MAGPIX and FLEXMAP 3D Luminex platforms for direct detection of miR-122-5p through dynamic chemical labelling.

MicroRNAs (miRs) have emerged as promising biomarkers for diagnosing and predicting the prognosis of liver injury. This study aimed to compare the performance of two Luminex platforms, MAGPIX and FLEXMAP 3D, utilizing the innovative Dynamic Chemical Labelling (DCL) technology for direct detection and analysis of miR-122-5p in serum samples from patients with liver injury. Serum samples were collected from four patients with liver injury and four healthy controls. The levels of miR-122-5p were measured using the DCL method on both MAGPIX and FLEXMAP 3D platforms. The performance evaluation included the limit of detection (LOD), intra-assay and inter-assay precision, as well as accuracy. The results demonstrated that both platforms exhibited high sensitivity and specificity in detecting miR-122-5p in serum samples from patients with liver injury. However, FLEXMAP 3D indicated a lower LOD compared to MAGPIX. The precision of miR-122-5p detection was similar between the two platforms. In conclusion, both MAGPIX and FLEXMAP 3D Luminex platforms, in conjunction with DCL reagents, proved to be reliable and sensitive tools for detecting miR-122-5p in serum samples from patients with liver injury. Although both platforms were effective, FLEXMAP 3D exhibited slightly better performance, suggesting its preference for miR detection in clinical settings. These findings offer valuable insights for selecting the appropriate Luminex platform for miR detection in patients with liver injury and beyond.

Direct Detection of miR-122 in Hepatotoxicity Using Dynamic Chemical Labeling Overcomes Stability and isomiR Challenges.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.

New Platform for the Direct Profiling of microRNAs in Biofluids.

Circulating microRNAs have been identified as potential biomarkers for early detection, prognosis, and prediction of several diseases. Their use in clinical diagnostics has been limited by the lack of suitable detection techniques. Most of the current technologies suffer from requiring complex protocols, not yet able to deliver robust and cost-effective assays in the field of clinical diagnostics. In this work, we report the development of a breakthrough platform for profiling circulating microRNAs. The platform comprises a novel silicon photomultiplier-based reader in conjunction with a chemical-based method for nucleic acid detection. Accurate microRNAs profiling without extraction, pre-amplification, or pre-labeling of the target is now achievable. We designed and synthesized a set of reagents that combined the chemical-based method with a chemiluminescent reaction. The signals generated were read out using a novel, compact silicon photomultiplier-based reader. The platform sensitivity was determined by measuring known concentrations of hsa-miR-21-5p spike-ins. The limit of detection was calculated as 4.7 pmol/L. The platform was also successfully used to directly detect hsa-miR-21-5p in eight non-small cell lung cancer plasma samples. Levels of plasma hsa-miR-21-5p expression were also measured via TaqMan RT-qPCR. The successful integration of the unique chemical-based method for nucleic acid detection with the novel silicon photomultiplier-based reader created an innovative product (ODG platform) with diagnostic utility, for the direct qualitative and quantitative analysis of microRNA biomarkers in biological fluids.

A PCR-free technology to detect and quantify microRNAs directly from human plasma.

A novel sensitive, specific and rapid method for the detection and quantification of microRNAs without requiring extraction from their biological sources is now available using a novel chemical based, PCR-free technology for nucleic acid testing. In this study, we both demonstrate how this method can be used to profile miR-451a, an important miRNA in erythropoiesis, and compare with the gold standard RT-qPCR.

A comprehensive review of Dynamic Chemical Labelling on Luminex xMAP technology: a journey towards Drug-Induced Liver Injury testing.

Drug-Induced Liver Injury (DILI) is a grave global adverse event that can result in fatal consequences, causing drug failures, market withdrawals, and regulatory warnings, leading to substantial financial losses. The early detection of DILI remains a significant challenge in global healthcare. Although circulating microRNAs (miRs) show promise as clinical biomarkers for DILI, the current analytical methods for their measurement are insufficient. There is a pressing need for rapid and reliable miR detection methods that eliminate the need for nucleic acid extraction and PCR-based amplification. This review highlights recent advancements achieved by integrating Dynamic Chemical Labelling (DCL) with Luminex xMAP technology. This powerful combination has resulted in groundbreaking bead-based assays that allow (1) the direct, multiplex detection of miRs, and (2) the simultaneous testing of miR and protein biomarkers. This triple capability enables a comprehensive assessment that significantly enhances the detection and analysis of crucial biomarkers, thus improving the understanding and diagnosis of DILI. In conclusion, this review offers valuable insights into the capabilities and potential applications of these groundbreaking assays in DILI research, as well as their potential use in other diagnostic and research domains that require direct or multiplex analysis of miRs or analysis of miRs in combination with proteins.

Direct detection of alpha satellite DNA with single-base resolution by using abasic Peptide Nucleic Acids and Fluorescent in situ Hybridization.

The detection of repetitive sequences with single-base resolution is becoming increasingly important aiming to understand the biological implications of genomic variation in these sequences. However, there is a lack of techniques to experimentally validate sequencing data from repetitive sequences obtained by Next-Generation Sequencing methods, especially in the case of Single-Nucleotide Variations (SNVs). That is one of the reasons why repetitive sequences have been poorly studied and excluded from most genomic studies. Therefore, in addition to sequencing data, there is an urgent need for efficient validation methods of genomic variation in these sequences. Herein we report the development of chemFISH, an alternative method for the detection of SNVs in repetitive sequences. ChemFISH is an innovative method based on dynamic chemistry labelling and abasic Peptide Nucleic Acid (PNA) probes to detect in situ the α-satellite DNA, organized in tandem repeats, with single-base resolution in a direct and rapid reaction. With this approach, we detected by microscopy the α-satellite DNA in a variety of human cell lines, we quantified the detection showing a low coefficient of variation among samples (13.16%-25.33%) and we detected single-base specificity with high sensitivity (82.41%-88.82%). These results indicate that chemFISH can serve as a rapid method to validate previously detected SNVs in sequencing data, as well as to find novel SNVs in repetitive sequences. Furthermore, the versatile chemistry behind chemFISH can lead to develop novel molecular assays for the in situ detection of nucleic acids.

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app.

The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

Novel biochip platform for nucleic acid analysis.

This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top "footprint" requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.

Amplification-free profiling of microRNA-122 biomarker in DILI patient serums, using the luminex MAGPIX system.

Dynamic chemical labelling is a single-base specific method to enable detection and quantification of micro-Ribonucleic Acids in biological fluids without extraction and pre-amplification. In this study, dynamic chemical labelling was combined with the Luminex MAGPIX system to profile levels of microRNA-122 biomarker in serum from patients with Drug-Induced Liver Injury.

A cooperative polymer-DNA microarray approach to biomaterial investigation.

In this study, polymer microarrays were used for the rapid identification of polymer substrates upon which a suspension cell line would both adhere and proliferate giving a detailed and rapid understanding of cell-biomaterial interactions. Analysis demonstrated that suspension K562 human erythroleukemic cells, which normally grow in suspension, adhered and proliferated on several different polymers. Phenotypic and transcriptomic analysis techniques allowed examination of the interaction between cells and polymers permitting the elucidation of putative links between phenotypic responses to cell-biomaterial interactions and global gene expression.

A colorimetric strategy based on dynamic chemistry for direct detection of Trypanosomatid species.

Leishmaniasis and Chagas disease are endemic in many countries, and re-emerging in the developed countries. A rapid and accurate diagnosis is important for early treatment for reducing the duration of infection as well as for preventing further potential health complications. In this work, we have developed a novel colorimetric molecular assay that integrates nucleic acid analysis by dynamic chemistry (ChemNAT) with reverse dot-blot hybridization in an array format for a rapid and easy discrimination of Leishmania major and Trypanosoma cruzi. The assay consists of a singleplex PCR step that amplifies a highly homologous DNA sequence which encodes for the RNA component of the large ribosome subunit. The amplicons of the two different parasites differ between them by single nucleotide variations, known as "Single Nucleotide Fingerprint" (SNF) markers. The SNF markers can be easily identified by naked eye using a novel micro Spin-Tube device "Spin-Tube", as each of them creates a specific spot pattern. Moreover, the direct use of ribosomal RNA without requiring the PCR pre-amplification step is also feasible, further increasing the simplicity of the assay. The molecular assay delivers sensitivity capable of identifying up to 8.7 copies per µL with single mismatch specificity. The Spin-Tube thus represents an innovative solution providing benefits in terms of time, cost, and simplicity, all of which are crucial for the diagnosis of infectious disease in developing countries.

miR-122 direct detection in human serum by time-gated fluorescence imaging.

A simple method for direct detection of microRNAs (miRs) in human serum without the use of polymerase amplification is presented, achieving low miR-122 concentrations and importantly, discerning effectively single-base sequence mutations. The method is based on the capture of target miRs with synthetic peptide nucleic acid oligomers, dynamic chemical labelling, separation with quaternary amine microplatforms and detection using time-gated fluorescence imaging.

A soft 3D polyacrylate hydrogel recapitulates the cartilage niche and allows growth-factor free tissue engineering of human articular cartilage.

Cartilage degeneration or damage treatment is still a challenge, but, tissue engineering strategies, which combine cell therapy strategies, which combine cell therapy and scaffolds, and have emerged as a promising new approach. In this regard, polyurethanes and polyacrylates polymers have been shown to have clinical potential to treat osteochondral injuries. Here, we have used polymer microarrays technology to screen 380 different polyurethanes and polyacrylates polymers. The top polymers with potential to maintain chondrocyte viability were selected, with scale-up studies performed to evaluate their ability to support chondrocyte proliferation during long-term culture, while maintaining their characteristic phenotype. Among the selected polymers, poly (methylmethacrylate-co-methacrylic acid), showed the highest level of chondrogenic potential and was used to create a 3D hydrogel. Ultrastructural morphology, microstructure and mechanical testing of this novel hydrogel revealed robust characteristics to support chondrocyte growth. Furthermore, in vitro and in vivo biological assays demonstrated that chondrocytes cultured on the hydrogel had the capacity to produce extracellular matrix similar to hyaline cartilage, as shown by increased expression of collagen type II, aggrecan and Sox9, and the reduced expression of the fibrotic marker's collagen type I. In conclusion, hydrogels generated from poly (methylmethacrylate-co-methacrylic acid) created the appropriate niche for chondrocyte growth and phenotype maintenance and might be an optimal candidate for cartilage tissue-engineering applications. SIGNIFICANCE STATEMENT: Articular cartilage has limited self-repair ability due to its avascular nature, therefore tissue engineering strategies have emerged as a promising new approach. Synthetic polymers displaygreat potential and are widely used in the clinical setting. In our study, using the polymer microarray technique a novel type of synthetic polyacrylate was identified, that was converted into hydrogels for articular cartilage regeneration studies. The hydrogel based on poly (methylmethacrylate-co-methacrylic acid-co-PEG-diacrylate) had a controlable ultrastructural morphology, microstructure (porosity) and mechanical properties (stiffness) appropriate for cartilage engineering. Our hydrogel created the optimal niche for chondrocyte growth and phenotype maintenance for long-term culture, producing a hyaline-like cartilage extracellular matrix. We propose that this novel polyacrylate hydrogel could be an appropriate support to help in the treatment efficient cartilage regeneration.

PCR-free and chemistry-based technology for miR-21 rapid detection directly from tumour cells.

miRNAs are well known for being implicated in a myriad of biological situations, including those related to serious diseases. Amongst miRNAs, miRNA-21 has the spotlight as it is reported to be up-regulated in multiple severe pathological conditions, being its quantification a key point in medicine. To date, most of the techniques for miRNA quantification have shown to be less effective than expected; thus, we herein present a novel, rapid, cost-effective, robust and PCR-free approach, based on dynamic chemistry, for the identification and quantification of miRNA directly from tumour cells using both FACS and a fluorescent microplate. This dynamic chemistry novel application involves bead based reagents and allows quantifying the number of miR-21 molecules presented in MDA-MB-468 and H1975 tumour cells.

Identification of Trypanosomatids by detecting Single Nucleotide Fingerprints using DNA analysis by dynamic chemistry with MALDI-ToF.

Protozoan parasites of the Trypanosomatidae family can cause devastating diseases in humans and animals, such as Human African Trypanosomiasis or Sleeping Sickness, Chagas disease and Leishmaniasis. Currently, there are molecular assays for detecting parasitic infections and their post-treatment monitoring based on nucleic acid amplification, but there are still certain limitations which limit the development of assays that can detect and discriminate between parasite infections with a single test. Here, we present the development of a novel molecular assay for the rapid identification of Trypanosomatids, integrating DNA analysis by dynamic chemistry in conjunction with Matrix-Assisted Laser Desorption Ionization - Time-of-Flight Mass Spectrometry (MALDI-ToF). Differentiation of Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp. is now possible using a single reaction tube, and enables rapid identification of Trypanosomatids. The test is based on a singleplex PCR, using a specific primer pair that amplifies a 155 base pair segment of the 28S ribosomal RNA gene, within a conserved homology region of Trypanosomatidae species. Amplified fragments are analysed by dynamic chemistry using two abasic PNA probes and the four reactive nucleobases - containing an aldehyde functional group - with MALDI-ToF to identify unique molecular patterns created by each specie due to their single base differences (Single Nucleotide Fingerprint 'SNF') in this highly homologous region. This novel assay offers the possibility to expand routine diagnostic testing for Trypanosomatids, and monitoring of therapeutic responses to these infectious diseases.

Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury.

We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA) probe-containing a reactive amine instead of a nucleotide at a specific position in the sequence-for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase-containing an aldehyde group-that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-ß-galactosidase (SßG), the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122)-an established biomarker of liver toxicity-extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use.

Poly(ethylmethacrylate-co-diethylaminoethyl acrylate) coating improves endothelial re-population, bio-mechanical and anti-thrombogenic properties of decellularized carotid arteries for blood vessel replacement.

Decellularized vascular scaffolds are promising materials for vessel replacements. However, despite the natural origin of decellularized vessels, issues such as biomechanical incompatibility, immunogenicity risks and the hazards of thrombus formation, still need to be addressed. In this study, we coated decellularized vessels obtained from porcine carotid arteries with poly (ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) with the purpose of improving endothelial coverage and minimizing platelet attachment while enhancing the mechanical properties of the decellularized vascular scaffolds. The polymer facilitated binding of endothelial cells (ECs) with high affinity and also induced endothelial cell capillary tube formation. In addition, platelets showed reduced adhesion on the polymer under flow conditions. Moreover, the coating of the decellularized arteries improved biomechanical properties by increasing its tensile strength and load. In addition, after 5 days in culture, ECs seeded on the luminal surface of 8g7-coated decellularized arteries showed good regeneration of the endothelium. Overall, this study shows that polymer coating of decellularized vessels provides a new strategy to improve re-endothelialization of vascular grafts, maintaining or enhancing mechanical properties while reducing the risk of thrombogenesis. These results could have potential applications in improving tissue-engineered vascular grafts for cardiovascular therapies with small caliber vessels.

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

Novel bead-based platform for direct detection of unlabelled nucleic acids through Single Nucleobase Labelling.

Over the last decade, circulating microRNAs have received attention as diagnostic and prognostic biomarkers. In particular, microRNA122 has been demonstrated to be an early and more sensitive indicator of drug-induced liver injury than the widely used biomarkers such as alanine aminotransferase and aspartate aminotransferase. Recently, microRNA122 has been used in vitro to assess the cellular toxicity of new drugs and as a biomarker for the development of a rapid test for drug overdose/liver damage. In this proof-of-concept study, we report a PCR-free and label-free detection method that has a limit of detection (3 standard deviations) of 15 fmoles of microRNA122, by integrating a dynamic chemical approach for "Single Nucleobase Labelling" with a bead-based platform (Luminex®) thereby, in principle, demonstrating the exciting prospect of rapid and accurate profiling of any microRNAs related to diseases and toxicology.

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.