Faculty of Engineering Technology

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 readout was presented which could rapidly and at low cost determine histamine concentrations in buffer solutions within pH 7−9. 1,2 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.

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

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-p...

In search for a better way to monitor the hybridization and denaturation of DNA onto a diamond based sensor, precise knowledge about the conditions of the immediate surroundings is very critical. One of the factors that have a great influence on the stability of the measurements is the temperature of the liquid environment in which these measurements take place. With this as a focal point, the design of a precise temperature regulator based on a boron doped diamond thin film is a key factor to achieve accurate measurements on a standalone basis. In this work temperature control is achieved making use of a thin boron doped nanocrystalline diamond (B-NCD) film, which, in combination with a proportional-integral-derivative-control (PID), is able to maintain a stable temperature with an accuracy better than 0.1 8C. By letting the B-NCD-layer act as a resistor together with the appropriate control it is possible to maintain a stable temperature in a range going from room temperature till 70 8C, with an accuracy exceeding a temperature variation 0.1 8C. The first prototype makes use of a reference temperature sensor, to verify the accuracy of the results.

A new impedance spectroscopy unit is developed, fully customized to become a vital part of label free biosensor arrays with possible applications in DNA and protein sensing. Test measurements are conducted to explore the accuracy and specificity of the system, both under electronic lab conditions as well as under wet cell conditions with synthetic-diamond based sensor electrodes. The impedance of seven resistors was monitored for 17 h and a maximum error <0.02% was found. Furthermore, the impedance of PBS at different concentrations was monitored for 60 min per concentration and a different impedance for each concentration was detected. The impedance is also monitored for NaOH, PBS and nuclease free water at different temperatures with a total duration of 60 min per fluid. Systematically different impedances for each temperature per fluid were found and the temperature coefficients were determined. All test measurements lead to results well within specification.

In this article, we report on the heat-transfer resistance at interfaces as a novel, denaturation-based method to detect single-nucleotide polymorphisms in DNA. We observed that a molecular brush of double-stranded DNA grafted onto synthetic diamond surfaces does not notably affect the heattransfer resistance at the solid-to-liquid interface. In contrast to this, molecular brushes of singlestranded DNA cause, surprisingly, a substantially higher heat-transfer resistance and behave like a thermally insulating layer. This effect can be utilized to identify ds-DNA melting temperatures via the switching from low-to high heat-transfer resistance. The melting temperatures identified with this method for different DNA duplexes (29 base pairs without and with built-in mutations) correlate nicely with data calculated by modeling. The method is fast, label-free (without the need for fluorescent or radioactive markers), allows for repetitive measurements, and can also be extended toward array formats. Reference measurements by confocal fluorescence microscopy and impedance spectroscopy confirm that the switching of heat-transfer resistance upon denaturation is indeed related to the thermal on-chip denaturation of DNA.

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-ofprinciple 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.

A new method is presented for smartphone-based impedance spectroscopy, especially fine-tuned for biomimetic sensor readout. Complete user control is given by means of an app while the on-board audio hardware of the smartphone or tablet PC is used to perform impedance measurements. This considerably limits the required external hardware. Disposable test strips can be mounted for convenient readout of various sensors. The system is verified on passive components and a synthetic molecularly imprinted polymer histamine sensor. The prototype design could prove a useful step toward the development of home-diagnostics biosensing applications.

In this article, we report on the electronic monitoring of DNA denaturation by NaOH using electrochemical impedance spectroscopy in combination with fluorescence imaging as a reference technique. The probe DNA consisting of a 36-mer fragment was covalently immobilized on nanocrystalline-diamond electrodes and hybridized with different types of 29-mer target DNA (complementary, single-nucleotide defects at two different positions, and a non-complementary random sequence). The mathematical separation of the impedimetric signals into the time constant for NaOH exposure and the intrinsic denaturation-time constants gives clear evidence that the denaturation times reflect the intrinsic stability of the DNA duplexes. The intrinsic time constants correlate with calculated DNA-melting temperatures. The impedimetric method requires minimal instrumentation, is label-free and fast with a typical time scale of minutes and is highly reproducible. The sensor electrodes can be used repetitively. These elements suggest that the monitoring of chemically induced denaturation at room temperature is an interesting approach to measure DNA duplex stability as an alternative to thermal denaturation at elevated temperatures, used in DNA-melting experiments and single nucleotide polymorphism (SNP) analysis.

In this article we describe the integration of impedance spectroscopy (EIS) and surface plasmon resonance (SPR) into one surface analytic device. A polydimethylsiloxane (PDMS) flow cell is created, matching the dimensions of a commercially available sensor chip used for SPR measurements. This flow cell allowed simultaneous measurements between an EIS and a SPR setup. After a successful integration, a proof of principle study was conducted to investigate any signs of interference between the two systems during a measurement. The flow cell was rinsed with 10 mM Tris-HCl and 1× PBS buffer in an alternating manner, while impedance and shifts of the resonance angle were monitored. After achieving a successful proof of principle, a usability test was conducted. It was assessed whether simultaneous detection occurred when: (i) Protein A is adsorbed to the gold surface of the chip; (ii) The non-occupied zone is blocked with BSA molecules and (iii) IgG1 is bound to the Protein A. The results indicate a successful merge between SPR and EIS.

The need for more advanced, accurate and lower cost sensor platforms is constantly growing. However, for certain applications the already existing sensing systems based on biological recognition elements have sometimes restrictions, which limit their use. As a result, sensors with synthetic recognition elements, such as molecular imprinted polymers (MIPs), can be interesting alternatives. Molecular imprinting leads to the formation of inert polymer particles with nanocavities, which can exhibit similar selectivity and specificity to target molecules as antibodies or enzymes. It is demonstrated that MIPs can be readily incorporated into two different sensor platforms for the detection of histamine in aqueous media. The first platform is based on electrochemical impedance spectroscopy and allows for the accurate detection of histamine in the nanomolar range. The second sensing technique is based on microgravimetry and allows for the detection of histamine in the micromolar range. Using the analogous molecule histidine, it is demonstrated that both sensor platforms are specific for the detection of histamine.

Bridging the gap between state of the art consumer electronics and bio-analytical setups designed for lab environments, an embedded, miniaturised, stand-alone measurement device is developed. The presented impedance analyser incorporates most of the data communication features present in current-generation smartphones and is specifically optimised for low-frequency impedance based biosensor readout. The compact unit operates fully stand-alone with a touchscreen. Its functionality was tested and verified. In this work the unit is tested by using a biomimetic sensing device for the detection of L-nicotine. The combination between the handheld, embedded design of this specialised measurement equipment and a sensor layout fine-tuned for specific applications could mean significant advances in point-of-care systems.