Kasper Eersels

Selective Identification of Macrophages and Cancer Cells Based on Thermal Transport through Surface-Imprinted Polymer Layers

In this article, we describe a novel straightforward method for the specific identification of viable cells (macrophages and cancer cell lines MCF-7 and Jurkat) in a buffer solution. The detection of the various cell types is based on changes of the heat transfer resistance at the solid-liquid interface of a thermal sensor device induced by binding of the cells to a surface-imprinted polymer layer covering an aluminum chip. We observed that the binding of cells to the polymer layer results in a measurable increase of heat transfer resistance, meaning that the cells act as a thermally insulating layer. The detection limit was found to be on the order of 10(4) cells/mL, and mutual cross-selectivity effects between the cells and different types of imprints were carefully characterized. Finally, a rinsing method was applied, allowing for the specific detection of cancer cells with their respective imprints while the cross-selectivity toward peripheral blood mononuclear cells was negligible. The concept of the sensor platform is fast and low-cost while allowing also for repetitive measurements.

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.

Heat-Transfer-Method-Based Cell Culture Quality Assay through Cell Detection by Surface Imprinted Polymers

Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

Heat-transfer resistance measurement method (HTM)-based cell detection at trace levels using a progressive enrichment approach with highly selective cell-binding surface imprints.

Surface-imprinted polymers allow for specific cell detection based on simultaneous recognition of the cell shape, cell size, and cell membrane functionalities by macromolecular cell imprints. In this study, the specificity of detection and the detection sensitivity for target cells within a pool of non-target cells were analyzed for a cell-specific surface-imprinted polymer combined with a heat-transfer-based read-out technique (HTM). A modified Chinese hamster ovarian cell line (CHO-ldlD) was used as a model system on which the transmembrane protein mucin-1 (MUC1) could be excessively expressed and for which the occurrence of MUC1 glycosylation could be controlled. In specific cancer cells, the overexpressed MUC1 protein typically shows an aberrant apical distribution and glycosylation. We show that surface-imprinted polymers discriminate between cell types that (1) only differ in the expression of a specific membrane protein (MUC1) or (2) only differ in the membrane protein being glycosylated or not. Moreover, surface-imprinted polymers of cells carrying different glycoforms of the same membrane protein do target both types of cells. These findings illustrate the high specificity of cell detection that can be reached by the structural imprinting of cells in polymer layers. Competitiveness between target and non-target cells was proven to negatively affect the detection sensitivity of target cells. Furthermore, we show that the detection sensitivity can be increased significantly by repetitively exposing the surface to the sample and eliminating non-specifically bound cells by flushing between consecutive cell exposures.

Label-Free Detection of Small Organic Molecules by Molecularly Imprinted Polymer Functionalized Thermocouples: Toward In Vivo Applications

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.

Biomimetic Bacterial Identification Platform Based on Thermal Wave Transport Analysis (TWTA) through Surface-Imprinted Polymers

This paper introduces a novel bacterial identification assay based on thermal wave analysis through surface-imprinted polymers (SIPs). Aluminum chips are coated with SIPs, serving as synthetic cell receptors that have been combined previously with the heat-transfer method (HTM) for the selective detection of bacteria. In this work, the concept of bacterial identification is extended toward the detection of nine different bacterial species. In addition, a novel sensing approach, thermal wave transport analysis (TWTA), is introduced, which analyzes the propagation of a thermal wave through a functional interface. The results presented here demonstrate that bacterial rebinding to the SIP layer resulted in a measurable phase shift in the propagated wave, which is most pronounced at a frequency of 0.03 Hz. In this way, the sensor is able to selectively distinguish between the different bacterial species used in this study. Furthermore, a dose–response curve was constructed to determine a limit of detection of 1 × 104 CFU mL–1, indicating that TWTA is advantageous over HTM in terms of sensitivity and response time. Additionally, the limit of selectivity of the sensor was tested in a mixed bacterial solution, containing the target species in the presence of a 99-fold excess of competitor species. Finally, a first application for the sensor in terms of infection diagnosis is presented, revealing that the platform is able to detect bacteria in clinically relevant concentrations as low as 3 × 104 CFU mL–1 in spiked urine samples.

Single-Shot Detection of Neurotransmitters in Whole-Blood Samples by Means of the Heat-Transfer Method in Combination with Synthetic Receptors

Serotonin is an important neurotransmitter that plays a major role in the pathogenesis of a variety of conditions, including psychiatric disorders. The detection of serotonin typically relies on high-performance liquid chromatography (HPLC), an expensive technique that requires sophisticated equipment and trained personnel, and is not suitable for point-of-care applications. In this contribution, we introduce a novel sensor platform that can measure spiked neurotransmitter concentrations in whole blood samples in a fast and low-cost manner by combining synthetic receptors with a thermal readout technique—the heat-transfer method. In addition, the design of a miniaturized version of the sensing platform is presented that aims to bridge the gap between measurements in a laboratory setting and point-of-care measurements. This fully automated and integrated, user-friendly design features a capillary pumping unit that is compatible with point-of-care sampling techniques such as a blood lancet device (sample volume—between 50 µL and 300 µL). Sample pre-treatment is limited to the addition of an anti-coagulant. With this fully integrated setup, it is possible to successfully discriminate serotonin from a competitor neurotransmitter (histamine) in whole blood samples. This is the first demonstration of a point-of-care ready device based on synthetic receptors for the screening of neurotransmitters in complex matrices, illustrating the sensor’s potential application in clinical research and diagnosis of e.g., early stage depression.

Molecularly Imprinted Polymers: Synthetic Receptors for Diagnostic Medical Devices

In recent years, molecularly imprinted polymers (MIPs) have gained increasing interest in the bioanalytical field because they are extremely suitable for detecting chemical targets in complex matrices. Their incorporation into sensing devices remains challenging; however, we will present a straightforward sensor platform that is compatible with electrochemical impedance spectroscopy and a novel readout technique, the heat-transfer method. Impedimetric readout allows for specific detection of neurotransmitters in buffer solutions in the subnanomolar regime and was also evaluated for its use in biological samples such as blood plasma and bowel fluid. The heat-transfer method offers a more straightforward analysis compared with impedance spectroscopy, with similar detection limits in buffer solutions, although detection in biological samples still needs to be analyzed. We show a versatile MIP-based sensor platform that can detect small organic molecules in a fast and low-cost manner, opening the door to applications in biological samples.

A Novel Biomimetic Tool for Assessing Vitamin K Status Based on Molecularly Imprinted Polymers

Vitamin K was originally discovered as a cofactor required to activate clotting factors and has recently been shown to play a key role in the regulation of soft tissue calcification. This property of vitamin K has led to an increased interest in novel methods for accurate vitamin K detection. Molecularly Imprinted Polymers (MIPs) could offer a solution, as they have been used as synthetic receptors in a large variety of biomimetic sensors for the detection of similar molecules over the past few decades, because of their robust nature and remarkable selectivity. In this article, the authors introduce a novel imprinting approach to create a MIP that is able to selectively rebind vitamin K1. As the native structure of the vitamin does not allow for imprinting, an alternative imprinting strategy was developed, using the synthetic compound menadione (vitamin K3) as a template. Target rebinding was analyzed by means of UV-visible (UV-VIS) spectroscopy and two custom-made thermal readout techniques. This analysis reveals that the MIP-based sensor reacts to an increasing concentration of both menadione and vitamin K1. The Limit of Detection (LoD) for both compounds was established at 700 nM for the Heat Transfer Method (HTM), while the optimized readout approach, Thermal Wave Transport Analysis (TWTA), displayed an increased sensitivity with a LoD of 200 nM. The sensor seems to react to a lesser extent to Vitamin E, the analogue under study. To further demonstrate its potential application in biochemical research, the sensor was used to measure the absorption of vitamin K in blood serum after taking vitamin K supplements. By employing a gradual enrichment strategy, the sensor was able to detect the difference between baseline and peak absorption samples and was able to quantify the vitamin K concentration in good agreement with a validation experiment using High-Performance Liquid Chromatography (HPLC). In this way, the authors provide a first proof of principle for a low-cost, straightforward, and label-free vitamin K sensor.

Studying the Drug Delivery Kinetics of a Nanoporous Matrix Using a MIP-Based Thermal Sensing Platform

The implementation of Molecularly Imprinted Polymers (MIPs) into sensing systems has been demonstrated abundantly over the past few decades. In this article, a novel application for an MIP-based thermal sensing platform is introduced by using the sensor to characterize the drug release kinetics of a nanoporous silver-organic framework. This Ag nanoporous matrix was loaded with acetylsalicylic acid (aspirin) which was used as a model drug compound in this study. The drug elution properties were studied by placing the nanoporous matrix in phosphate buffered saline solution for two days and measuring the drug concentration at regular time intervals. To this extent, an acrylamide-based MIP was synthesized that was able to detect aspirin in a specific and selective manner. Rebinding of the template to the MIP was analyzed using a thermal sensor platform. The results illustrate that the addition of aspirin into the sensing chamber leads to a concentration-dependent increase in the phase shift of a thermal wave that propagates through the MIP-coated sensor chip. After constructing a dose-response curve, this system was used to study the drug release kinetics of the nanoporous matrix, clearly demonstrating that the metalorganic framework releases the drug steadily over the course of the first hour, after which the concentration reaches a plateau. These findings were further confirmed by UV–Visible spectroscopy, illustrating a similar time-dependent release in the same concentration range, which demonstrates that the MIP-based platform can indeed be used as a low-cost straightforward tool to assess the efficacy of drug delivery systems in a lab environment.