A High-Throughput Screening Strategy for Synthesizing Molecularly Imprinted Polymer Nanoparticles Selectively Targeting Tumors.

Molecularly imprinted polymers (MIPs) show significant promise as effective alternatives to antibodies in disease diagnosis and therapy. However, the challenging process of screening extensive libraries of monomer combinations and synthesis conditions to identify formulations with enhanced selectivity and affinity presents a notable time constraint. The need for expedient methods becomes clear in accelerating the strategic development of MIPs tailored for precise molecular recognition purposes. In this study, an innovative high-throughput screening methodology designed to identify the optimal MIP formulation for targeting tumors is presented. Employing a microtiter plate format, over 100 polymer syntheses are conducted, incorporating diverse combinations of functional monomers. Evaluation of binding performance utilizes fluorescence-based assays, focusing on an epitope of the epidermal growth factor receptor (EGFR). Through this meticulously structured screening process, synthesis conditions that produced MIP nanoparticles exhibiting substantial specificity for EGFR targeting (KD = 10-12 m) are identified. These "bionic antibodies" demonstrate selective recognition of cancer cells in whole blood samples, even at concentrations as low as 5 cells mL-1. Further validation through fluorescent imaging confirms the tumor-specific localization of the MIPs in vivo. This highly efficient screening approach facilitates the strategic synthesis of imprinted polymers functioning as precision bioprobes.

Double Imprinted Nanoparticles for Sequential Membrane-to-Nuclear Drug Delivery.

Efficient and site-specific delivery of therapeutics drugs remains a critical challenge in cancer treatment. Traditional drug nanocarriers such as antibody-drug conjugates are not generally accessible due to their high cost and can lead to serious side effects including life-threatening allergic reactions. Here, these problems are overcome via the engineering of supramolecular agents that are manufactured with an innovative double imprinting approach. The developed molecularly imprinted nanoparticles (nanoMIPs) are targeted toward a linear epitope of estrogen receptor alfa (ERα) and loaded with the chemotherapeutic drug doxorubicin. These nanoMIPs are cost-effective and rival the affinity of commercial antibodies for ERα. Upon specific binding of the materials to ERα, which is overexpressed in most breast cancers (BCs), nuclear drug delivery is achieved via receptor-mediated endocytosis. Consequentially, significantly enhanced cytotoxicity is elicited in BC cell lines overexpressing ERα, paving the way for precision treatment of BC. Proof-of-concept for the clinical use of the nanoMIPs is provided by evaluating their drug efficacy in sophisticated three-dimensional (3D) cancer models, which capture the complexity of the tumor microenvironment in vivo without requiring animal models. Thus, these findings highlight the potential of nanoMIPs as a promising class of novel drug compounds for use in cancer treatment.

Boosting Electrochemical Sensing Performances Using Molecularly Imprinted Nanoparticles.

Nanoparticles of molecularly imprinted polymers (nanoMIPs) combine the excellent recognition ability of imprinted polymers with specific properties related to the nanosize, such as a high surface-to-volume ratio, resulting in highly performing recognition elements with surface-exposed binding sites that promote the interaction with the target and, in turn, binding kinetics. Different synthetic strategies are currently available to produce nanoMIPs, with the possibility to select specific conditions in relation to the nature of monomers/templates and, importantly, to tune the nanoparticle size. The excellent sensing properties, combined with the size, tunability, and flexibility of synthetic protocols applicable to different targets, have enabled the widespread use of nanoMIPs in several applications, including sensors, imaging, and drug delivery. The present review summarizes nanoMIPs applications in sensors, specifically focusing on electrochemical detection, for which nanoMIPs have been mostly applied. After a general survey of the most widely adopted nanoMIP synthetic approaches, the integration of imprinted nanoparticles with electrochemical transducers will be discussed, representing a key step for enabling a reliable and stable sensor response. The mechanisms for electrochemical signal generation will also be compared, followed by an illustration of nanoMIP-based electrochemical sensor employment in several application fields. The high potentialities of nanoMIP-based electrochemical sensors are presented, and possible reasons that still limit their commercialization and issues to be resolved for coupling electrochemical sensing and nanoMIPs in an increasingly widespread daily-use technology are discussed.

Sensing Approaches Exploiting Molecularly Imprinted Nanoparticles and Lossy Mode Resonance in Polymer Optical Fibers.

In this work, two different lossy mode resonance (LMR) platforms based on plastic optical fibers (POFs) are developed and tested in a biochemical sensing scenario. The LMR platforms are based on the combination of two metal oxides (MOs), i.e., zirconium oxide (ZrO2) and titanium oxide (TiO2), and deposited on the exposed core of D-shaped POF chips. More specifically, two experimental sensor configurations were obtained by swapping the mutual position of the Mos films over to the core of the D-shaped POF probe. The POF-LMR sensors were first characterized as refractometers, proving the bulk sensitivities. Then, both the POF-LMR platforms were functionalized using molecularly imprinted nanoparticles (nanoMIPs) specific for human transferrin (HTR) in order to carry out binding tests. The achieved results report a bulk sensitivity equal to about 148 nm/RIU in the best sensor configuration, namely the POF-TiO2-ZrO2. In contrast, both optical configurations combined with nanoMIPs showed an ultra-low detection limit (fM), demonstrating excellent efficiency of the used receptor (nanoMIPs) and paving the way to disposable POF-LMR biochemical sensors that are easy-to-use, low-cost, and highly sensitive.

Imprinted Hydrogel Nanoparticles for Protein Biosensing: A Review.

Over the past decade, molecular imprinting (MI) technology has made tremendous progress, and the advancements in nanotechnology have been the major driving force behind the improvement of MI technology. The preparation of nanoscale imprinted materials, i.e., molecularly imprinted polymer nanoparticles (MIP NPs, also commonly called nanoMIPs), opened new horizons in terms of practical applications, including in the field of sensors. Currently, hydrogels are very promising for applications in bioanalytical assays and sensors due to their high biocompatibility and possibility to tune chemical composition, size (microgels, nanogels, etc.), and format (nanostructures, MIP film, fibers, etc.) to prepare optimized analyte-responsive imprinted materials. This review aims to highlight the recent progress on the use of hydrogel MIP NPs for biosensing purposes over the past decade, mainly focusing on their incorporation on sensing devices for detection of a fundamental class of biomolecules, the peptides and proteins. The review begins by directing its focus on the ability of MIPs to replace biological antibodies in (bio)analytical assays and highlight their great potential to face the current demands of chemical sensing in several fields, such as disease diagnosis, food safety, environmental monitoring, among others. After that, we address the general advantages of nanosized MIPs over macro/micro-MIP materials, such as higher affinity toward target analytes and improved binding kinetics. Then, we provide a general overview on hydrogel properties and their great advantages for applications in the field of Sensors, followed by a brief description on current popular routes for synthesis of imprinted hydrogel nanospheres targeting large biomolecules, namely precipitation polymerization and solid-phase synthesis, along with fruitful combination with epitope imprinting as reliable approaches for developing optimized protein-imprinted materials. In the second part of the review, we have provided the state of the art on the application of MIP nanogels for screening macromolecules with sensors having different transduction modes (optical, electrochemical, thermal, etc.) and design formats for single use, reusable, continuous monitoring, and even multiple analyte detection in specialized laboratories or in situ using mobile technology. Finally, we explore aspects about the development of this technology and its applications and discuss areas of future growth.

Preparation of a Molecularly Imprinted Silica Nanoparticles Embedded Microfiltration Membrane for Selective Separation of Tetrabromobisphenol A from Water.

The ubiquitous presence of tetrabromobisphenol A (TBBPA) in aquatic environments has caused severe environmental and public health concerns; it is therefore of great significance to develop effective techniques to remove this compound from contaminated waters. Herein, a TBBPA imprinted membrane was successfully fabricated via incorporating imprinted silica nanoparticles (SiO2 NPs). The TBBPA imprinted layer was synthesized on the 3-(methacryloyloxy) propyltrimethoxysilane (KH-570) modified SiO2 NPs via surface imprinting. Eluted TBBPA molecularly imprinted nanoparticles (E-TBBPA-MINs) were incorporated onto a polyvinylidene difluoride (PVDF) microfiltration membrane via vacuum-assisted filtration. The obtained E-TBBPA-MINs embedded membrane (E-TBBPA-MIM) showed appreciable permeation selectivity toward the structurally analogous to TBBPA (i.e., 6.74, 5.24 and 6.31 of the permselectivity factors for p-tert-butylphenol (BP), bisphenol A (BPA) and 4,4'-dihydroxybiphenyl (DDBP), respectively), far superior to the non-imprinted membrane (i.e., 1.47, 1.17 and 1.56 for BP, BPA and DDBP, respectively). The permselectivity mechanism of E-TBBPA-MIM could be attributed to the specific chemical adsorption and spatial complementation of TBBPA molecules by the imprinted cavities. The resulting E-TBBPA-MIM exhibited good stability after five adsorption/desorption cycles. The findings of this study validated the feasibility of developing nanoparticles embedded molecularly imprinted membrane for efficient separation and removal of TBBPA from water.

A selectivity-enhanced fluorescence imprinted sensor based on yellow-emission peptide nanodots for sensitive and visual smart detection of λ-cyhalothrin.

The development of precise and efficient detection technologies to recognize λ-cyhalothrin (LC) in agricultural products has attracted attention worldwide due to its widespread use and notable toxic effects on humans. Herein, a novel fluorescence biomimetic nanosensor was elaborately designed based on Zn(II)-doped cyclo-ditryptophan (c-WW)-type peptide nanodots and incorporating molecularly imprinted polymer (c-WW/Zn-PNs@MIP) for LC assays. C-WW/Zn-PNs obtained by self-assembly with aromatic cyclic dipeptides as basic building blocks and coordination with Zn(II) have low-toxicity, photostability, and bright yellow fluorescence emission, as a sensitive signal transducer. High-affinity imprinting sites further endow c-WW/Zn-PNs@MIP with superior selectivity and reusability. Based on prominent merits, c-WW/Zn-PNs@MIP demonstrated a good linear range (1-360 µg/L) with a low limit of detection (LOD) (0.93 µg/L), fast kinetics in target capture (10 min), and strong practicability in the capture of LC from real samples (spiked recovery of 81.0-107.7%). Additionally, to attain onsite profiling of LC, a visual platform was developed by integrating c-WW/Zn-PNs@MIP with a smartphone-assisted optical device. This smart evaluation system can capture concentration-dependent fluorescent images and accurately digitize them, enabling quantitative analysis of LC. This study developed a fluorescent c-WW/Zn-PNs@MIP-based smart evaluation system as a novel platform for LC monitoring applications, which not only has enormous economic value but also great environmental health significance.

Solid-Phase Screening and Synthesis of Molecularly Imprinted Nanoparticles for Selective Recognition and Detection of Brain Natriuretic Peptide.

A new recognition method is explored for the rapid detection of B-type natriuretic peptide (BNP) based on the rational design and solid-phase synthesis of molecularly imprinted nanoparticles (nanoMIP) encapsulated with carbon dots. The nanosized magnetic template is first prepared by attaching the epitope of BNP on amino-functionalized magnetic carriers. High-dilution polymerization of monomers in the presence of magnetic template generates lightly crosslinked imprinted nanoparticles. To obtain the optimal MIP formulation, a new combinatorial screening approach is developed by a competitive fluorescence assay using the magnetic template. The resultant nanoMIP exhibits high affinity and selectivity toward BNP with an equilibrium dissociation constant (KD ) of ≈10-11  m. The proposed assay allows fast BNP detection within ≈7 min with a linear range of its concentration from 0.25 to 5000 pg mL-1 and a limit of detection of 0.208 pg mL-1 (S/N = 3). To demonstrate its practicability in clinical diagnosis, unknown real serum samples from 160 individuals are analyzed and the relative standard deviation is less than 4.43%. Compared with the routine electrochemiluminescence detection method that is widely used in hospital, the relative error is less than 4.98% and the correlation coefficient is 0.994.

Bioselective PES Membranes Based on Chitosan Functionalization and Virus-Imprinted NanoMIPs for Highly Efficient Separation of Human Pathogenic Viruses from Water.

Waterborne viruses are a public health concern due to relatively small infection doses. Particularly, adenoviruses (AdVs) are more resistant than RNA viruses to water purification treatments in terms of ultraviolet (UV) irradiation, pH, and chlorination tolerance. Moreover, AdVs are one of the most predominant waterborne viruses. Membrane separations have proven superior removal capabilities of waterborne pathogens over other separation methods. However, virus removal at ultratrace levels is still a significant challenge for current membrane technology. This study successfully addressed this challenge by developing a bioselective polyethersulfone (PES) membrane by a joint strategy involving chitosan hydrophilic surface modification and the immobilization of adenovirus-specific molecularly imprinted nanoparticles (nanoMIPs). The topological and chemical changes taking place on the membrane surface were characterized by using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Furthermore, hydrophilicity and membrane performance were investigated in terms of swelling behavior, permeation flux, and surface fouling studies. The membrane efficacy was evaluated by filtration experiments, where the virus concentration of the loading solution before filtration and the permeates after filtration was quantified. The novel bioselective membrane showed excellent virus removal capabilities by separating 99.99% of the viruses from the water samples.

Molecularly Designed Ion-Imprinted Nanoparticles for Real-Time Sensing of Cu(II) Ions Using Quartz Crystal Microbalance.

A molecularly designed imprinting method was combined with a gravimetric nanosensor for the real-time detection Cu(II) ions in aqueous solutions without using expensive laboratory devices. Thus, 1:1 and 2:1 mol-ratio-dependent coordination modes between Cu(II), N-methacyloly-L histidine methyl ester (MAH) functional monomer complexes, and their four-fold and six-fold coordinations were calculated by means of density functional theory molecular modeling. Cu(II)-MIP1 and Cu(II)-MIP2 nanoparticles were synthesized in the size range of 80-100 nm and characterized by SEM, AFM and FTIR. Cu(II)-MIP nanoparticles were then conducted to a quartz crystal microbalance sensor for the real-time detection of Cu(II) ions in aqueous solutions. The effects of initial Cu(II) concentration, selectivity, and imprinting efficiency were investigated for the optimization of the nanosensor. Linearity of 99% was obtained in the Cu(II) ion linear concentration range of 0.15-1.57 µM with high sensitivity. The LOD was obtained as 40.7 nM for Cu(II)-MIP2 nanoparticles. The selectivity and the imprinting efficiency of the QCM nanosensor were obtained significantly in the presence of competitive ion samples (Co(II), Ni(II), Zn(II), and Fe(II)). The results are promising for sensing Cu(II) ions as environmental toxicants in water by combining molecularly designed ion-imprinted nanoparticles and a gravimetric sensor.

Selective capture and determination of doxycycline in marine sediments by using magnetic imprinting dispersive solid-phase extraction coupled with high performance liquid chromatography.

Antibiotics are frequently used in aquaculture as feed additives and finally enter the marine environment that can pose potential threat to humans. In this study, magnetic molecularly imprinted nanocomposites were prepared by surface imprinting and applied as selective sorbents for specific capture of doxycycline. A multivariate approach based on response surface methodology with Box-Behnken design was adopted to optimize the dispersive solid-phase extraction of doxycycline from marine sediment. Three key parameters, including adsorbent amount and type of washing/eluting solvent, were screened. Under optimum conditions, the limit of detection was 0.03 µg g-1 with good linearity from 0.5 to 20 µg g-1 followed by HPLC detection. Finally, two sediment samples were analysed and satisfactory recoveries between 90.60 % and 93.76 % were obtained with acceptable relative standard deviations (≤4.12 %), suggesting a promising applicability of the developed method for efficient extraction and sensitive quantification of antibiotics in complex marine environmental matrix.

Molecularly Imprinted Nanoparticles towards MMP9 for Controlling Cardiac ECM after Myocardial Infarction: A Predictive Experimental-Computational Chemistry Investigation.

The recent advances in nanotechnology are revolutionizing preventive and therapeutic approaches to treating cardiovascular diseases. Controlling the extracellular matrix metalloproteinase (MMP) activation and expression in the failing human left ventricular myocardium represents a significant therapeutic target for heart disease. In this study, we used molecularly imprinting polymers (MIPs) to restore the correct balance between MMPs and their tissue inhibitors (TIMPs), and explored the potential of this technique exhaustively through chemical synthesis, physicochemical and biological characterizations, and computational chemistry methods. By molecular dynamics simulations based on classical force fields, we simulated the early stages of the imprinting process in solution disclosing the pivotal interaction established between the monomers and the MMP9 protein template. The average interaction energies of methacrylic acid (MAA) and poly (ethylene glycol) ethyl ether methacrylate (PEG) units were in the ranges 17-22 and 30-37 kcal/mol, respectively. At low coverage, the PEG monomers seemed firmly anchored to the protein surface and were not displaced by water, while only about 20% of MAA was replaced by water. The synthesis of MIPs was successfully with a monomer conversion higher than 99% and the production of spherical particles with average diameter of 344 ± 33 nm. HPLC analysis showed a specific recognition factor of MMP9 on MIPs of about 1.3. FT-IR Chemical Imaging confirmed the mechanisms necessary to generate a "selective memory" of the MIPs towards the enzyme. HPLC results indicated that the rebound amount of both TIMP1 and MMP2 to MIPs is lower than that of the template, showing a selectivity factor of 2.1 and 2.3, respectively. Preliminary tests on the effect of MIPs on H9C2 cells revealed that this treatment has no cytotoxic effects.

A Plasmonic Biosensor Based on Light-Diffusing Fibers Functionalized with Molecularly Imprinted Nanoparticles for Ultralow Sensing of Proteins.

Plasmonic bio/chemical sensing based on optical fibers combined with molecularly imprinted nanoparticles (nanoMIPs), which are polymeric receptors prepared by a template-assisted synthesis, has been demonstrated as a powerful method to attain ultra-low detection limits, particularly when exploiting soft nanoMIPs, which are known to deform upon analyte binding. This work presents the development of a surface plasmon resonance (SPR) sensor in silica light-diffusing fibers (LDFs) functionalized with a specific nanoMIP receptor, entailed for the recognition of the protein human serum transferrin (HTR). Despite their great versatility, to date only SPR-LFDs functionalized with antibodies have been reported. Here, the innovative combination of an SPR-LFD platform and nanoMIPs led to the development of a sensor with an ultra-low limit of detection (LOD), equal to about 4 fM, and selective for its target analyte HTR. It is worth noting that the SPR-LDF-nanoMIP sensor was mounted within a specially designed 3D-printed holder yielding a measurement cell suitable for a rapid and reliable setup, and easy for the scaling up of the measurements. Moreover, the fabrication process to realize the SPR platform is minimal, requiring only a metal deposition step.

Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis.

The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to Gi/o proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 ± 10.5 nm at pH 3 (25 ºC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 ± 15.2 nm at 35 °C. Lower critical solution temperature of this polymer was found to be ≈ 33.4 °C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1414-472 and GST-CB1414-442 were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR).

Colorimetric detection of human alpha-2-macroglobulin by janus imprinted nanoparticles constructed dual molecular imprinting immunosandwich strategy.

Simple and rapid detection of disease-related bio-markers are significant for early clinical diagnosis and can potentially improve the survival rate. However, establishing a high-specificity colorimetric detection method for bio-markers are still challenges due to their inevitable natural antibody used or enzymatic labeling. Herein, a cost-efficient and easy-to-use approach, which called dual molecular imprinting immunosandwich colorimetric strategy (DMI-ICS) was constructed for detection alpha-2-macroglobulin (α2MG) by janus imprinted nanoparticles. The unique detection principle was contained with two mimic antibody parts, the first part was α2MG glass slides molecularly imprinted material (GS-MIP) as a "Separation antibody", which can specifically rapid separate the protein in the complex sample; Another part was asymmetrically modified janus molecularly imprinted gold nanoparticles nanozyme (J-GNPs-MIP) as a "Detection antibody", which has the properties of specific recognition and catalytic substrate color performance at the same time. The concentration of α2MG can be determined by the substrate color changes and observed with naked eyes. Under the optimized conditions, the DMI-ICS had a great performance and offering lower relative standard deviation (RSD, 7.69%), good linear range (0.297-130 µg/mL, R2 = 0.994), high imprinting factor (IF: 3.74) with lower detection limit (0.089 µg/mL). This strategy provides an easy operation and low cost signal readout method for direct detection and separation of α2MG in human serum samples, which is a versatile tool for point-of-care diagnosis, while also offering a new perspective on antibody simulation technology, multifunctional antibody preparation and contribute to detection of disease-related bio-marker in nonspecialized laboratory infrastructure.

Synthesis and evaluation of mesoporous silica/mesoporous molecularly imprinted nanoparticles as adsorbents for detection and selective removal of imidacloprid in food samples.

The double-mesoporous-layer imprinted polymer of mesoporous silica/mesoporous molecularly imprinted nanoparticles (MIP), with high specific surface area, rich porosity, excellent mass transfer rate and selectivity, were synthesized using imidacloprid (IDP) as a template. Under the optimal conditions of pH, contact time, concentration and temperature, MIP showed high adsorption capacity of 13.86 µg·mg-1 toward IDP and the imprinting factor reached 3.5. The adsorption process model including binding isotherm and kinetics was investigated. MIP exhibited excellent regeneration and its adsorption and selectivity were outstanding among its structurally pesticide analogues. The recovery of spiked IDP for MIP in fortified real samples can reach 96.0 ± 8.5% for cabbage and 105.0 ± 9.9% for apple. The limit of detection of the enrichment method can be as low as 0.037 µg·mL-1 with a good linear relationship (R2 = 0.996) from 0.30 to 10.0 µg·mL-1. The results indicated that the proposed method allowed class-specific detection of IDP in food samples.

Selective recognition of a cyclic peptide hormone in human plasma by hydrazone bond-oriented surface imprinted nanoparticles.

As a kind of artificial recognition material, molecularly imprinted polymers (MIPs) offer a promising perspective to be developed as synthetic chemical binders capable of selectively recognize biomacromolecules. However, owing to the large size and conformational flexibility of proteins and peptides, imprinting of these biomacromolecules remains a challenge. Novel imprinting strategies still need exploration for the improvement of recognition performance of MIPs. Herein, we developed a hydrazone bond-oriented surface imprinting strategy for an endogenous peptide hormone, human atrial natriuretic peptide (ANP). Surface-oriented imprinting of peptide via reversible covalent bond anchoring approach increased the orientation homogeneity of imprinted cavities as well as the utility of templates. The prepared nanoparticles exhibited high selectivity and fast recognition kinetics for ANP epitope. The dissociation constant between ANP epitope and MIP was measured as 5.3 µM. The applicability of the material in real samples was verified by the selective magnetic extraction of ANP from human plasma samples. This hydrazone bond-oriented surface imprinting strategy provides an alternative approach for the separation of peptides or proteins in complex bio-samples.

Molecularly imprinted curcumin nanoparticles decorated paper for electrochemical and fluorescence dual-mode sensing of bisphenol A.

A molecularly imprinted paper-based analytical device (MIP-µPAD) was developed for the sensing of bisphenol A (BPA). The platform was screen-printed onto a filter paper support, where the electrodes and the fluorescence µPADs were designed. Owing to its dual electrochemical and fluorescence responses, molecularly imprinted curcumin nanoparticles were used to sense BPA. The µPAD design was characterized by transmission electron microscopy, scanning electron microscopy, fluorescence spectroscopy, and electrochemical techniques. The sensor design comprised a wide linear range from 1 to 200 µg L-1 with limits of detection of 0.47 ± 0.2 and 0.62 ± 0.3 µg L-1 (LOD, S/N = 3) for electrochemical and fluorescence sensing, respectively. Furthermore, the system showed good analytical performance such as selectivity, stability, and reproducibility. The feasibility of the MIP-µPAD was demonstrated for the sensing of BPA in seawater, foods, and polycarbonate plastic packaged water with recovery values of 97.2 and 101.8%.

Cellular reprogramming with multigene activation by the delivery of CRISPR/dCas9 ribonucleoproteins via magnetic peptide-imprinted chitosan nanoparticles.

Induced pluripotent stem cells are usually derived by reprogramming transcription factors (OSKM), such as octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and cellular proto-oncogene (c-Myc). However, the genomic integration of transcription factors risks the insertion of mutations into the genome of the target cells. Recently, the clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system has been used to edit genomes. In this work, dCas9-VPR (dCas9 with a gene activator, VP64-p65-Rta (VPR), fused to its c-terminus) and guide RNA (gRNA) combined to form ribonucleoproteins, which were immobilized on magnetic peptide-imprinted chitosan nanoparticles. These were then used to activate OSKM genes in human embryonic kidney (HEK) 293T cells. Four pairs of gRNAs were used for the binding site recognition to activate the OSKM genes. Transfected HEK293T cells were then prescreened for the high expression of OSKM proteins by immunohistochemistry images. The optimal gRNAs for OSKM expression were identified using quantitative real-time polymerase chain reaction and the staining of OSKM proteins. Finally, we found that the activated expression of one of the OSKM genes is up to three-fold higher than that of the other genes, enabling precise control of the cell differentiation.

Molecularly Imprinted Nanoparticles (NanoMIPs) Selective for Proteins: Optimization of a Protocol for Solid-Phase Synthesis Using Automatic Chemical Reactor.

Molecularly imprinted polymer nanoparticles (nanoMIPs) are receiving broad interest as robust and highly selective synthetic receptors for a variety of molecules. Due to their stability, inexpensive synthesis and easy implementation, they are considered a promising alternative to antibodies in sensors, diagnostics and separation applications. The most challenging targets for the production of synthetic receptors are proteins due to their fragile nature and the multitude of possible binding sites in their structure. Herein, we describe the modification and optimization of the protocol for synthesis of nanoMIPs with specificity for proteins using the prototype of an automated solid-phase synthesizer. Using an automated system gives an advantage for the simple, fast and fully controlled, reproducible production of nanoMIPs. The molecular imprinting in the reactor is performed using a template covalently immobilized on a solid support, in mild conditions suitable for preserving protein native structure. The validation of the protocol was made by assessing the ability to regenerate a solid-phase, and by measuring affinity and specificity of nanoparticles. As a model protein, we have chosen trypsin since its enzymatic activity can be easily monitored by using a commercial colorimetric assay. Different protocols were tested for their ability to improve the yield of high affinity nanoparticles in the final elution.