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 heat-transfer resistance at the solid-to-liquid interface. In contrast to this, molecular brushes of single-stranded 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.
Serotonin is an important signaling molecule in the human body. The detection of serotonin is commonly performed by high performance liquid chromatography (HPLC), which is costly and time consuming due to extensive sample preparation. We will show that these problems can be overcome by using molecularly imprinted polymers (MIPs) as synthetic receptors in combination with impedance spectroscopy as readout technique. The MIPs were prepared with several blends of the underlying monomers and the best performing MIP material was selected by optical batch-rebinding experiments. MIP microparticles were then integrated in an impedimetric sensor cell and dose-response curves were measured in PBS buffer and in non-diluted blood plasma. The sensor provides reliable data in the physiologically relevant concentration regime as an independent validation by HPLC measurements demonstrates. Finally, we show that the impedimetric response upon serotonin binding can be attributed to a capacitive effect at the interface between the MIP particles and the plasma.
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.
A straightforward protocol for the covalent functionalization of boron‐doped diamond electrodes with either ferrocene or single‐stranded deoxyribonucleic acid (DNA) is reported. The functionalization method is based on a combination of diazonium salt electrografting and click chemistry. An azide‐terminated organic layer is first electrografted onto the diamond surface by electrochemical reduction of 4‐azidophenyldiazonium chloride. The azidophenyl‐modified surface then reacts rapidly and efficiently with molecules bearing a terminal alkyne moiety by means of CuI‐catalyzed alkyne–azide cycloaddition. Covalent attachment of ferrocene moieties was analyzed by X‐ray photoelectron spectroscopy and cyclic voltammetry, whereas impedance spectroscopy was applied for the characterization of the immobilized DNA.
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