Scanning force microscopy based amperometric biosensors

Cantilever-based biosensing has developed into an important research area especially for biomedical and clinical analysis. In particular, the possibility to scan miniaturized amperometric biosensors with high-fidelity distance control across biomedically relevant sample surfaces enables the determination of pertinent analytes such as, e.g., adenosine 5′-triphosphate (ATP). ATP is involved in a wide variety of important regulatory cellular mechanisms, and quantification of ATP has therefore been the focus of extensive research in recent years. Usually, scanning force microscopy-based biosensing relates to detection principles based on either mass-sensitive mechanical transduction, which can be detected as a frequency change of the cantilever, or a change in the force constant due to the absorption of molecules onto the sensor surface, as shown in Fig. 1a. Alternatively, a bimetallic cantilever can be used as a temperature sensing device for detecting calorimetric changes due to specific binding to an immobilized receptor, or due to absorption of molecules. Advanced microfabrication in combination with sophisticated surface functionalization schemes results in sensing interfaces tailored to specifically interact with analyte molecules, thus providing specificity and sensitivity of such miniaturized devices to the picomolar and even femtomolar concentration range [1]. Conventional cantilevertype biosensors do not provide a sharp tip, and are typically not applied for laterally resolved measurements in an imaging mode, which requires that the sensor is scanned across, e.g., a biological specimen at a controlled distance. Recently, the National Heart Lung and Blood Institute has published a report identifying demands for future developments related to diagnosis and therapy of cardiovascular, pulmonary, and hematologic diseases, which identifies in vivo nanosensors as a promising prospective application of nanotechnology for real-timemonitoring of biological signals in response to cardiac or inflammatory events [2]. Hence, besides measurements in complex background matrices, the analytical challenges encompass the ability of biosensors to provide information on structural changes along with chemical information on, e.g., regulatory processes at single cells or cell ensembles, ideally at a nanometer scale. Consequently, miniaturization of the transducer and sensing interface, along with positioning of such miniaturized sensors in close proximity to the sample surface providing laterally resolved quantitative information is a prerequisite. Alternatively to mass-sensitive cantilever-based sensing in bulk solution, atomic force microscopy (AFM) probes can be chemically functionalized, which enables detection and quantification of forces associated with single-molecule binding events at ambient conditions. Recently, this concept has evolved into a combined tool providing laterally resolved information along with force mapping, also known as topography and recognition imaging [3], which enables the visualization of single receptor binding sites at biological surfaces. The present article, however, focuses on the detection of chemical signaling events involving the secretion of transmitter molecules into the extracellular space, and associated efforts to detect such molecules and their concentration for enhancing fundamental understanding on disease-related cellular processes at a molecular level. Anal Bioanal Chem (2008) 390:239–243 DOI 10.1007/s00216-007-1670-8