Optimización de la inmovilización de glucosa oxidasa en microgeles de poliacrilamida

The synthesis of polyacrylamide microgels using the concentrated emulsion pathway (W/O), without and with entrapped glucose oxidase from Aspergillus niger is proposed. The effect of initiator concentration in the maximum temperature of polymerization and the conversion rate of the monomer as a function of time were studied in the microparticles without enzyme. As a result of the polymerization, spherical micropart iculas were obtained in all the cases with a range of size between 2.5 ƒÝm and 6.2 ƒÝm of diameter In the microgels with enzyme the effect of the cross-linking content in the kinetic parameters was studied.. The use of the microgels with enzyme as the biological material of a amperometric biosensor allowed to evaluate the enzymatic activity of the glucose oxidase entrapped in the polymer. The catalytic activity of the entrapped enzyme and the biosensor response were affected by the cross-linking content of the microgel. The biosensor response was also affected by the quantity of microparticles immobilized in the surface of the electrode that acts as transducer of the biosensor.Se propone la sintesis de microgeles de poliacrilamida usando el metodo de polimerizacion en emulsion (W/O) concentrada, sin y con glucosa oxidasa de Aspergillus niger atrapada en su red polimerica. En las microparticulas sin enzima se estudio la influencia del agente iniciador en la temperatura maxima de polimerizacion y el grado de conversion del monomero en funcion del tiempo. Como resultado de la polimerizacion se obtuvieron en todos los casos microparticulas esfericas con un tamano comprendido entre 2,5ƒÝm y 6,2ƒÝm de diametro. En los microgeles con enzima se estudio la influencia del agente reticulante en diversos parametros cineticos. La utilizacion de las microparticulas con enzima como material biologico de un biosensor amperometrico permitio evaluar la actividad enzimatica de la glucosa oxidasa atrapada en su red polimerica. La actividad catalitica del enzima atrapado se vio afectada por el grado de reticulacion del microgel y como consecuencia la respuesta del biosensor sobre la que tambien influye la cantidad de microparticulas inmovilizadas en la superficie del electrodo que actua como transductor del biosensor

A brighter era for silver chalcogenide semiconductor nanocrystals

Silver chalcogenide semiconductor nanocrystals (Ag2E SNCs) have become a household name in the biomedical field, where they are used as contrast agents in bioimaging, photothermal therapy agents, and luminescent nanothermometers. The prominent position they have come to occupy in this field stems from a unique combination of features, above all near-infrared excitation and emission alongside low cytotoxicity. However, the first reports on Ag2E SNCs showed that a great limitation of these luminescent nanomaterials resided in their low photoluminescence quantum yield, which results in reduced brightness: a crippling feature in bioimaging and biosensing. In this article, we provide an overview of the strategies developed to overcome this hurdle. These strategies aim to remedy the presence of defects in the SNC core and/or surface, the presence of metallic silver, and off-stoichiometric composition. These features stem from the high mobility and redox potential of Ag+ ions, alongside the difficulty in controlling the nucleation and growth rate of Ag2E SNCs. The effectiveness of each approach is discussed. Lastly, a perspective on future research efforts to make Ag2E SNCs even brighter – and thus more effective in biomedical applications – is provided, with the hope of inspiring further investigation on these nanomaterials with a rich, complex set of physicochemical and spectroscopic propertiesThis work was financed by the Spanish Ministerio de Ciencia e Innovacion under project NANONERV PID2019-106211RB-I00, NANOGRANZ PID2021-123318OB-I00, PID2021-122806OB-I00 and TED2021-132317-I00B, by the Instituto de Salud Carlos III (PI19/ 00565), by the Comunidad Autonoma de Madrid (P2022/BMD-7403 RENIM-CM) and co-financed by the European structural and investment fund. R.M. is grateful to the Spanish Ministerio de Ciencia e Innovación for support to research through a Ramón y Cajal Fellowship (RYC2021- 032913-I). I.Z.-G. thanks UCM-Santander for a predoctoral contract (CT63/19-CT64/19). L.M. acknowledges a scholarship from the China Scholarship Council (No. 202108350018

Neural networks push the limits of luminescence lifetime nanosensing

Luminescence lifetime-based sensing is ideally suited to monitor biological systems due to its minimal invasiveness and remote working principle. Yet, its applicability is limited in conditions of low signal-to-noise ratio (SNR) induced by, e.g., short exposure times and presence of opaque tissues. Herein this limitation is overcome by applying a U-shaped convolutional neural network (U-NET) to improve luminescence lifetime estimation under conditions of extremely low SNR. Specifically, the prowess of the U-NET is showcased in the context of luminescence lifetime thermometry, achieving more precise thermal readouts using Ag2S nanothermometers. Compared to traditional analysis methods of decay curve fitting and integration, the U-NET can extract average lifetimes more precisely and consistently regardless of the SNR value. The improvement achieved in the sensing performance using the U-NET is demonstrated with two experiments characterized by extreme measurement conditions: thermal monitoring of free-falling droplets, and monitoring of thermal transients in suspended droplets through an opaque medium. These results broaden the applicability of luminescence lifetime-based sensing in fields including in vivo experimentation and microfluidics, while, hopefully, spurring further research on the implementation of machine learning (ML) in luminescence sensingThis work was financed by the Spanish Ministerio de Innovación y Ciencias under Project Nos. RTI2018-101050-J-I00, NANONERV PID2019-106211RB-I00, NANOGRANZ PID2021-123318OB-I00, TED2021-132317- I00B, and EIN2020-112419. Additional funding was provided by the European Union Horizon 2020 FETOpen project NanoTBTech (Grant No. 801305) and by the Comunidad Autónoma de Madrid (S2022/BMD7403 REMIN-CM). R.M. is grateful to the Spanish Ministerio de Ciencia e Innovación for support to research through a Ramón y Cajal Fellowship (RYC2021-032913-I). L.M. acknowledges a scholarship from the China Scholarship Council (No. 202108350018). I.Z.-G. thanks UCM-Santander for a predoctoral contract (CT63/19-CT64/19). P.R.-S. is grateful for a Juan de la Cierva-Incorporación scholarship (Grant No. IJC2019-041915-I

Clickable albumin nanoparticles for pretargeted drug delivery toward PD-L1 overexpressing tumors in combination immunotherapy

We present a simple methodology to design a pretargeted drug delivery system, based on clickable anti-programmed death ligand 1 (anti-PD-L1) antibodies (Abs) and clickable bovine serum albumin (BSA) nanoparticles (NPs). Pretargeted drug delivery is based on the decoupling of a targeting moiety and a drug-delivering vector which can then react in vivo after separate injections. This may be key to achieve active targeting of drug-delivering NPs toward cancerous tissue. In pretargeted approaches, drug-delivering NPs were observed to accumulate in a higher amount in the targeted tissue due to shielding-related enhanced blood circulation and size-related enhanced tissue penetration. In this work, BSA NPs were produced using the solvent precipitation methodology that renders colloidally stable NPs, which were subsequently functionalized with a clickable moiety based on chlorosydnone (Cl-Syd). Those reactive groups are able to specifically react with dibenzocyclooctyne (DBCO) groups in a click-type fashion, reaching second-order reaction rate constants as high as 1.9 M-1·s-1, which makes this reaction highly suitable for in vivo applications. The presence of reactive Cl-Syd was demonstrated by reacting the functionalized NPs with a DBCO-modified sulfo-cyanine-5 dye. With this reaction, it was possible to infer the number of reactive moieties per NPs. Finally, and with the aim of demonstrating the suitability of this system to be used in pretargeted strategies, functionalized fluorescent NPs were used to label H358 cells with a clickable anti-PD-L1 Ab, applying the reaction between Cl-Syd and DBCO as corresponding clickable groups. The results of these experiments demonstrate the bio-orthogonality of the system to perform the reaction in vitro, in a period as short as 15 mi

Thermoresponsive Polymeric Nanolenses Magnify the Thermal Sensitivity of Single Upconverting Nanoparticles

Lanthanide-based upconverting nanoparticles (UCNPs) are trustworthy workhorses in luminescent nanothermometry. The use of UCNPs-based nanothermometers has enabled the determination of the thermal properties of cell membranes and monitoring of in vivo thermal therapies in real time. However, UCNPs boast low thermal sensitivity and brightness, which, along with the difficulty in controlling individual UCNP remotely, make them less than ideal nanothermometers at the single-particle level. In this work, it is shown how these problems can be elegantly solved using a thermoresponsive polymeric coating. Upon decorating the surface of NaYF4:Er3+,Yb3+ UCNPs with poly(N-isopropylacrylamide) (PNIPAM), a >10-fold enhancement in optical forces is observed, allowing stable trapping and manipulation of a single UCNP in the physiological temperature range (20–45 °C). This optical force improvement is accompanied by a significant enhancement of the thermal sensitivity— a maximum value of 8% °C+1 at 32 °C induced by the collapse of PNIPAM. Numerical simulations reveal that the enhancement in thermal sensitivity mainly stems from the high-refractive-index polymeric coating that behaves as a nanolens of high numerical aperture. The results in this work demonstrate how UCNP nanothermometers can be further improved by an adequate surface decoration and open a new avenue toward highly sensitive single-particle nanothermometryThis work was supported by the Ministerio de Ciencia e Innovación de España (PID2019-106211RB-I00 PID2019-105195RA-I00 and MAT2017- 83111R), by the Comunidad de Madrid (S2017/BMD-3867 RENIM-CM), co-financed by European Structural and Investment Fund and by the Universidad Autónoma de Madrid and Comunidad Autónoma de Madrid (SI1/PJI/2019-00052 and PR38/21-36 ANTICIPA-CM). D.L. acknowledges a scholarship from the China Scholarship Council (201808350097). J.R.B. acknowledges the support from Carl Tryggers Foundation (CTS18:229). M.I.M acknowledges financial support from the Spanish Ministerio de Ciencia e Innovación, through the “María de Maeztu” Programme for Units of Excellence in R&D (CEX2018-000805-M) and the MELODIA PGC2018-095777-B-C22 proje

Lanthanide doped nanoheaters with reliable and absolute temperature feedback

The development of selective and controlled photo-thermal therapies requires luminescent nanoparticles capable of simultaneous heating and contactless thermal sensing. Until now, thermal therapies have suffered from a lack of control over the absolute temperature of the treated tissue because the nanothermometers used for thermal feedback, based on a spectral analysis of emitted radiation, were affected by the inhomogeneous extinction of the tissues. This work shows how this deficiency can be overcome by using core-shell-shell nanostructures doped with lanthanide ions (Nd3+ and Yb3+). Thermal reading was achieved from the analysis of the Yb3+ luminescence lifetime whereas simultaneous heating was achieved thanks to the non-radiative deexcitations of Nd3+ ions. Simple proof-of-concept experiments show the great potential of these lanthanide-doped nanostructures for the development of in vivo photo-thermal treatments with absolute and reliable thermal feedbac

Oligonucleotide Sensor Based on Selective Capture of Upconversion Nanoparticles Triggered by Target-Induced DNA Interstrand Ligand Reaction

We present a sensor that exploits the phenomenon of upconversion luminescence to detect the presence of specific sequences of small oligonucleotides such as miRNAs among others. The sensor is based on NaYF4:Yb,Er@SiO2 nanoparticles functionalized with ssDNA that contain azide groups on the 3' ends. In the presence of a target sequence, interstrand ligation is possible via the click-reaction between one azide of the upconversion probe and a DBCO-ssDNA-biotin probe present in the solution. As a result of this specific and selective process, biotin is covalently attached to the surface of the upconversion nanoparticles. The presence of biotin on the surface of the nanoparticles allows their selective capture on a streptavidin-coated support, giving a luminescent signal proportional to the amount of target strands present in the test samples. With the aim of studying the analytical properties of the sensor, total RNA samples were extracted from healthy mosquitoes and were spiked-in with a specific target sequence at different concentrations. The result of these experiments revealed that the sensor was able to detect 10-17 moles per well (100 fM) of the target sequence in mixtures containing 100 ng of total RNA per well. A similar limit of detection was found for spiked human serum samples, demonstrating the suitability of the sensor for detecting specific sequences of small oligonucleotides under real conditions. In contrast, in the presence of noncomplementary sequences or sequences having mismatches, the luminescent signal was negligible or conspicuously reduced.The authors are grateful for the financial support from the Bill & Melinda Gates Foundation, with Grant OPP1128411, Asociación Española Contra el Cáncer, Santander-Universidad Complutense project PR26/16-12B-3, and from the Spanish MINECO for the projects MAT2014-55065-R, SAF2014-56763-R, and FIS2013-41709-P.S

Reaching Deeper: Absolute In Vivo Thermal Reading of Liver by Combining Superbright Ag S Nanothermometers and In Silico Simulations

Luminescent nano-thermometry is a fast-developing technique with great potential for in vivo sensing, diagnosis, and therapy. Unfortunately, it presents serious limitations. The luminescence generated by nanothermometers, from which thermal readout is obtained, is strongly distorted by the attenuation induced by tissues. Such distortions lead to low signal levels and entangle absolute and reliable thermal monitoring of internal organs. Overcoming both limitations requires the use of high-brightness luminescent nanothermometers and adopting more complex approaches for temperature estimation. In this work, it is demonstrated how superbright Ag2S nanothermometers can provide in vivo, reliable, and absolute thermal reading of the liver during laser-induced hyperthermia. For that, a new procedure is designed in which thermal readout is obtained from the combination of in vivo transient thermometry measurements and in silico simulations. The synergy between in vivo and in silico measurements has made it possible to assess relevant numbers such as the efficiency of hyperthermia processes, the total heat energy deposited in the liver, and the relative contribution of Ag2S nanoparticles to liver heating. This work provides a new way for absolute thermal sensing of internal organs with potential application not only to hyperthermia processes but also to advanced diagnosis and therapy.This work was supported by the Spanish Ministry of Economy and Competitiveness under projects MAT2016-75362-C3-1-R, MAT2017-83111R, and MAT2017-85617-R, by the Instituto de Salud Carlos III (PI16/00812), by the Comunidad Autonoma de Madrid (B2017/BMD-3867 RENIM-CM), and cofinanced by the European Structural and investment fund. Additional funding was provided by the European Union's Horizon 2020 FET Open programme (Grant Agreement No. 801305, NanoTBTech), the Fundacion para la Investigacion Biomedica del Hospital Universitario Ramon y Cajal project IMP18_38 (2018/0265), and also by COST action CA17140. Y.S. acknowledges a scholarship from the China Scholarship Council (No. 201806870023). I.Z.G. thanks UCM-Santander for a predoctoral contract (CT63/19-CT64/19). D.O. and I.R. acknowledge financial support from the Community of Madrid under Contract No. PEJD-2017-PRE/IND-3663, and from the Spanish Ministry of Science and Innovation through the Ramon y Cajal grant RYC2018-025253-I, Research Networks grant RED2018-102626-T and the PID2019-106211RB-I00 grant as well as the Ministry of Economy and Competitiveness through the grants MAT2017-85617-R, MAT2017-88148R and the "Severo Ochoa" Program for Centers of Excellence in R&D (SEV-2016-0686). D.O. and I.R. also acknowledge support from the "NoCanTher" project, which has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 685795. E.X. is grateful for a Juan de la Cierva Formacion scholarship (FJC2018-036734-I)

Boosting the near-infrared emission of Ag2S nanoparticles by a controllable surface treatment for bioimaging applications

Ag2S nanoparticles are the staple for high-resolution preclinical imaging and sensing owing to their photochemical stability, low toxicity, and photoluminescence (PL) in the second near-infrared biological window. Unfortunately, Ag2S nanoparticles exhibit a low PL efficiency attributed to their defective surface chemistry, which curbs their translation into the clinics. To address this shortcoming, we present a simple methodology that allows to improve the PL quantum yield from 2 to 10%, which is accompanied by a PL lifetime lengthening from 0.7 to 3.8 μs. Elemental mapping and X-ray photoelectron spectroscopy indicate that the PL enhancement is related to the partial removal of sulfur atoms from the nanoparticle's surface, reducing surface traps responsible for nonradiative de-excitation processes. This interpretation is further backed by theoretical modeling. The acquired knowledge about the nanoparticles' surface chemistry is used to optimize the procedure to transfer the nanoparticles into aqueous media, obtaining water-dispersible Ag2S nanoparticles that maintain excellent PL properties. Finally, we compare the performance of these nanoparticles with other near-infrared luminescent probes in a set of in vitro and in vivo experiments, which demonstrates not only their cytocompatibility but also their superb optical properties when they are used in vivo, affording higher resolution image