Open Access

Beim Ausbau nachhaltiger, regenerativer Energieversorgung hat die Umwandlung von organischer Biomasse in Biogas ein großes Potential. Der zugrundeliegende, komplexe biologische Prozess wird noch immer unzureichend verstanden und bedarf systematischer Untersuchungen der Prozessparameter, um einen hohen Ertrag bei guter Gasqualität zu ermöglichen. Die Fragestellungen zur Entschlüsselung des Prozesses sind sowohl verfahrenstechnischer als auch mikrobiologischer Natur. Aus mikrobiologischer Sicht ist die Kenntnis der tatsächlich beteiligten prozesstragenden Mikroorganismen von erheblicher Bedeutung, aus verfahrenstechnischer Sicht die Kenntnis der physikalischen und chemischen Faktoren, welche die mikrobiologischen Prozesse und kontrollieren. Im Zusammenspiel aller dieser Parameter wird die Biogasbildung befördert oder behindert, bis zum Abbruch des Prozesses. Eine mögliche Kontrollmethode ist die Messung der metabolischen Aktivität prozesstragender Organismen. Diese soll, beruhend auf fundierten Prozessdaten, gewonnen durch eine Parallelanlage, mit einem lichtadressierbaren potentiometrischen Sensor-System (LAPS) realisiert werden. Dieser Sensor ist in der Lage, pH-Wert-änderungen zu detektieren, die durch den Stoffwechsel der auf dem Chip immobilisierten Organismen hervorgerufen werden, um eine Online-Überwachung von Biogasanlagen zu ermöglichen.

A multi-spot (4 × 4 spots) light-addressable potentiometric sensor (MLAPS) consisting of an Al–p-Si–SiO2 structure has been applied for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization by the intrinsic molecular charge for the first time. Single-stranded probe ssDNA molecules (20 bases) were covalently immobilized onto the silanized SiO2 gate surface. The unspecific adsorption of mismatch ssDNA on the MLAPS gate surface was blocked by bovine serum albumin molecules. To reduce the screening effect and to achieve a high sensor signal, the measurements were performed in a low ionic-strength solution. The photocurrent–voltage (I–V) curves were simultaneously recorded on all 16 spots after each surface functionalization step. Large shifts of I–V curves of 25 mV were registered after the DNA immobilization and hybridization event. In contrast, a small potential shift (∼5 mV) was observed in case of mismatch ssDNA, revealing good specificity of the sensor. The obtained results demonstrate the potential of the MLAPS as promising transducer platform for the multi-spot label-free electrical detection of DNA molecules by their intrinsic molecular charge.

Light-addressable potentiometric sensors (LAPS) consisting of a p-Si-SiO2 and p-Si-SiO2-Au structure, respectively, have been tested for a label-free electrical detection of DNA (deoxyribonucleic acid) hybridization. Three different strategies for immobilizing single-stranded probe DNA (ssDNA) molecules on a LAPS surface have been studied and compared: (a) immobilization of thiol-modified ssDNA on the patterned Au surface via gold-thiol bond, (b) covalent immobilization of amino-modified ssDNA onto the SiO2 surface functionalized with 3-aminopropyltriethoxysilane and (c) layer-by-layer adsorption of negatively charged ssDNA on a positively charged weak polyelectrolyte layer of poly(allylamine hydrochloride).

A multi-spot (16 spots) light-addressable potentiometric sensor (MLAPS) consisting of an Al–p-Si–SiO2 structure modified with a weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was applied for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization by the intrinsic molecular charge for the first time. To achieve a preferentially flat orientation of DNA strands and thus, to reduce the distance between the DNA charge and MLAPS surface, the negatively charged probe single-stranded DNAs (ssDNA) were electrostatically adsorbed onto the positively charged PAH layer using a simple layer-by-layer (LbL) technique. In this way, more DNA charge can be positioned within the Debye length, yielding a higher sensor signal. The surface potential changes in each spot induced due to the surface modification steps (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), non-specific adsorption of mismatched ssDNA) were determined from the shifts of photocurrent–voltage curves along the voltage axis. A high sensor signal of 83 mV was registered after immobilization of probe ssDNA onto the PAH layer. The hybridization signal increases from 5 mV to 32 mV with increasing the concentration of cDNA from 0.1 nM to 5 μM. In contrast, a small signal of 5 mV was recorded in the case of non-specific adsorption of fully mismatched ssDNA (5 μM). The obtained results demonstrate the potential of the MLAPS in combination with the simple and rapid LbL immobilization technique as a promising platform for the future development of multi-spot light-addressable label-free DNA chips with direct electrical readout.