RESUMEN
BACKGROUND: Among mechanoporation techniques for intracellular delivery, microfluidic approaches succeed in high delivery efficiency and throughput. However, especially the entry of large cargoes (e.g. DNA origami, mRNAs, organic/inorganic nanoparticles) is currently impaired since it requires large cell membrane pores with the need to apply multi-step processes and high forces, dramatically reducing cell viability. RESULTS: Here, HiViPore presents as a microfluidic viscoelastic contactless compression for one-step cell mechanoporation to produce large pores while preserving high cell viability. Inducing an increase of curvature at the equatorial region of cells, formation of a pore with a size of ~ 1 µm is obtained. The poration is coupled to an increase of membrane tension, measured as a raised fluorescence lifetime of 12% of a planarizable push-pull fluorescent probe (Flipper-TR) labelling the cell plasma membrane. Importantly, the local disruptions of cell membrane are transient and non-invasive, with a complete recovery of cell integrity and functions in ~ 10 min. As result, HiViPore guarantees cell viability as high as ~ 90%. In such conditions, an endocytic-free diffusion of large nanoparticles is obtained with typical size up to 500 nm and with a delivery efficiency up to 12 times higher than not-treated cells. CONCLUSIONS: The proposed one-step contactless mechanoporation results in an efficient and safe approach for advancing intracellular delivery strategies. In detail, HiViPore solves the issues of low cell viability when multiple steps of poration are required to obtain large pores across the cell plasma membrane. Moreover, the compression uses a versatile, low-cost, biocompatible viscoelastic fluid, thus also optimizing the operational costs. With HiViPore, we aim to propose an easy-to-use microfluidic device to a wide range of users, involved in biomedical research, imaging techniques and nanotechnology for intracellular delivery applications in cell engineering.
Asunto(s)
Membrana Celular , Supervivencia Celular , Microfluídica , Nanopartículas , Nanopartículas/química , Humanos , Microfluídica/métodos , Membrana Celular/metabolismo , Sistemas de Liberación de Medicamentos/métodosRESUMEN
In this work, we compare the role of two different uptake mechanisms in the effectiveness of a nanoformulated drug, specifically insulin. Insulin is activated by interacting with insulin receptors exposed on the liver cell membrane that triggers the uptake and storage of glucose. To prove that the uptake mechanism of a delivery system can interfere directly with the effectiveness of the delivered drug, two extremely different delivery systems are tested. In detail, hydrogel-based NPs (cHANPs) and natural lipid vesicles (EVs) encapsulating insulin are used to trigger the activation of this hormone in 3D liver microtissues (µTs) based on their different uptake mechanisms. Results demonstrated that the fusion mechanism of Ins-EVs mediates faster and more pronounced insulin activation with respect to the endocytic mechanism of Ins-cHANPs. Indeed, the fusion causes an increased reduction in glucose concentration in the culture medium EV-treated l-µTs with respect to free insulin-treated tissues. The same effect is not observed for Ins-cHANPs that, taken up by endocytosis, can only equal the reduction in glucose concentration produced by free insulin in 48 h. Overall, these results demonstrate that the effectiveness of nanoformulated drugs depends on the identity they acquire in the biological context (biological identity). Indeed, the nanoparticle (NP) biological identity, such as the uptake mechanism, triggers a unique set of nano-bio-interactions that is ultimately responsible for their fate both in the extracellular and intracellular compartments.
Asunto(s)
Vesículas Extracelulares , Nanopartículas , Insulina , Polímeros/metabolismo , Vesículas Extracelulares/metabolismo , Nanopartículas/metabolismo , Hígado , Glucosa/metabolismoRESUMEN
Current 3D cancer models (in vitro) fail to reproduce complex cancer cell extracellular matrices (ECMs) and the interrelationships occurring (in vivo) in the tumor microenvironment (TME). Herein, we propose 3D in vitro colorectal cancer microtissues (3D CRC µTs), which reproduce the TME more faithfully in vitro. Normal human fibroblasts were seeded onto porous biodegradable gelatin microbeads (GPMs) and were continuously induced to synthesize and assemble their own ECMs (3D Stroma µTs) in a spinner flask bioreactor. Then, human colon cancer cells were dynamically seeded onto the 3D Stroma µTs to achieve the 3D CRC µTs. Morphological characterization of the 3D CRC µTs was performed to assess the presence of different complex macromolecular components that feature in vivo in the ECM. The results showed the 3D CRC µTs recapitulated the TME in terms of ECM remodeling, cell growth, and the activation of normal fibroblasts toward an activated phenotype. Then, the microtissues were assessed as a drug screening platform by evaluating the effect of 5-Fluorouracil (5-FU), curcumin-loaded nanoemulsions (CT-NE-Curc), and the combination of the two. When taken together, the results showed that our microtissues are promising in that they can help clarify complex cancer-ECM interactions and evaluate the efficacy of therapies. Moreover, they may be combined with tissue-on-chip technologies aimed at addressing further studies in cancer progression and drug discovery.
Asunto(s)
Neoplasias del Colon , Matriz Extracelular , Humanos , Sistemas de Liberación de Medicamentos , Fluorouracilo/farmacología , Microambiente TumoralRESUMEN
Supramolecular assemblies of short peptides are experiencing a stimulating flowering. Herein, we report a novel class of bioinspired pentapeptides, not bearing Phe, that form hydrogels with fibrillar structures. The inherent sequence comes from the fragment 269-273 of nucleophosmin 1 protein, that is normally involved in liquid-liquid phase separation processes into the nucleolus. By means of rheology, spectroscopy, and scanning microscopy the crucial roles of the extremities in the modulation of the mechanical properties of hydrogels were elucidated. Three of four peptide showed a typical shear-thinning profile and a self-assembly into hierarchical nanostructures fibers and two of them resulted biocompatible in MCF7 cells. The presence of an amide group at C-terminal extremity caused the fastest aggregation and the major content of structured intermediates during gelling process. The tunable mechanical and structural features of this class of hydrogels render derived supramolecular systems versatile and suitable for future biomedical applications.
Asunto(s)
Nanoestructuras , Péptidos , Péptidos/química , Hidrogeles/química , Nanoestructuras/química , Reología , ProteínasRESUMEN
The growth of (bio)sensors in analytical chemistry is mainly attributable to the development of affordable, effective, portable, and user-friendly analytical tools. In the field of sensors, paper-based devices are gaining a relevant position for their outstanding features including foldability, ease of use, and instrument-free microfluidics. Herein, a multifarious use of filter paper to detect copper ions in bodily fluids is reported by exploiting this eco-friendly material to (i) synthesize AuNPs without the use of reductants and/or external stimuli, (ii) print the electrodes, (iii) load the reagents for the assay, (iv) filter the gross impurities, and (v) preconcentrate the target analyte. Copper ions were detected down to 3 ppb with a linearity up to 400 ppb in standard solutions. The applicability in biological matrices, namely, sweat and serum, was demonstrated by recovery studies and by analyzing these biofluids with the paper-based platform and the reference method (atomic absorption spectroscopy), demonstrating satisfactory accuracy of the novel eco-designed analytical tool.
Asunto(s)
Técnicas Biosensibles , Nanopartículas del Metal , Cobre , Oro , Iones , SudorRESUMEN
The development of assays for protein biomarkers in complex matrices is a demanding task that still needs implementation of new approaches. Antibodies as capture agents have been largely used in bioassays but their low stability, low-efficiency production, and cross-reactivity in multiplex approaches impairs their larger applications. Instead, synthetic peptides, even with higher stability and easily adapted amino acid sequences, still remain largely unexplored in this field. Here, we provide a proof-of-concept of a microfluidic device for direct detection of biomarker overexpression. The multichannel microfluidic polydimethylsiloxane (PDMS) device was first derivatized with PAA (poly(acrylic acid)) solution. CRP-1, VEGF-114, and ΦG6 peptides were preliminarily tested to respectively bind the biomarkers, C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α). Each PDMS microchannel was then respectively bioconjugated with a specific peptide (CRP-1, VEGF-114, or ΦG6) to specifically capture CRP, VEGF, and TNF-α. With such microdevices, a fluorescence bioassay has been set up with sensitivity in the nanomolar range, both in buffered solution and in human serum. The proposed multiplex assay worked with a low amount of sample (25 µL) and detected biomarker overexpression (above nM concentration), representing a noninvasive and inexpensive screening platform.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Péptidos/química , Biomarcadores/análisis , Humanos , Inflamación/diagnóstico , Dispositivos Laboratorio en un ChipRESUMEN
Peptide hydrogels, deriving from natural protein fragments, present unique advantages as compatibility and low cost of production that allow their wide application in different fields as wound healing, cell delivery and tissue regeneration. To engineer new biomaterials, the change of the chirality of single amino acids demonstrated a powerful approach to modulate the self-assembly mechanism. Recently we unveiled that a small stretch spanning residues 268-273 in the C-terminal domain (CTD) of Nucleophosmin 1 (NPM1) is an amyloid sequence. Herein, we performed a systematic D-scan of this sequence and analyzed the structural properties of obtained peptides. The conformational and kinetic features of self-aggregates and the morphologies of derived microstructures were investigated by means of different biophysical techniques, as well as the compatibility of hydrogels was evaluated in HeLa cells. All the investigated hexapeptides formed hydrogels even if they exhibited different conformational intermediates during aggregation, and they structural featured are finely tuned by introduced chiralities.
Asunto(s)
Hidrogeles/química , Nucleofosmina/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Hidrogeles/toxicidad , Nucleofosmina/toxicidad , Oligopéptidos/toxicidad , Fragmentos de Péptidos/toxicidad , Multimerización de Proteína , EstereoisomerismoRESUMEN
Herein we describe the development of a mix-read bioassay based on a three-dimensional (3D) poly ethylene glycol-(PEG)-hydrogel microparticles for the detection of oligonucleotides in complex media. The key steps of hydrogels synthesis and molecular recognition in a 3D polymer network are elucidated. The design of the DNA probes and their density in polymer network were opportunely optimized. Furthermore, the diffusion into the polymer was tuned adjusting the polymer concentration and consequently the characteristic mesh size. Upon parameters optimization, 3D-PEG-hydrogels were synthetized in a microfluidic system and provided with fluorescent probe. Target detection occurred by double strand displacement assay associated to fluorescence depletion within the hydrogel microparticle. Proposed 3D-PEG-hydrogel microparticles were designed for miR-143-3p detection. Results showed 3D-hydrogel microparticles with working range comprise between 10-6-10-12 M, had limit of detection of 30 pM and good specificity. Moreover, due to the anti-fouling properties of PEG-hydrogel, the target detection occurred in human serum with performance comparable to that in buffer. Due to the approach versatility, such design could be easily adapted to other short oligonucleotides detection.
Asunto(s)
Hidrogeles , MicroARNs , Bioensayo , Sondas de ADN , Humanos , MicroARNs/genética , PolietilenglicolesRESUMEN
In recent decades, endogenous nanocarrier-exosomes have received considerable scientific interest as drug delivery systems. The unique proteo-lipid architecture allows the crossing of various natural barriers and protects exosomes cargo from degradation in the bloodstream. However, the presence of this bilayer membrane as well as their endogenous content make loading of exogenous molecules challenging. In the present work, we will investigate how to promote the manipulation of vesicles curvature by a high-pressure microfluidic system as a ground-breaking method for exosomes encapsulation. Exosomes isolated from Uppsala 87 Malignant Glioma (U87-MG) cell culture media were characterized before and after the treatment with high-pressure homogenization. Once their structural and biological stability were validated, we applied this novel method for the encapsulation in the lipidic exosomal bilayer of the chemotherapeutic Irinotecan HCl Trihydrate-CPT 11. Finally, we performed in vitro preliminary test to validate the nanobiointeraction of exosomes, uptake mechanisms, and cytotoxic effect in cell culture model.
Asunto(s)
Antineoplásicos/química , Sistemas de Liberación de Medicamentos , Exosomas/química , Glioma/tratamiento farmacológico , Antineoplásicos/farmacología , Medios de Cultivo/química , Glioma/patología , Ensayos Analíticos de Alto Rendimiento , Humanos , Microfluídica , Medicina de PrecisiónRESUMEN
The investigation of conformational features of regions of amyloidogenic proteins are of great interest to deepen the structural changes and consequent self-aggregation mechanisms at the basis of many neurodegenerative diseases. Here we explore the effect of ß-hairpin inducing motifs on regions of prion protein covering strands S1 and S2. In detail, we unveiled the structural and functional features of two model chimeric peptides in which natural sequences are covalently linked together by two dipeptides (l-Pro-Gly and d-Pro-Gly) that are known to differently enhance ß-hairpin conformations but both containing N- and the C-terminal aromatic cap motifs to further improve interactions between natural strands. Spectroscopic investigations at solution state indicate that primary assemblies of the monomers of both constructs follow different aggregativemechanisms during the self-assembly: these distinctions, evidenced by CD and ThT emission spectroscopies, reflect into great morphological differences of nanostructures and suggest that rigid ß-hairpin conformations greatly limit amyloid-like fibrillogenesis. Overall data confirm the important role exerted by the ß-structure of regions S1 and S2 during the aggregation process and lead to speculate to its persistence even in unfolding conditions.
Asunto(s)
Amiloide/química , Dipéptidos/química , Proteínas Priónicas/química , Agregado de Proteínas , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Conformación Proteica , Conformación Proteica en Lámina betaRESUMEN
Human cytomegalovirus (hCMV) infection is the leading cause of birth defects in newborns and death in immunosuppressed people. Traditional techniques require time-consuming and costly analyses, and sometimes result in false positive results; thus, a rapid and accurate detection for hCMV infection is necessary. Recently, hcmv-miR-US4-5p was selected as the biomarker for cytomegalovirus diagnosis and follow-up. Herein, we propose a bioassay based on microgels endowed with optical fluorescent oligonucleotide probes for the detection of circulating endogenous hcmv-microRNAs. In particular, a double strand probe, based on the fluorescence recovery after target capture, was conjugated on microgels and the probe density was opportunely optimised. Then, the microgels were directly mixed with the sample. The fluorescence read-out was measured as a function of target concentration at a fixed number of microgels per tube. As a bead-based assay, the performances of optical detection in terms of dynamic working range and limit of detection could be finely tuned by tuning the number of microgels per tube. The limit of detection of the assay could be tuned in the range from 39.1 fM to 156 aM by changing the microgel concentration from 50 µg mL-1 to 0.5 µg mL-1, respectively. The assay results specific for the selected target were stable over a one-year time span and they were not affected by the presence of human serum. Therefore, this bioassay based on microgels might represent a flexible platform that should be able to predict, identify and follow-up several diseases by monitoring freely circulating oligonucleotides in body fluids.
Asunto(s)
Bioensayo/métodos , Citomegalovirus/aislamiento & purificación , Colorantes Fluorescentes/química , MicroARNs/análisis , Sondas de Oligonucleótidos/química , ARN Viral/análisis , Secuencia de Bases , Infecciones por Citomegalovirus/virología , Geles , Humanos , Límite de Detección , Espectrometría de FluorescenciaRESUMEN
Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.
Asunto(s)
Linfoma de Células B/tratamiento farmacológico , Imagen Multimodal/métodos , Nanopartículas/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Nanomedicina Teranóstica , Animales , Quitosano/química , Humanos , Ácido Hialurónico/química , Linfoma de Células B/diagnóstico por imagen , Linfoma de Células B/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/química , Fragmentos de Péptidos/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Strategies to enhance the relaxometric properties of gadolinium (Gd)-based contrast agents (CAs) for magnetic resonance imaging (MRI), without the chemical modification of chelates, have recently had a strong impact on the diagnostic field. We have taken advantage of the interaction between Gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA) and the hydrogel structure of hyaluronic acid to design cross-linked hyaluronic acid nanoparticles down to 35 nm for use in MRI applications. The proposed bioformulations enable the control of the relaxometric properties of CAs, thus boosting the relaxation rate of T1. Our results led us to identify this approach as an adjustable scenario to design intravascularly injectable hydrogel nanoparticles entrapping Gd-DTPA. This approach overcomes the general drawbacks of clinically approved CAs having poor relaxivity and toxic effects.
Asunto(s)
Ácido Hialurónico/química , Imagen por Resonancia Magnética/instrumentación , Microfluídica/métodos , Nanopartículas/química , Medios de Contraste/química , Gadolinio/químicaRESUMEN
Dual imaging dramatically improves detection and early diagnosis of cancer. In this work we present an oil in water (O/W) nano-emulsion stabilized with lecithin and loaded with cobalt ferrite oxide (Co0.5Fe2.5O4) nanocubes for photo-acoustic and magnetic resonance dual imaging. The nanocarrier is responsive in in vitro photo-acoustic and magnetic resonance imaging (MRI) tests. A clear and significant time-dependent accumulation in tumor tissue is shown in in vivo photo-acoustic studies on a murine melanoma xenograft model. The proposed O/W nano-emulsion exhibits also high values of r2/r1 (ranging from 45 to 85, depending on the magnetic field) suggesting a possible use as T2 weighted image contrast agents. In addition, viability and cellular uptake studies show no significant cytotoxicity on the fibroblast cell line. We also tested the O/W nano-emulsion loaded with curcumin against melanoma cancer cells demonstrating a significant cytotoxicity and thus showing possible therapeutic effects in addition to the in vivo imaging.
Asunto(s)
Cobalto/química , Medios de Contraste , Imagen por Resonancia Magnética , Melanoma/diagnóstico por imagen , Nanopartículas/química , Técnicas Fotoacústicas , Células 3T3 , Animales , Emulsiones/química , Humanos , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Óxidos/químicaRESUMEN
Fundamental understanding of thermodynamic of phase separation plays a key role in tuning the desired features of biomedical devices. In particular, phase separation of ternary solution is of remarkable interest in processes to obtain biodegradable and biocompatible architectures applied as artificial devices to repair, replace, or support damaged tissues or organs. In these perspectives, thermally induced phase separation (TIPS) is the most widely used technique to obtained porous morphologies and, in addition, among different ternary systems, polylactic acid (PLLA)/dioxane/water has given promising results and has been largely studied. However, to increase the control of TIPS-based processes and architectures, an investigation of the basic energetic phenomena occurring during phase separation is still required. Here we propose an experimental investigation of the selected ternary system by using isothermal titration calorimetric approach at different solvent/antisolvent ratio and a thermodynamic explanation related to the polymer-solvents interactions in terms of energetic contribution to the phase separation process. Furthermore, relevant information about the phase diagrams and interaction parameters of the studied systems are furnished in terms of liquid-liquid miscibility gap. Indeed, polymer-solvents interactions are responsible for the mechanism of the phase separation process and, therefore, of the final features of the morphologies; the knowledge of such data is fundamental to control processes for the production of membranes, scaffolds and several nanostructures. The behavior of the polymer at different solvent/nonsolvent ratios is discussed in terms of solvation mechanism and a preliminary contribution to the understanding of the role of the hydrogen bonding in the interface phenomena is also reported. It is the first time that thermodynamic data of a ternary system are collected by mean of nano-isothermal titration calorimetry (nano-ITC). Supporting Information is available.
Asunto(s)
Dioxanos/química , Ácido Láctico/química , Nanoestructuras/química , Polímeros/química , Solventes/química , Agua/química , Poliésteres , Porosidad , Ingeniería de TejidosRESUMEN
Three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes. However, diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. Recently, a simple holographic approach, based on shape from silhouette algorithm, has been demonstrated for accurate calculation of cells biovolume and displaying their 3D shapes. Such approach has been adopted in combination with holographic optical tweezers and successfully applied to cells with convex shape. Nevertheless, unfortunately, the method fails in case of specimen with concave surfaces. Here, we propose an effective approach to achieve correct 3D shape measurement that can be extended in case of cells having concave surfaces, thus overcoming the limit of the previous technique. We prove the new procedure for healthy red blood cells (RBCs) (i.e., discocytes) having a concave surface in their central region. Comparative analysis of experimental results with a theoretical 3D geometrical model of RBC is discussed in order to evaluate accuracy of the proposed approach. Finally, we show that the method can be also useful to classify, in terms of morphology, different varieties of RBCs.
Asunto(s)
Eritrocitos/citología , Holografía/métodos , Algoritmos , Humanos , Modelos TeóricosRESUMEN
In "situ" tissue engineering is a promising approach in regenerative medicine, envisaging to potentiate the physiological tissue repair processes by recruiting the host's own cellular progenitors at the lesion site by means of bioactive materials. Despite numerous works focused the attention in characterizing novel chemoattractant molecules, only few studied the optimal way to present signal in the microenvironment, in order to recruit cells more effectively. In this work, we have analyzed the effects of gradients of stromal derived factor-1 (SDF-1) on the migratory behavior of human mesenchymal stem cells (MSCs). We have characterized the expression of the chemokine-associated receptor, CXCR4, using cytofluorimetric and real-time PCR analyses. Gradients of SDF-1 were created in 3D collagen gels in a chemotaxis chamber. Migration parameters were evaluated using different chemoattractant concentrations. Our results show that cell motion is strongly affected by the spatio-temporal features of SDF-1 gradients. In particular, we demonstrated that the presence of SDF-1 not only influences cell motility but alters the cell state in terms of SDF-1 receptor expression and productions, thus modifying the way cells perceive the signal itself. Our observations highlight the importance of a correct stimulation of MSCs by means of SDF-1 in order to implement on effective cell recruitment. Our results could be useful for the creation of a "cell instructive material" that is capable to communicate with the cells and control and direct tissue regeneration. Biotechnol. Bioeng. 2014;111: 2303-2316. © 2014 Wiley Periodicals, Inc.
Asunto(s)
Movimiento Celular , Quimiocina CXCL12/metabolismo , Quimiotaxis , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR4/biosíntesisRESUMEN
The stabilization of oil in water nano-emulsions by means of a polymer coating is extremely important; it prolongs the shelf life of the product and makes it suitable for a variety of applications ranging from nutraceutics to cosmetics and pharmaceutics. To date, an effective methodology to assess the best formulations in terms of thermodynamic stability has yet to be designed. Here, we perform a complete physicochemical characterization based on isothermal titration calorimetry (ITC) compared to conventional dynamic light scattering (DLS) to identify polymer concentration domains that are thermodynamically stable and to define the degree of stability through thermodynamic functions depending upon any relevant parameter affecting the stability itself, such as type of polymer coating, droplet distance, etc. For instance, the method was proven by measuring the energetics in the case of two different biopolymers, chitosan and poly-L-lysine, and for different concentrations of the emulsion coated with poly-L-lysine.
Asunto(s)
Calorimetría/métodos , Emulsiones/química , Polímeros/química , Termodinámica , Quitosano/química , Polilisina/químicaRESUMEN
Chemically modified oligonucleotides can solve biosensing issues for the development of capture probes, antisense, CRISPR/Cas, and siRNA, by enhancing their duplex-forming ability, their stability against enzymatic degradation, and their specificity for targets with high sequence similarity as microRNA families. However, the use of modified oligonucleotides such as locked nucleic acids (LNA) for biosensors is still limited by hurdles in design and from performances on the material interface. Here we developed a fluorogenic biosensor for non-coding RNAs, represented by polymeric PEG microgels conjugated with molecular beacons (MB) modified with locked nucleic acids (MicroLOCK). By 3D modeling and computational analysis, we designed molecular beacons (MB) inserting spot-on LNAs for high specificity among targets with high sequence similarity (95%). MicroLOCK can reversibly detect microRNA targets in a tiny amount of biological sample (2 µL) at 25 °C with a higher sensitivity (LOD 1.3 fM) without any reverse transcription or amplification. MicroLOCK can hybridize the target with fast kinetic (about 30 min), high duplex stability without interferences from the polymer interface, showing high signal-to-noise ratio (up to S/N = 7.3). MicroLOCK also demonstrated excellent resistance to highly nuclease-rich environments, in real samples. These findings represent a great breakthrough for using the LNA in developing low-cost biosensing approaches and can be applied not only for nucleic acids and protein detection but also for real-time imaging and quantitative assessment of gene targeting both in vitro and in vivo.