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In recent years, melamine-sensing technologies have increasingly gained attention, mainly due to the misuse of the molecule as an adulterant in milk and other foods. Molecularly imprinted polymers (MIPs) are ideal candidates for the recognition of melamine in real-life samples. The prepared MIP particles were incorporated into a thermally conductive layer via micro-contact deposition and its response towards melamine was analyzed using the heat-transfer method (HTM). The sensor displayed an excellent selectivity when analyzing the thermal response to other chemicals commonly found in foods, and its applicability in food safety was demonstrated after evaluation in untreated milk samples, demonstrating a limit of detection of 6.02 μM. As the EU/US melamine legal limit in milk of 2.5 mg/kg falls within the linear range of the sensor, it can offer an innovative solution for routine screening of milk samples in order to detect adulteration with melamine. The results shown in this work thus demonstrate the great potential of a low-cost thermal platform for the detection of food adulteration in complex matrices. [ABSTRACT FROM AUTHOR]

In recent years, melamine-sensing technologies have increasingly gained attention, mainly due to the misuse of the molecule as an adulterant in milk and other foods. Molecularly imprinted polymers (MIPs) are ideal candidates for the recognition of melamine in real-life samples. The prepared MIP particles were incorporated into a thermally conductive layer via micro-contact deposition and its response towards melamine was analyzed using the heat-transfer method (HTM). The sensor displayed an excellent selectivity when analyzing the thermal response to other chemicals commonly found in foods, and its applicability in food safety was demonstrated after evaluation in untreated milk samples, demonstrating a limit of detection of 6.02 μM. As the EU/US melamine legal limit in milk of 2.5 mg/kg falls within the linear range of the sensor, it can offer an innovative solution for routine screening of milk samples in order to detect adulteration with melamine. The results shown in this work thus demonstrate the great potential of a low-cost thermal platform for the detection of food adulteration in complex matrices.

Highlights • Preparation and optimization of MIPs for detection of caffeine. • First evaluation and optimization of the temperature dependence of the HTM. • Thermal detection caffeine with HTM and TWTA. • First proof of concept for measurements in complicated samples such as beverages. • Ability to fine-tune limits of detection by changing the temperature. Abstract Molecularly Imprinted Polymers (MIPs) are synthesized for the selective detection of caffeine. The polymerization process, monomer and crosslinker monomer composition are varied to determine the optimal synthesis procedure via batch rebinding experiments evaluated with optical detection. The selectivity is tested by comparing the response of caffeine to compounds with similar chemical structures (theophylline and theobromine) and dopamine, another neurotransmitter. Subsequently, the MIP polymer particles are integrated into bulk modified MIP screen-printed electrodes (MIP-modified SPEs). The sensors are used to measure caffeine content in various samples employing the Heat-Transfer Method (HTM), a low-cost and simple thermal detection method that is based on differences in thermal resistance at the solid-liquid interface. At first, the noise is minimized by adjusting the settings of temperature feedback loop. Second, the response of the MIP-modified SPE is studied at various temperatures ranging from 37 to 50 and 85 °C. The binding to MIP-modified SPEs has never been studied at elevated temperatures since most biomolecules are not stable at those temperatures. Using caffeine as proof-of-concept, it is demonstrated that at 85 °C the detection limit is significantly enhanced due to higher signal to noise ratios and enhanced diffusion of the biomolecule. Thermal wave transport analysis (TWTA) is also optimized at 85 °C producing a limit of detection of ∼1 nM. Next, MIP-modified SPEs are used to measure the caffeine concentration in complex samples including caffeinated beverages, spiked tap water and waste water samples. The use of MIP-modified SPEs combined with thermal detection provides sensors that can be used for fast and low-cost detection performed on-site, which holds great potential for the determination of contaminants in environmental samples. The platform is generic and by adapting the MIP layer, we can expand to this a range of relevant targets. [ABSTRACT FROM AUTHOR]

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.

Molecularly Imprinted Polymers (MIPs) are synthesized for the neurotransmitter noradrenaline with the optimal composition and binding conditions being determined via optical batch rebinding experiments. Next, the obtained MIP polymer particles are mixed within screen-printed inks to produce mass-producible bulk modified MIPs screen-printed electrodes (MIP-SPEs). In this contribution, the supporting surface which the MIP-SPEs are screen-printed upon are explored to deviate from conventional polyester, to polyvinylchloride, tracing paper and household-printing paper. The performance of the MIP-SPEs are measured using the Heat-Transfer Method (HTM), a straightforward and low-cost detection technique based on thermal resistance. At first, the noise on the signal is minimized by adjusting the settings of the temperature feedback loop. Second, the response of the MIP-SPEs to noradrenaline is measured and compared for the different substrate materials. Sensors printed onto paper are considered in further experiments as their response to noradrenaline is the highest and advantageous material properties, including sustainability and flexibility of the material. Subsequently, dose-response curves are determined by simultaneously measuring HTM and Thermal Wave Transport Analysis (TWTA). The latter is a new thermal detection method that relies on the use of thermal waves and has the advantage of a short measurement time (2 min). With these thermal methods, it is possible to specifically detect noradrenaline in aqueous solutions and quantify it at relevant concentrations. In summary, by combining synthetic receptors with thermal measurement techniques it is possible to develop a portable sensor platform that is capable of low-cost and straightforward detection of biomolecules. Through exploring novel SPE substrates, a system is designed that is flexible and holds potential for the use in commercial biomedical devices and complex sensor architectures. [ABSTRACT FROM AUTHOR]

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.

A novel procedure is developed for producing bulk modified Molecularly Imprinted Polymer (MIP) screen-printed electrodes (SPEs), which involves the direct mixing of the polymer particles within the screen-printed ink. This allowed reduction of the sample preparation time from 45 min to 1 min, and resulted in higher reproducibility of the electrodes. The samples are measured with a novel detection method, namely, thermal wave transport analysis (TWTA), relying on the analysis of thermal waves through a functional interface. As a first proof-of-principle, MIPs for dopamine are developed and successfully incorporated within a bulk modified MIP SPE. The detection limits of dopamine within buffer solutions for the MIP SPEs are determined via three independent techniques. With cyclic voltammetry this was determined to be 4.7 x 10-6 M, whereas by using the heat-transfer method (HTM) 0.35 x 10-6 M was obtained, and with the novel TWTA concept 0.26 x 10-6 M is possible. This TWTA technique is measured simultaneously with HTM and has the benefits of reducing measurement time to less than 5 min and increasing effect size by nearly a factor of two. The two thermal methods are able to enhance dopamine detection by one order of magnitude compared to the electrochemical method. In previous research, it was not possible to measure neurotransmitters in complex samples with HTM, but with the improved signal-to-noise of TWTA for the first time, spiked dopamine concentrations were determined in a relevant food sample. In summary, novel concepts are presented for both the sensor functionalization side by employing screen-printing technology, and on the sensing side, the novel TWTA thermal technique is reported. The developed bio-sensing platform is cost-effective and suitable for mass-production due to the nature of screen-printing technology, which makes it very interesting for neurotransmitter detection in clinical diagnostic applications. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a ubiquitous multi-drug-resistant bacterium, capable of causing serious illnesses and infections. This research focuses on the development of a thermal sensor for the indirect detection of P. aeruginosa infection using molecularly imprinted polymers (MIPs). This was achieved by developing MIPs for the detection of pyocyanin, the main toxin secreted by P. aeruginosa . To this end, phenazine was used as a dummy template, evaluating several polymeric compositions to achieve a selective MIP for pyocyanin recognition. The sensitivity of the synthesized MIPs was investigated by UV-vis analysis, with the best composition having a maximum rebinding capacity of 30 μmol g -1 and an imprinting factor (IF) of 1.59. Subsequently, the MIP particles were immobilized onto planar aluminum chips using an adhesive layer, to perform thermal resistance measurements at clinically relevant concentrations of pyocyanin (1.4-9.8 μM), achieving a limit of detection (LoD) of 0.347 ± 0.027 μM. The selectivity of the sensor was also scrutinized by subjecting the receptor to potential interferents. Furthermore, the rebinding was demonstrated in King's A medium, highlighting the potential of the sensor for the indirect detection of P. aeruginosa in complex fluids. The research culminates in the demonstration of the MIP-based sensor's applicability for clinical diagnosis. To achieve this goal, an experiment was performed in which the sensor was exposed to pyocyanin-spiked saliva samples, achieving a limit of detection of 0.569 ± 0.063 μM and demonstrating that this technology is suitable to detect the presence of the toxin even at the very first stage of its production.

In this article, we increased the sensitivity of the heat-transfer method (HTM) for the detection of breast cancer cells (ZR-75-1 cells, see figure) in phosphate buffered saline (PBS). The effect of small technological changes on the limit of detection (LoD) of the methodology was examined. To this extent, polished aluminum substrates with a mirror finish were used, replacing the unpolished chips used in previous studies. These chips were coated with a polyurethane layer and imprinted for the target cell type, creating a so-called surface imprinted-polymer (SIP). Binding of target cells to the SIP resulted in an increase of the thermal resistance at the solid-liquid interface under study. Background thermal resistance measurements were performed with polished and unpolished aluminum substrates. In addition, the effect of using silver paste as thermal coupling between the aluminum chip and the copper heat provider was analyzed. The results of these experiments reveal that optimal thermal contact is achieved when directly coupling the copper heat provider to the polished side of the aluminum substrate as evidenced by a decrease in the baseline thermal resistance. In addition, noise levels on the heat-transfer resistance ( Rth) signal decreased by a factor in the optimal configuration. Dose-response curves were obtained using the optimized methodology and were compared with results obtained with the original substrates. These quantitative experiments demonstrated an improvement of the LoD by approximately thirty percent. ZR-75-1 cells applied onto a home-made rubber stamp. [ABSTRACT FROM AUTHOR]

A novel procedure is developed for producing bulk modified Molecularly Imprinted Polymer (MIP) screen-printed electrodes (SPEs), which involves the direct mixing of the polymer particles within the screen-printed ink. This allowed reduction of the sample preparation time from 45 min to 1 min, and resulted in higher reproducibility of the electrodes. The samples are measured with a novel detection method, namely, thermal wave transport analysis (TWTA), relying on the analysis of thermal waves through a functional interface. As a first proof-of-principle, MIPs for dopamine are developed and successfully incorporated within a bulk modified MIP SPE. The detection limits of dopamine within buffer solutions for the MIP SPEs are determined via three independent techniques. With cyclic voltammetry this was determined to be 4.7 × 10−6 M, whereas by using the heat-transfer method (HTM) 0.35 × 10−6 M was obtained, and with the novel TWTA concept 0.26 × 10−6 M is possible. This TWTA technique is measured simultaneously with HTM and has the benefits of reducing measurement time to less than 5 min and increasing effect size by nearly a factor of two. The two thermal methods are able to enhance dopamine detection by one order of magnitude compared to the electrochemical method. In previous research, it was not possible to measure neurotransmitters in complex samples with HTM, but with the improved signal-to-noise of TWTA for the first time, spiked dopamine concentrations were determined in a relevant food sample. In summary, novel concepts are presented for both the sensor functionalization side by employing screen-printing technology, and on the sensing side, the novel TWTA thermal technique is reported. The developed bio-sensing platform is cost-effective and suitable for mass-production due to the nature of screen-printing technology, which makes it very interesting for neurotransmitter detection in clinical diagnostic applications.

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. [ABSTRACT FROM AUTHOR]

In 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. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We demonstrate that a novel functionalized interface, where molecularly imprinted polymer nanoparticles (nanoMIPs) are attached to screen-printed graphite electrodes (SPEs), can be utilized for the thermal detection of the cardiac biomarker troponin I (cTnI). The ultrasensitive detection of the unique protein cTnI can be utilized for the early diagnosis of myocardial infraction (i.e., heart attacks), resulting in considerably lower patient mortality and morbidity. Our developed platform presents an innovative route to develop accurate, low-cost, and disposable sensors for the diagnosis of cardiovascular diseases, specifically myocardial infraction. A reproducible and advantageous solid-phase approach was utilized to synthesize high-affinity nanoMIPs (average size = 71 nm) for cTnI, which served as synthetic receptors in a thermal sensing platform. To assess the performance and commercial potential of the sensor platform, various approaches were used to immobilize nanoMIPs onto thermocouples or SPEs: dip coating, drop casting, and a covalent approach relying on electrografting with an organic coupling reaction. Characterization of the nanoMIP-functionalized surfaces was performed with electrochemical impedance spectroscopy, atomic force microscopy, and scanning electron microscopy. Measurements from an in-house designed thermal setup revealed that covalent functionalization of nanoMIPs onto SPEs led to the most reproducible sensing capabilities. The proof of application was provided by measuring buffered solutions spiked with cTnI, which demonstrated that through monitoring changes in heat transfer at the solid-liquid interface, we can measure concentrations as low as 10 pg L -1 , resulting in the most sensitive test of this type. Furthermore, preliminary data are presented for a prototype platform, which can detect cTnI with shorter measurement times and smaller sample volumes. The excellent sensor performance, versatility of the nanoMIPs, and reproducible and low-cost nature of the SPEs demonstrate that this sensor platform technology has a clear commercial route with high potential to contribute to sustainable healthcare.

Molecularly imprinted polymers (MIPs), synthetic polymeric receptors, have been combined successfully with thermal transducers for the detection of small molecules in recent years. However, up until now they have been combined with planar electrodes which limits their use for in vivo applications. In this work, a new biosensor platform is developed by roll-coating MIP particles onto thermocouples, functionalized with polylactic acid (PLLA). As a first proof-of-principle, MIPs for the neurotransmitter dopamine were incorporated into PLLA-coated thermocouples. The response of the synthetic receptor layer to an increasing concentration of dopamine in buffer was analyzed using a homemade heat-transfer setup. Binding of the template to the MIP layer blocks the heat transport through the thermocouple, leading to less heat loss to the environment and an overall higher temperature in the measuring chamber. The measured temperature increase is correlated to the neurotransmitter concentration, which enables measurement of dopamine levels in the micromolar regime. To demonstrate the general applicability of the proposed biosensor platform, thermocouples were functionalized with similar MIPs for cortisol and serotonin, indicating a similar response and limit-of-detection. As the platform does not require planar electrodes, it can easily be integrated in, e.g., a catheter. In this way, it is an excellent fit for the current niche in the market of therapeutics and diagnostics. Moreover, the use of a biocompatible and disposable PLLA-layer further illustrates its potential for in vivo diagnostics.

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.