Marloes Peeters

How controlled and versatile is N-carboxy anhydride (NCA) polymerization at 0 °C? Effect of temperature on homo-, block- and graft (co)polymerization

We have investigated the polymerization of various amino acid N-carboxyanhydrides (NCAs) at 0 °C. Detailed MALDI-ToF analysis of homopolymerizations of three amino acid NCAs clearly confirms that frequently occurring end-group termination and other side-reactions are absent at 0 °C. The polymerization is thus controlled and homo and copolypeptides with low polydispersities, around 1.1, were obtained for soluble polypeptides. MALDI-ToF contour plot analysis confirmed the randomness of the copolymers. The controlled character of the low temperature NCA polymerization was further verified by the successful block copolymer synthesis from polypeptide macroinitiators, which confirms the availability of the amino end-groups for chain extension. Moreover, graft copolymers were obtained by grafting of benzyl-L-glutamate NCA from lysine-containing copolymers. The formation of gels from the graft and corresponding block copolymers was investigated. While for the block copolymers interconnected polymer particles were observed by optical microscopy, the graft copolymers form an open cell structure.

Selective enzymatic degradation of self-assembled particles from amphiphilic block copolymers obtained by the combination of N-carboxyanhydride and nitroxide-mediated polymerization.

Combining controlled radical polymerizations and a controlled polypeptide synthetic technique, such as N-carboxyanhydride (NCA) ring-opening polymerization, enables the generation of well-defined block copolymers to be easily accessible. Here we combine NCA polymerization with the nitroxide-mediated radical polymerization of poly(n-butyl acrylate) (PBA) and polystyrene (PS), using a TIPNO and SG1-based bifunctional initiator to create a hybrid block copolymer. The polypeptide block consists of (block) copolymers of poly(L-glutamic acid) embedded with various quantities of L-alanine. The formed superstructures (vesicles and micelles) of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, resulting in controlled enzymatic degradation dictated by the polypeptide composition. The PBA containing block copolymers possessing 50% L-alanine in the polypeptide block showed a high degradation response compared to polymers containing lower L-alanine quantities. The particles stabilized by copolypeptides with L-alanine near the hydrophobic block showed full degradation within 4 days. Particles containing polystyrene blocks revealed no appreciable degradation under the same conditions, highlighting the specificity of the system and the importance of synthetic polymer selection. However, when the degradation temperature was increased to 70 °C, degradation could be achieved due to the higher block copolymer exchange between the particle and the solution. A number of novel biohybrid structures are disclosed that show promise as enzyme-responsive materials with potential use as payload release vehicles, following their controlled degradation by specific, target, enzymes.

Impedimetric detection of histamine in bowel fluids using synthetic receptors with pH-optimized binding characteristics

Histamine is a biogenic amine that is indispensable in the efficient functioning of various physiological systems. In previous work, a molecularly imprinted polymer (MIP) based sensor platform with impedimetric read-out was presented which could rapidly and at low cost determine histamine concentrations in buffer solutions within pH 7-9. For diagnostic applications, histamine should be detectable in a wider pH range as it mostly occurs in mildly acidic environments. To understand this pH-dependent response of the MIP sensor, we propose a statistical binding analysis model. Within this model, we predict the theoretical performance of MIP based on acrylic acid in the required pH range and verify these results experimentally by UV-vis spectroscopy, microgravimetry, and impedance spectroscopy. Using impedimetric read-out, specific and selective detection of histamine in the physiologically relevant nanomolar concentration range is possible in neutral and mildly acidic phosphate buffer. Finally, this sensor platform was used to analyze the histamine concentration of mildly acidic bowel fluid samples of several test persons. We show that this sensor provides reliable data in the relevant concentration regime, which was validated independently by enzyme-linked immuno sorbent assay (ELISA) tests.

Heat-transfer-based detection of L-nicotine, histamine, and serotonin using molecularly imprinted polymers as biomimetic receptors

AbstractIn this work, we will present a novel approach for the detection of small molecules with molecularly imprinted polymer (MIP)-type receptors. This heat-transfer method (HTM) is based on the change in heat-transfer resistance imposed upon binding of target molecules to the MIP nanocavities. Simultaneously with that technique, the impedance is measured to validate the results. For proof-of-principle purposes, aluminum electrodes are functionalized with MIP particles, and l-nicotine measurements are performed in phosphate-buffered saline solutions. To determine if this could be extended to other templates, histamine and serotonin samples in buffer solutions are also studied. The developed sensor platform is proven to be specific for a variety of target molecules, which is in agreement with impedance spectroscopy reference tests. In addition, detection limits in the nanomolar range could be achieved, which is well within the physiologically relevant concentration regime. These limits are comparable to impedance spectroscopy, which is considered one of the state-of-the-art techniques for the analysis of small molecules with MIPs. As a first demonstration of the applicability in biological samples, measurements are performed on saliva samples spiked with l-nicotine. In summary, the combination of MIPs with HTM as a novel readout technique enables fast and low-cost measurements in buffer solutions with the possibility of extending to biological samples. FigureHeat-transfer based detection with molecularly imprinted polymers

The Heat-Transfer Method: A Versatile Low-Cost, Label-Free, Fast, and User-Friendly Readout Platform for Biosensor Applications

In recent years, biosensors have become increasingly important in various scientific domains including medicine, biology, and pharmacology, resulting in an increased demand for fast and effective readout techniques. In this Spotlight on Applications, we report on the recently developed heat-transfer method (HTM) and illustrate the use of the technique by zooming in on four established bio(mimetic) sensor applications: (i) mutation analysis in DNA sequences, (ii) cancer cell identification through surface-imprinted polymers, (iii) detection of neurotransmitters with molecularly imprinted polymers, and (iv) phase-transition analysis in lipid vesicle layers. The methodology is based on changes in heat-transfer resistance at a functionalized solid-liquid interface. To this extent, the device applies a temperature gradient over this interface and monitors the temperature underneath and above the functionalized chip in time. The heat-transfer resistance can be obtained by dividing this temperature gradient by the power needed to achieve a programmed temperature. The low-cost, fast, label-free and user-friendly nature of the technology in combination with a high degree of specificity, selectivity, and sensitivity makes HTM a promising sensor technology.

Label-free Protein Detection Based on the Heat-Transfer Method-A Case Study with the Peanut Allergen Ara h 1 and Aptamer-Based Synthetic Receptors

Aptamers are an emerging class of molecules that, because of the development of the systematic evolution of ligands by exponential enrichment (SELEX) process, can recognize virtually every target ranging from ions, to proteins, and even whole cells. Although there are many techniques capable of detecting template molecules with aptamer-based systems with high specificity and selectivity, they lack the possibility of integrating them into a compact and portable biosensor setup. Therefore, we will present the heat-transfer method (HTM) as an interesting alternative because this offers detection in a fast and low-cost manner and has the possibility of performing experiments with a fully integrated device. This concept has been demonstrated for a variety of applications including DNA mutation analysis and screening of cancer cells. To the best our knowledge, this is the first report on HTM-based detection of proteins, in this case specifically with aptamer-type receptors. For proof-of-principle purposes, measurements will be performed with the peanut allergen Ara h 1 and results indicate detection limits in the lower nanomolar regime in buffer liquid. As a first proof-of-application, spiked Ara h 1 solutions will be studied in a food matrix of dissolved peanut butter. Reference experiments with the quartz-crystal microbalance will allow for an estimate of the areal density of aptamer molecules on the sensor-chip surface.

Molecularly imprinted polymers as synthetic receptors for the QCM-D-based detection of l -nicotine in diluted saliva and urine samples

AbstractMolecularly imprinted polymers (MIPs) are synthetic receptors that are able to specifically bind their target molecules in complex samples, making them a versatile tool in biosensor technology. The combination of MIPs as a recognition element with quartz crystal microbalances (QCM-D with dissipation monitoring) gives a straightforward and sensitive device, which can simultaneously measure frequency and dissipation changes. In this work, bulk-polymerized l-nicotine MIPs were used to test the feasibility of l-nicotine detection in saliva and urine samples. First, l-nicotine-spiked saliva and urine were measured after dilution in demineralized water and 0.1× phosphate-buffered saline solution for proof-of-concept purposes. l-nicotine could indeed be detected specifically in the biologically relevant micromolar concentration range. After successfully testing on spiked samples, saliva was analyzed, which was collected during chewing of either nicotine tablets with different concentrations or of smokeless tobacco. The MIPs in combination with QCM-D were able to distinguish clearly between these samples: This proves the functioning of the concept with saliva, which mediates the oral uptake of nicotine as an alternative to the consumption of cigarettes. FigureSchematics of the sample-preparation procedure for l-nicotine spiked saliva- and urine samples with various concentration levels

Fluorescein functionalized random amino acid copolymers in the biomimetic synthesis of CaCO3

In nature we find a large variety of biocomposites with complex structures to fulfill structural functions such as skeletal support and protection of soft tissues. For calcium carbonate based biominerals, acidic proteins have been reported to be instrumental in controlling the mineral formation process, including the control over nucleation, growth and polymorph selection. To unravel the crucial physicochemical characteristics of these biopolymers, we have synthesized random amino acid copolymers with varying Glu/Asp/Ala ratios using N-carboxy anhydride ring opening polymerization (NCA ROP) and a post-polymerization modification with fluorescein. As these polymers lack a specific order in their amino acid sequence, as well as a defined secondary structure under mineralization conditions, they can only influence the mineralization reactions through their amino acid composition which decides the Glu/Asp ratio and the hydrophilic/hydrophobic balance. Where P(Asp-co-Ala) produced dog-bone like crystals elongated along the crystallographic c-axis, P(Glu-co-Ala) led to the formation of rounded calcite. Also for P(Glu-co-Asp-co-Ala) the calcite crystals were elongated along the c-axis but they were overgrown with rounded calcite rhombs, crystallographically aligned with the elongated core. Hence, the ter-copolymer combines the effects of the two di-copolymers, leading to single crystals with unusual complex morphologies. The presence of a fluorescent group allowed us to study the location of the polymers in the mineral phase using fluorescence microscopy. This demonstrated that while controlling the nucleation and growth of calcite, all polymers were also incorporated within the crystals.

Array formatting of the heat-transfer method (HTM) for the detection of small organic molecules by molecularly imprinted polymers.

In this work we present the first steps towards a molecularly imprinted polymer (MIP)-based biomimetic sensor array for the detection of small organic molecules via the heat-transfer method (HTM). HTM relies on the change in thermal resistance upon binding of the target molecule to the MIP-type receptor. A flow-through sensor cell was developed, which is segmented into four quadrants with a volume of 2.5 μL each, allowing four measurements to be done simultaneously on a single substrate. Verification measurements were conducted, in which all quadrants received a uniform treatment and all four channels exhibited a similar response. Subsequently, measurements were performed in quadrants, which were functionalized with different MIP particles. Each of these quadrants was exposed to the same buffer solution, spiked with different molecules, according to the MIP under analysis. With the flow cell design we could discriminate between similar small organic molecules and observed no significant cross-selectivity. Therefore, the MIP array sensor platform with HTM as a readout technique, has the potential to become a low-cost analysis tool for bioanalytical applications.

Optimizing the thermal read-out technique for MIP-based biomimetic sensors: towards nanomolar detection limits.

In previous work, the novel heat-transfer method (HTM) for the detection of small molecules with Molecularly Imprinted Polymers (MIP)-type receptors was presented. In this study we focus on optimization of this sensor performance, with as final aim to lower the detection limit by reducing the noise level. It was determined that the noise originates foremost from the power supply, which can be controlled by varying the PID parameters. Therefore, the effect of the individual parameters was evaluated by tuning P, I and D separately at a temperature of 37 °C, giving a first indication of the optimal configuration. Next, a temperature profile was programmed and the standard deviation of the heat-transfer resistance over the entire regime was studied for a set of parameters. The optimal configuration, P1-I6-D0, reduced the noise level with nearly a factor of three compared to the original parameters of P10-I5-D0. With the optimized settings, the detection of L-nicotine in buffer solutions was studied and the detection limit improved significantly from 100 nM to 35 nM. Summarizing, optimization of the PID parameters and thereby improving the detection limit is a key parameter for first applications of the HTM-method for MIP receptors in analytical research.