Synthesis of obovatol and related neolignan analogues as α-glucosidase and α-amylase inhibitors.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy), enzymes responsible for the hydrolysis of carbohydrates. In recent decades, many natural compounds and their bioinspired analogues have been studied as α-Glu and α-Amy inhibitors. However, no studies have been devoted to the evaluation of α-Glu and α-Amy inhibition by the neolignan obovatol (1). In this work, we report the synthesis of 1 and a library of new analogues. The synthesis of these compounds was achieved by implementing methodologies based on: phenol allylation, Claisen/Cope rearrangements, methylation, Ullmann coupling, demethylation, phenol oxidation and Michael-type addition. Obovatol (1) and ten analogues were evaluated for their in vitro inhibitory activity towards α-Glu and α-Amy. Our investigation highlighted that the naturally occurring 1 and four neolignan analogues (11, 22, 26 and 27) were more effective inhibitors than the hypoglycemic drug acarbose (α-Amy: 34.6 µM; α-Glu: 248.3 µM) with IC5O value of 6.2-23.6 µM toward α-Amy and 39.8-124.6 µM toward α-Glu. Docking investigations validated the inhibition outcomes, highlighting optimal compatibility between synthesized neolignans and both the enzymes. Concurrently circular dichroism spectroscopy detected the conformational changes in α-Glu induced by its interaction with the studied neolignans. Detailed studies through fluorescence measurements and kinetics of α-Glu and α-Amy inhibition also indicated that 1, 11, 22, 26 and 27 have the greatest affinity for α-Glu and 1, 11 and 27 for α-Amy. Surface plasmon resonance imaging (SPRI) measurements confirmed that among the compounds studied, the neolignan 27 has the greater affinity for both enzymes, thus corroborating the results obtained by kinetics and fluorescence quenching. Finally, in vitro cytotoxicity of the investigated compounds was tested on human colon cancer cell line (HCT-116). All these results demonstrate that these obovatol-based neolignan analogues constitute promising candidates in the pursuit of developing novel hypoglycemic drugs.

Plasmonic aptasensor with antifouling dual-functional surface layer for lysozyme detection in food.

Antifouling coatings are critically necessary for optical biosensors for various analytical application sectors, from medical diagnostics to foodborne pathogen detection. They help avoid non-specific protein/cell attachment on the active biosensor surface and catch the analytes directly in the complex media. Advances in antifouling plasmonic surfaces have been mainly focused on detecting clinical biomarkers in real biofluids, whereas developing antifouling coatings for direct analysis of analytes in complex media has been scarcely investigated for food quality control and safety. Herein, we propose a new low-fouling poly-l-lysine (PLL)-based surface layer for directly detecting an allergen protein, lysozyme, in the food matrix using surface plasmon resonance. The PLL-based polymer contains densely immobilized anionic oligopeptide side chains to create an electric charge-balanced layer able to repel the non-specific adsorption of undesired molecules on the biosensor surface. It also includes sparsely attached aptamer probes for capturing lysozyme directly in food sources with no pre-analytical sample treatment. We optimized the surface layer fabrication condition and tested the dual-functional surface to evaluate its ability to detect the target protein selectively. The developed analytical approach allowed for achieving a limit of detection of 0.04 µg mL-1 (2.95 nM) and a limit of quantification of 0.13 µg mL-1 (8.95 nM). Lysozyme was successfully quantified in milk samples using the plasmonic dual-functional aptasensor without sample pre-treatment or target isolation, illustrating the device's utility.

Isothermal circular strand displacement-based assay for microRNA detection in liquid biopsy.

Extracellular miRNAs are promising targets for developing new assays for the early diagnosis and prognosis of diseases based on liquid biopsy. The detection of miRNAs in liquid biopsies is challenged by their short sequence length, low concentration, and interferences with bodily fluid components. Isothermal circular strand displacement polymerization has emerged as a convenient method for nucleic acid amplification and detection. Herein, we describe an innovative strategy for microRNA detection directly from biological fluids based on hairpin probe-assisted isothermal amplification reaction. We designed and optimized the assay to detect target analytes in 1 µL of the complex media's biological matrix using a microfluidic device for the straightforward analysis of multiple samples. We validated the assay to detect circulating miR-127-5p in synovial fluid, recently indicated as a predictive biomarker for osteoarthritis disease. The combined use of a mutant polymerase operating with high yield and a primer incorporating locked nucleic acid nucleosides allowed detection of miR-127-5p with 34 fmol L-1 LOD. We quantified circulating miR-127-5p directly in synovial fluid, thus demonstrating that the assay may be employed for the convenient detection of 4.3 ± 0.5 pmol L-1 concentrated miRNAs in liquid biopsy samples.

Nanoparticle-Enhanced Surface Plasmon Resonance Imaging Enables the Ultrasensitive Detection of Non-Amplified Cell-Free Fetal DNA for Non-Invasive Prenatal Testing.

Although many potential applications in early clinical diagnosis have been proposed, the use of a surface plasmon resonance imaging (SPRI) technique for non-invasive prenatal diagnostic approaches based on maternal blood analysis is confined. Here, we report a nanoparticle-enhanced SPRI strategy for a non-invasive prenatal fetal sex determination based on the detection of a Y-chromosome specific sequence (single-gene SRY) in cell-free fetal DNA from maternal plasma. The SPR assay proposed here allows for detection of male DNA in mixtures of 2.5 aM male and female genomic DNAs with no preliminary amplification of the DNA target sequence, thus establishing an analytical protocol that does not require costly, time-consuming, and prone to sample contamination PCR-based procedures. Afterward, the developed protocol was successfully applied to reveal male cell-free fetal DNA in the plasma of pregnant women at different gestational ages, including early gestational ages. This approach would pave the way for the establishment of faster and cost-effective non-invasive prenatal testing.

Recent Advances in Antifouling Materials for Surface Plasmon Resonance Biosensing in Clinical Diagnostics and Food Safety.

Strategies to develop antifouling surface coatings are crucial for surface plasmon resonance (SPR) sensing in many analytical application fields, such as detecting human disease biomarkers for clinical diagnostics and monitoring foodborne pathogens and toxins involved in food quality control. In this review, firstly, we provide a brief discussion with considerations about the importance of adopting appropriate antifouling materials for achieving excellent performances in biosensing for food safety and clinical diagnosis. Secondly, a non-exhaustive landscape of polymeric layers is given in the context of surface modification and the mechanism of fouling resistance. Finally, we present an overview of some selected developments in SPR sensing, emphasizing applications of antifouling materials and progress to overcome the challenges related to the detection of targets in complex matrices relevant for diagnosis and food biosensing.

Detection of Tumor DNA in Human Plasma with a Functional PLL-Based Surface Layer and Plasmonic Biosensing.

Standard protocols for the analysis of circulating tumor DNA (ctDNA) include the isolation of DNA from the patient's plasma and its amplification and analysis in buffered solutions. The application of such protocols is hampered by several factors, including the complexity and time-constrained preanalytical procedures, risks for sample contamination, extended analysis time, and assay costs. A recently introduced nanoparticle-enhanced surface plasmon resonance imaging-based assay has been shown to simplify procedures for the direct detection of tumor DNA in the patient's plasma, greatly simplifying the cumbersome preanalytical phase. To further simplify the protocol, a new dual-functional low-fouling poly-l-lysine (PLL)-based surface layer has been introduced that is described herein. The new PLL-based layer includes a densely immobilized CEEEEE oligopeptide to create a charge-balanced system preventing the nonspecific adsorption of plasma components on the sensor surface. The layer also comprises sparsely attached peptide nucleic acid probes complementary to the sequence of circulating DNA, e.g., the analyte that has to be captured in the plasma from cancer patients. We thoroughly investigated the contribution of each component of the dual-functional polymer to the antifouling properties of the surface layer. The low-fouling property of the new surface layer allowed us to detect wild-type and KRAS p.G12D-mutated DNA in human plasma at the attomolar level (∼2.5 aM) and KRAS p.G13D-mutated tumor DNA in liquid biopsy from a cancer patient with almost no preanalytical treatment of the patient's plasma, no need to isolate DNA from plasma, and without PCR amplification of the target sequence.

Label free detection of miRNA-21 with electrolyte gated organic field effect transistors (EGOFETs).

We report a dual gate/common channel organic transistor architecture designed for quantifying the concentration of one of the strands of miRNA-21 in solution. The device allows one to measure the differential response between two gate electrodes, viz. one sensing and one reference, both immersed in the electrolyte above the transistor channel. Hybridization with oligonucleotide in the picomolar regime induces a sizable reduction of the current flowing through the transistor channel. The device signal is reported at various gate voltages, showing maximum sensitivity in the sublinear regime, with a limit of detection as low as 35 pM. We describe the dose curves with an analytical function derived from a thermodynamic model of the reaction equilibria relevant in our experiment and device configuration, and we show that the apparent Hill dependence on analyte concentration, whose exponent lies between 0.5 and 1, emerges from the interplay of the different equilibria. The binding free energy characteristic of the hybridization on the device surface is found to be approximately 20% lower with respect to the reaction in solution, hinting to partially inhibiting effect of the surface and presence of competing reactions. Impedance spectroscopy and surface plasmon resonance (SPR) performed on the same oligonucleotide pair were correlated to the electronic current transduced by the EGOFET, and confirmed the selectivity of the biorecognition probe covalently bound on the gold surface.

Novel nucleic acid origami structures and conventional molecular beacon-based platforms: a comparison in biosensing applications.

Nucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami.

A new ultralow fouling surface for the analysis of human plasma samples with surface plasmon resonance.

Surface plasmon resonance (SPR) has been widely used to detect a variety of biomolecular systems, but only a small fraction of applications report on the analysis of patients' samples. A critical barrier to the full implementation of SPR technology in molecular diagnostics currently exists for its potential application to analyze blood plasma or serum samples. Such capability is mostly hindered by the non-specific adsorption of interfering species present in the biological sample at the functional interface of the biosensor, often referred to as fouling. Suitable polymeric layers having a thickness ranging from 15 and about 70 nm are usually deposited on the active surface of biosensors to introduce antifouling properties. A similar approach is not fully adequate for SPR detection where the exponential decay of the evanescent plasmonic field limits the thickness of the layer beyond the SPR metallic sensor surface for which a sensitive detection can be obtained. Here, a triethylene glycol (PEG(3))-pentrimer carboxybetaine system is proposed to fabricate a new surface coating bearing excellent antifouling properties with a thickness of less than 2 nm, thus compatible with sensitive SPR detection. The high variability of experimental conditions described in the literature for the quantitative assessment of the antifouling performances of surface layers moved us to compare the superior antifouling capacity of the new pentrimeric system with that of 4-aminophenylphosphorylcholine, PEG-carboxybetaine and sulfobetaine-modified surface layers, respectively, using undiluted and diluted pooled human plasma samples. The use of the new coating for the immunologic SPRI biosensing of human arginase 1 in plasma is also presented.

Direct plasmonic detection of circulating RAS mutated DNA in colorectal cancer patients.

RAS mutations in the blood of colorectal cancer (CRC) patients are emerging as biomarkers of acquired resistance to Epidermal Growth Factor Receptor therapy. Unfortunately, reliable assays granting fast, real-time monitoring of treatment response, capable of refining retrospective, tissue-based analysis, are still needed. Recently, several methods for detecting blood RAS mutations have been proposed, generally relying on multi-step and PCR-based, time-consuming and cost-ineffective procedures. By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting ~1 aM RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The assay does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 µL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS mutations are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. The assay was proven in plasma from CRC patients and healthy donors, and full discrimination between mutated DNA from patients over wild-type DNA from healthy volunteers was obtained thus demonstrating its promising avenue for cancer monitoring based on liquid biopsy.

Surface Plasmon Resonance for Biomarker Detection: Advances in Non-invasive Cancer Diagnosis.

Biomarker-based cancer analysis has great potential to lead to a better understanding of disease at the molecular level and to improve early diagnosis and monitoring. Unlike conventional tissue biopsy, liquid biopsy allows the detection of a large variety of circulating biomarkers, such as microRNA (miRNA), exosomes, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and proteins, in an easily accessible and minimally invasive way. In this review, we describe and evaluate the relevance and applicability of surface plasmon resonance (SPR) and localized SPR (LSPR)-based platforms for the detection of different classes of cancer biomarkers in liquid biopsy samples. Firstly, we critically discuss unsolved problems and issues in capturing and analyzing biomarkers. Secondly, we highlight current challenges which need to be resolved in applying SPR biosensors into clinical practice. Then, we mainly focus on applications of SPR-based platforms that process a patient sample aiming to detect and quantify biomarkers as a minimally invasive liquid biopsy tool for cancer patients appearing over the last 5 years. Finally, we describe the analytical performances of selected SPR biosensor assays and their significant advantages in terms of high sensitivity and specificity as well as accuracy and workflow simplicity.

Advanced methods for microRNA biosensing: a problem-solving perspective.

MicroRNAs (miRNAs) present several features that make them more difficult to analyze than DNA and RNA. For this reason, efforts have been made in recent years to develop innovative platforms for the efficient detection of microRNAs. The aim of this review is to provide an overview of the sensing strategies able to deal with drawbacks and pitfalls related to microRNA detection. With a critical perspective of the field, we identify the main challenges to be overcome in microRNA sensing, and describe the areas where several innovative approaches are likely to come for managing those issues that put limits on improvement to the performances of the current methods. Then, in the following sections, we critically discuss the contribution of the most promising approaches based on the peculiar properties of nanomaterials or nanostructures and other hybrid strategies which are envisaged to support the adoption of these new methods useful for the detection of miRNA as biomarkers of practical clinical utility. Graphical abstract ᅟ.

Peptide Nucleic Acid-Based Biosensors for Cancer Diagnosis.

The monitoring of DNA and RNA biomarkers freely circulating in the blood constitutes the basis of innovative cancer detection methods based on liquid biopsy. Such methods are expected to provide new opportunities for a better understanding of cancer disease at the molecular level, thus contributing to improved patient outcomes. Advanced biosensors can advance possibilities for cancer-related nucleic acid biomarkers detection. In this context, peptide nucleic acids (PNAs) play an important role in the fabrication of highly sensitive biosensors. This review provides an overview of recently described PNA-based biosensors for cancer biomarker detection. One of the most striking features of the described detection approaches is represented by the possibility to detect target nucleic acids at the ultra-low concentration with the capability to identify single-base mutations.

Droplet Microfluidic Device Fabrication and Use for Isothermal Amplification and Detection of MicroRNA.

Droplet microfluidics combined with the isothermal circular strand displacement polymerization (ICSDP) represents a powerful new technique to detect both single-stranded DNA and microRNA sequences. The method here described helps in overcoming some drawbacks of the lately introduced droplet polymerase chain reaction (PCR) amplification when implemented in microfluidic devices. The method also allows the detection of nanoliter droplets of nucleic acids sequences solutions, with a particular attention to microRNA sequences that are detected at the picomolar level. The integration of the ICSDP amplification protocol in droplet microfluidic devices reduces the time of analysis and the amount of sample required. In addition, there is also the possibility to design parallel analyses to be integrated in portable devices.

Atmospheric pressure MALDI for the noninvasive characterization of carbonaceous ink from Renaissance documents.

The analytical methods that are usually applied to determine the compositions of inks from ancient manuscripts usually focus on inorganic components, as in the case of iron gall ink. In this work, we describe the use of atmospheric pressure/matrix-assisted laser desorption ionization-mass spectrometry (AP/MALDI-MS) as a spatially resolved analytical technique for the study of the organic carbonaceous components of inks used in handwritten parts of ancient books for the first time. Large polycyclic aromatic hydrocarbons (L-PAH) were identified in situ in the ink of XVII century handwritten documents. We prove that it is possible to apply MALDI-MS as a suitable microdestructive diagnostic tool for analyzing samples in air at atmospheric pressure, thus simplifying investigations of the organic components of artistic and archaeological objects. The interpretation of the experimental MS results was supported by independent Raman spectroscopic investigations. Graphical abstract Atmospheric pressure/MALDI mass spectrometry detects in situ polycyclic aromatic hydrocarbons in the carbonaceous ink of XVII century manuscripts.

Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation.

Gold nanoparticles (AuNPs) exhibit unique properties that can be modulated through a tailored surface functionalization, enabling their targeted use in biochemical sensing and medical diagnostics. In particular, streptavidin-modified AuNPs are increasingly used for biosensing purposes. We report here a study of AuNPs surface-functionalized with streptavidin-biotinylated oligonucleotide, focussing on the role played by the oligonucleotide probes in the stabilization/destabilization of the functionalized nanoparticle dispersion. The behaviour of the modified AuNP dispersion as a consequence of the competitive displacement of the biotinylated oligonucleotide has been investigated and the critical role of displaced oligonucletides in triggering the quasi one-dimensional aggregation of nanoparticles is demonstrated for the first time. The thorough understanding of the fundamental properties of bioconjugated AuNPs is of great importance for the design of highly sensitive and reliable functionalized AuNP-based assays.

Cyclam glycoconjugates as lectin ligands and protective agents of metal-induced amyloid aggregation.

Transition metal ion complexes of a number of chelators have been widely investigated due to their biological properties. The sugar conjugation of metal complexes has resulted in improved properties of the systems, such as solubility and lectin recognition. In this paper, we report the synthesis, the characterization of new glucose and galactose conjugates of 1,4,8,11-tetraazacyclotetradecane (cyclam) and their Cu(II) complexes. The glycoconjugates were proved to be protective agents of metal-induced amyloid aggregation. The binding constants of the galactose conjugate KA1=(3.07±0.16)×10(7) and KA2=(2.13±0.04)×10(7)M(-1) with the lectin Ricinus communis agglutinin (RCA120) as a model of galactose-specific human lectin were obtained with surface plasmon resonance.

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

Peptide nucleic acid molecular beacons for the detection of PCR amplicons in droplet-based microfluidic devices.

The use of droplet-based microfluidics and peptide nucleic acid molecular beacons for the detection of polymerase chain reaction (PCR)-amplified DNA sequences within nanoliter-sized droplets is described in this work. The nanomolar-attomolar detection capabilities of the method were preliminarily tested by targeting two different single-stranded DNA sequences from the genetically modified Roundup Ready soybean and the Olea europaea genomes and detecting the fluorescence generated by peptide nucleic acid molecular beacons with fluorescence microscopy. Furthermore, the detection of 10 nM solutions of PCR amplicon of DNA extracted from leaves of O. europaea L. encapsulated in nanoliter-sized droplets was performed to demonstrate that peptide nucleic acid molecular beacons can discriminate O. europaea L. cultivar species carrying different single-nucleotide polymorphisms.

Surface plasmon resonance imaging for nucleic acid detection.

Surface plasmon resonance imaging (SPRI) is a powerful tool for simple, fast and cheap nucleic acid detection. Great efforts have been made during the last decade with the aim of developing even more sensitive and specific SPRI-based methods to be used for the direct detection of DNA and RNA. Here, after a description of the fundamentals of SPRI, the state of the art of recent platform and assay developments is presented, with special attention given to advances in SPRI signal enhancement procedures.