Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA - development of a novel assay for vancomycin

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop ELISA type assays is presented here for the first time. NanoMIPs were synthesized by a solid phase approach with immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering and electron microscopy. Immobilization, blocking and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a HRP-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was three orders of magnitude better than a previously described ELISA based on antibodies. In these experiments nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELIS

Surface plasmon resonance based sensor for the detection of glycopeptide antibiotics in milk using rationally designed nanoMIPs

Glycopeptide antibiotics are known as the last resort for the treatment of serious infections caused by Gram-positive bacteria. The use of milk products contaminated with these antibiotic residues leads to allergic reactions and sensitivity in human. Also, long-term consumption of milk products containing low levels of these antibiotics may cause the relevant bacteria to build up resistance to these last resort antibiotics. Sensitive, rapid and effective quantification and monitoring systems play a key role for their determination in milk products. Hence, molecularly imprinted nanostructures were rationally designed in this work to produce high affinity synthetic receptors to be coupled with a surface plasmon resonance sensor for the analysis of glycopeptide antibiotics in milk samples. The nanoMIP-SPR sensor enabled vancomycin quantification with the LODs of 4.1 ng mL−1 and 17.7 ng mL−1 using direct and competitive assays, respectively. The recoveries rates for two sensor methods ranged in 85–110% with RSDs below 7%. The affinity between the nanoMIP receptors and the target molecule (dissociation constant: 1.8 × 10−9 M) is mostly superior to natural receptors and other synthetic receptors. Unlike other methods commonly employed for the detection of milk contaminants this approach is extremely simple, fast and robust, and do not require pre-sample treatment.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli

Soft molecularly imprinted nanoparticles with simultaneous lossy mode and surface plasmon multi-resonances for femtomolar sensing of serum transferrin protein

: The simultaneous interrogation of both lossy mode (LMR) and surface plasmon (SPR) resonances was herein exploited for the first time to devise a sensor in combination with soft molecularly imprinting of nanoparticles (nanoMIPs), specifically entailed of the selectivity towards the protein biomarker human serum transferrin (HTR). Two distinct metal-oxide bilayers, i.e. TiO2-ZrO2 and ZrO2-TiO2, were used in the SPR-LMR sensing platforms. The responses to binding of the target protein HTR of both sensing configurations (TiO2-ZrO2-Au-nanoMIPs, ZrO2-TiO2-Au-nanoMIPs) showed femtomolar HTR detection, LODs of tens of fM and KDapp ~ 30 fM. Selectivity for HTR was demonstrated. The SPR interrogation was more efficient for the ZrO2-TiO2-Au-nanoMIPs configuration (sensitivity at low concentrations, S = 0.108 nm/fM) than for the TiO2-ZrO2-Au-nanoMIPs one (S = 0.061 nm/fM); while LMR was more efficient for TiO2-ZrO2-Au-nanoMIPs (S = 0.396 nm/fM) than for ZrO2-TiO2-Au-nanoMIPs (S = 0.177 nm/fM). The simultaneous resonance monitoring is advantageous for point of care determinations, both in terms of measurement's redundancy, that enables the cross-control of the measure and the optimization of the detection, by exploiting the individual characteristics of each resonance

Time-Resolved Fluorescence Spectroscopy of Molecularly Imprinted Nanoprobes as an Ultralow Detection Nanosensing Tool for Protein Contaminants

: Currently, optical sensors based on molecularly imprinted polymers (MIPs) have been attracting significant interest. MIP sensing relies on the combination of the MIP's selective capability, which is conveyed to the polymeric material by a template-assisted synthesis, with optical techniques that offer exquisite sensitivity. In this work, we devised an MIP nanoparticle optical sensor for the ultralow detection of serum albumin through time-resolved fluorescence spectroscopy. The Fluo-nanoMIPs (∅~120 nm) were synthetized using fluorescein-O-methacrylate (0.1×, 1×, 10× mol:mol versus template) as an organic fluorescent reporter. The ability of 0.1× and 1×Fluo-nanoMIPs to bind albumin (15 fM-150 nM) was confirmed by fluorescence intensity analyses and isothermal titration calorimetry. The apparent dissociation constant (Kapp) was 30 pM. Conversely, the 10× fluorophore content did not enable monitoring binding. Then, the time-resolved fluorescence spectroscopy of the nanosensors was studied. The 1×Fluo-nanoMIPs showed a decrease in fluorescence lifetime upon binding to albumin (100 fM-150 nM), Kapp = 28 pM, linear dynamic range 3.0-83.5 pM, limit of detection (LOD) 1.26 pM. Selectivity was confirmed testing 1×Fluo-nanoMIPs against competitor proteins. Finally, as a proof of concept, the nanosensors demonstrated detection of the albumin (1.5 nM) spiked in wine samples, suggesting a possible scaling up of the method in monitoring allergens in wines

Electrochemical and thermal detection of allergenic substance lysozyme with molecularly imprinted nanoparticles

Lysozyme (LYZ) is a small cationic protein which is widely used for medical treatment and in the food industry to act as an anti-bacterial agent; however, it can trigger allergic reactions. In this study, high-affinity molecularly imprinted nanoparticles (nanoMIPs) were synthesized for LYZ using a solid-phase approach. The produced nanoMIPs were electrografted to screen-printed electrodes (SPEs), disposable electrodes with high commercial potential, to enable electrochemical and thermal sensing. Electrochemical impedance spectroscopy (EIS) facilitated fast measurement (5–10 min) and is able to determine trace levels of LYZ (pM) and can discriminate between LYZ and structurally similar proteins (bovine serum albumin, troponin-I). In tandem, thermal analysis was conducted with the heat transfer method (HTM), which is based on monitoring the heat transfer resistance at the solid–liquid interface of the functionalized SPE. HTM as detection technique guaranteed trace-level (fM) detection of LYZ but needed longer analysis time compared to EIS measurement (30 min vs 5–10 min). Considering the versatility of the nanoMIPs which can be adapted to virtually any target of interest, these low-cost point-of-care sensors hold great potential to improve food safety

Synthesis of Molecularly Imprinted Polymer Nanoparticles for SARS-CoV-2 Virus Detection Using Surface Plasmon Resonance

COVID-19 caused by a SARS-CoV-2 infection was first reported from Wuhan, China, and later recognized as a pandemic on March 11, 2020, by the World Health Organization (WHO). Gold standard nucleic acid and molecular-based testing have largely satisfied the requirements of early diagnosis and management of this infectious disease; however, these techniques are expensive and not readily available for point-of-care (POC) applications. The COVID-19 pandemic of the 21st century has emphasized that medicine is in dire need of advanced, rapid, and cheap diagnostic tools. Herein, we report on molecularly imprinted polymer nanoparticles (MIP-NPs/nanoMIPs) as plastic antibodies for the specific detection of SARS-CoV-2 by employing a surface plasmon resonance (SPR) sensor. High-affinity MIP-NPs directed against SARS-CoV-2 were manufactured using a solid-phase imprinting method. The MIP-NPs were then characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM) prior to their incorporation into a label-free portable SPR device. Detection of SARS-CoV-2 was studied within a range of 104–106 PFU mL−1. The MIP-NPs demonstrated good binding affinity (KD = 0.12 pM) and selectivity toward SARS-CoV-2. The AFM, cyclic voltammetry, and square-wave voltammetry studies revealed the successful stepwise preparation of the sensor. A cross-reactivity test confirmed the specificity of the sensor. For the first time, this study demonstrates the potential of molecular imprinting technology in conjunction with miniaturized SPR devices for the detection of SARS-CoV-2 particles with high-affinity and specificity. Such sensors could help monitor and manage the risks related to virus contamination and infections also beyond the current pandemic

A Novel NanoMIP-SPR Sensor for the Point-of-Care Diagnosis of Breast Cancer

Simple, fast, selective, and reliable detection of human epidermal growth factor receptor 2 (HER2) is of utmost importance in the early diagnosis of breast cancer to prevent its high prevalence and mortality. Molecularly imprinted polymers (MIPs), also known as artificial antibodies, have recently been used as a specific tool in cancer diagnosis and therapy. In this study, a miniaturized surface plasmon resonance (SPR)-based sensor was developed using epitope-mediated HER2-nanoMIPs. The nanoMIP receptors were characterized using dynamic light scattering (DLS), zeta potential, Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), and fluorescent microscopy. The average size of the nanoMIPs was determined to be 67.5 ± 12.5 nm. The proposed novel SPR sensor provided superior selectivity to HER2 with a detection limit (LOD) of 11.6 pg mL-1 in human serum. The high specificity of the sensor was confirmed by cross-reactivity studies using P53, human serum albumin (HSA), transferrin, and glucose. The sensor preparation steps were successfully characterized by employing cyclic and square wave voltammetry. The nanoMIP-SPR sensor demonstrates great potential for use in the early diagnosis of breast cancer as a robust tool with high sensitivity, selectivity, and specificity

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

Modulation of EGFR activity by molecularly imprinted polymer nanoparticles targeting intracellular epitopes

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy