superfamily proteins in thyroid carcinoma and their associations with tumor immunity

BackgroundThe zinc-finger CCHC-type (ZCCHC) superfamily proteins are characterized by the shared sequence CX2-CX4-HX4-C and thought to own high affinity to single-stranded nucleic acids, particularly RNAs. In humans, a total of 24 ZCCHC proteins have been annotated in the HUGO Gene Nomenclature Committee (HGNC, https://www.genenames.org/) database with most of these members involved in multiple steps of RNA metabolism. Many studies have indicated that the ZCCHC genes play a regulatory role in the development and progression of solid tumors. To date, the expression pattern and prognostic value of ZCCHC factors in thyroid carcinomas have not been reported.MethodsBioinformatics analyses on the functions of ZCCHC factors in thyroid carcinoma (THCA) patients were performed based on various databases, i.e., TCGA, GEPIA, Kaplan-Meier Plotter, and TIMER.ResultsCompared with normal tissues, the expression of ZCCHC12 mRNA was significantly increased in THCA tissues. And it was associated with the overall survival of THCA patients, based on the Kaplan-Meier Plotter database. Furthermore, the expression levels of all ZCCHCs were correlated with tumor stages, implying its high relevance to THCA, specifically its immunity.ConclusionThe ZCCHC genes, represented by ZCCHC12, are differentially expressed in THCA staging. These genes are associated with immune infiltration of THCA and identified as the potential therapeutic targets for immunotherapy in THCA patients, which are possible novel biomarkers for the treatment of THCA.

Updating Ortho- and Metachromatic Acridine Orange Fluorescence in Cytochemical Chromosome Staining: A Proposal for Understanding Its Differential Fluorescence on Double- and Single-Stranded Nucleic Acids Substrates Based on Intercalation

Many fluorophores display interesting features that make them useful biological labels and chemosensors, in particular in Cell Biology. Changes in the absorption-emission spectra (ortho- and metachromasia) are accounted among them. Acridine orange (AO) is one such fluorochromes that shows a prototypical orthochromatic vs. metachromatic behavior depending on its concentration and binding mode to different cellular substrates. Here, we revisit the differential AO fluorescence that occurs in selected biological examples, which allows for the identification of single-stranded or double-stranded nucleic acids. Although known for long, the ultimate reason for this phenomenon has not been properly advanced. We provide a potential molecular mechanism that adequately accounts for the different aspects of the phenomenon. This theoretical mechanism implies a difference in the degree of overlap of excited state orbitals whenever AO molecules are interacting with a single-stranded or a double-stranded nucleic acid. In the first case, massive π-electron overlapping between bases and intercalated AO leads to a metachromatic red emission. On the contrary, no excited-state orbital overlapping in AO-intercalated DNA duplexes is possible due to excessive separation between AO molecules and compliancy to the nearest neighbor exclusion principle, which manifests as orthochromatic green fluorescence.

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.

A Universal Hydrogel Microneedle for on-Site Detection of Small Molecules, Proteins, and Ribonucleic Acids

Point-of-care testing (POCT) of clinical biomarkers is critical to health monitoring and timely treatment, yet biosensing assays capable of detecting biomarkers without the need for costly external equipment and reagents are limited. Blood-based assays are, specifically, challenging as blood collection is invasive, and several processing steps are required. A promising alternative to blood is interstitial fluid (ISF), which originates from blood and fills the extracellular space; thus, it has common biomarkers with plasma/serum while also containing biomarkers unique to the local cells. A wide range of metabolites, including amino acids, lipids, nucleotides, and protein biomarkers, can be detected in ISF, emphasizing the potential of ISF for health monitoring. Microneedle (MN)-based biosensors have recently emerged as a promising approach for investigating the ISF. They enable minimally invasive penetration through the skin for ISF access. Specifically, Hydrogel-based MNs have two main advantages; i) their fabrication is cost-effective and simple, ii) they are highly biocompatible. Therefore, they overcome the challenges with conventional solid MNs. However, Hydrogel Microneedles (HMN) capable of both extracting ISF and sensing an analyte on-microneedle (without any need for post-sampling processing) have been limited. Here, we report a versatile assay that employs HMNs to both extract (ISF) and perform on-MN sensing of a specific analyte/biomarker in a minimally invasive manner. Our assay incorporates graphene oxide-nucleic acid (GO.NA) optical sensors for sensing. The GO.NA optical sensor consists of GO nanosheets conjugated to fluorophore-modified nucleic acid (NA), in which GO acts as a quencher. Single-stranded NAs have high affinity for binding to GO, therefore, in the absence of the biomarker of interest the NAs are tightly bound to GO; in turn, their fluorophore tag is quenched. However, in the presence of a specific biomarker, the NAs bind to their target, inducing a conformational change that distances the fluorophore tag from GO, leading to fluorescence recovery and generation of an optical signal. GO also acts as a substrate for NA immobilization; therefore, it protects the NA from degradation and prevents any potential NA release from HMN-GO.NA. To enable microscope-free patch visualization and optical measurements for POCT, we developed a miniaturized smartphone-based system that captures fluorescence images of the HMN-GO.NA patches (Scheme 1A and 1B), which are then analyzed using freely available software (ImageJ). Our system could successfully measure six clinically important biomarkers (glucose, uric acid, insulin, and serotonin as well as microribonucleic acid 210 and 21) in-vitro (using phantom gel) and ex-vivo (using porcine skin). Further, we have been able to accurately detect glucose and uric acid in live diabetic animal models, proving the efficacy of our system. Figure 1

Fluorescent nanostructured carbon dot-aptasensor for chlorpyrifos detection: Elucidating the interaction mechanism for an environmentally hazardous pollutant

Chlorpyrifos (CPF) is a commonly used insecticide found in many water sources and is related to several health and environmental effects. Biosensors based on aptamers (single-stranded nucleic acid oligonucleotides) are promising alternatives to achieve the detection of CPF and other pesticides in natural waters. However, several challenges need to be addressed to promote the real application of functional aptasensing devices. In this work, an ssDNA aptamer (S1) is combined with carbon quantum dots (CD) and graphene oxide (GO) to produce a stable fluorescent aptasensor characterized through spectrophotometric and electrophoretic techniques. For a deeper understanding of the system, the mechanism of molecular interaction was studied through docking modeling using free bioinformatic tools like HDOCK, showing that the stem-loops and the higher guanine (G) content are crucial for better interaction. The model also suggests the possibility of generating a truncated aptamer to improve the binding affinity. The biosensor was evaluated for CPF detection, showing a low LOD of 0.01 μg L−1 and good specificity in tap water, even compared to other organophosphates pesticides (OPs) like profenofos. Finally, the recovery of the proposed aptasensor was evaluated in some natural water using spiked samples and compared with UPLC MS-MS chromatography as the gold standard, showing a good recovery above 2.85 nM and evidencing the need of protecting ssDNA aptamers from an erratic interaction with the aromatic groups of dissolved organic matter (humic substances). This work paves the way for a better aptasensors design and the on-site implementation of novel devices for environmental monitoring.

Surfactant-mediated colorimetric assay assisted with in-situ rolling circle amplification on magnetic beads

Gold nanoparticles (AuNPs) with localized surface plasmon resonance effect have been widely used for colorimetric detection based on the interparticle plasmon coupling during AuNPs aggregation. However, it is still challenging to develop portable and quantitative methods with good sensitivity and excellent selectivity. In this study, a smartphone-based colorimetric assay is developed on the principle of surfactant-mediated AuNPs aggregation assisted with rolling circle amplification (RCA) on magnetic beads (MBs). The detection of adenosine is demonstrated as an example. The cetyl trimethyl ammonium bromide (CTAB) causes the negatively charged AuNPs to aggregate, which results in the color change from red to blue. When adenosine is in solution, the RCA process is triggered on the MBs because of specific adenosine-aptamer recognition, resulting in prolongation of single-stranded nucleic acid (ssDNA). The solution color remains red due to the electrostatic interaction between CTAB and ssDNA. Using this method, the limit of detection (LOD) for adenosine can be as low as 16 pM. Besides, it also works well in human serum. In addition, a portable device integrated with in-situ RGB analysis software is developed for the detection with a smartphone. This study offers a new strategy to improve the sensitivity and selectivity for the AuNPs-based colorimetric assay, taking advantages of specific aptamer recognition, in-situ RCA on MBs, magnetic separation, and smartphone-based portable device.