Techniques for characterizing biofunctionalized surfaces for bioanalysis purposes

Surface biofunctionalization is an essential stage in the preparation of any bioassay affecting its analytical performance. However, a complete characterization of the biofunctionalized surface, considering studies of coverage density, distribution and orientation of biomolecules, layer thickness, and target biorecognition efficiency, is not met most of the time. This review is a critical overview of the main techniques and strategies used for characterizing biofunctionalized surfaces and the process in between. Emphasis is given to scanning force microscopies as the most versatile and suitable tools to evaluate the quality of the biofunctionalized surfaces in real-time dynamic experiments, highlighting the helpful of atomic force microscopy, Kelvin probe force microscopy, electrochemical atomic force microscopy and photo-induced force microscopy. Other techniques such as optical and electronic microscopies, quartz crystal microbalance, X-ray photoelectron spectroscopy, contact angle, and electrochemical techniques, are also discussed regarding their advantages and disadvantages in addressing the whole characterization of the biomodified surface. Scarce reviews point out the importance of practicing an entire characterization of the biofunctionalized surfaces. This is the first review that embraces this topic discussing a wide variety of characterization tools applied in any bioanalysis platform developed to detect both clinical and environmental analytes. This survey provides information to the analysts on the applications, strengths, and weaknesses of the techniques discussed here to extract fruitful insights from them. The aim is to prompt and help the analysts to accomplish an entire assessment of the biofunctionalized surface, and profit from the information obtained to enhance the bioanalysis output.