- Nanobiolithography techniques have the ability to fabricate structures of biomolecules as small as ∼40 nm. However, very few examples of working biosensors of these sizes have been demonstrated. These examples use substrates like Gold and Silicon, that are advantageous for fabrication purposes, but present disadvantages as far as signal detection is concerned. The preferred and standard substrates used in microarray research are fabricated on glass. On these surfaces, the binding site density varies between and within individual samples, and is largely not characterized. We report here on the fabrication of a fully functional immunochip with spots of ∼1 μm diameter and a signal to noise ratio (SNR) above 10, using Nano-fountain pen (NFP). To achieve this, we analyze the dominant parameters influencing SNR, develop a model that enables us to compare various types of surfaces and choose the most appropriate ones. We show that a miniaturized immunochip is feasible, yielding detection limit as low as 1.3 ng/ml and dynamic range well above 10 5 . Cross-reactivity of two different species is shown to be negligible. In addition, we study the binding mechanism of surfaces, show how to differentiate between 2D and 3D immobilization, and show that a hydrogel surface (using non-covalent immobilization strategy) yields higher intensities for the same target molecule concentrations, and higher dynamic range.