The ultrasensitive measurement of protein markers plays a pivotal role in the early diagnosis of infectious and progressive diseases. Recently, digital methods such as those enabled by the Simoa Planar Array technology (SP-X System) have made significant progress in reaching ultrasensitive detection with clinically relevant protein biomarkers. The elicited Simoa technology is based on the printing of high-density capturing antibodies layers on the bottom of the wells of a microtiter plate, followed by a standard sandwich-type immunometric chemiluminescent detection. The elicited assay, reaching limit-of-detections (LODs) in the low femtomolar range, can be conveniently customized via the immobilization of the capturing antibodies. This is accomplished through a pair of anchoring peptide tags printed on the bottom of each well. An optimized Simoa SP-X assay for detecting and quantifying immunoglobulin M (IgM, non-specific indicator of inflammation) is developed herein and optimized. A full factorial experimental design has been undertaken to optimize the assay, leading to a reduced experimental effort and increased quality of the information obtained concerning the traditional one-variable-at-a-time approach. The optimization process leads to an IgM LOD of 4 fM, that compares well with those achieved with commercially available Simoa® Planar Array kits. Remarkably, depositing both the capturing and detecting layer from a solution (0.1 g/mL) one order of magnitude less concentrated than in standard kits is needed, the assay's cost will be sizably reduced.
Biodetection using electrolyte gated field effect transistors has been mainly correlated to charge modulated transduction. Therefore, such platforms are designed and studied for limited applications involving relatively small charged species and much care is taken in the operating conditions particularly pH and salt concentration (ionic strength). However, there are several reports suggesting that the device conductance can also be very sensitive towards variations in the capacitance coupling. Understanding the sensing mechanism is important for further exploitation of these promising sensors in broader range of applications. In this paper, we present a thorough and in depth study of a multilayer protein system coupled to an electrolyte gated transistor. It is demonstrated that detection associated to a binding event taking place at a distance of 30 nm far from the organic semiconductor-electrolyte interface is possible and the device conductance is dominated by Donnan’s capacitance of anchored biomolecules.
Functional bio-interlayer organic field - effect transistors (FBI-OFET), embedding streptavidin, avidin and neutravidin as bio-recognition element, have been studied to probe the electronic properties of protein complexes. The threshold voltage control has been achieved modifying the SiO2 gate diaelectric surface by means of the deposition of an interlayer of bio-recognition elements. A threshold voltage shift with respect to the unmodified dielectric surface toward more negative potential values has been found for the three different proteins, in agreement with their isoelectric points. The relative responses in terms of source – drain current, mobility and threshold voltage upon exposure to biotin of the FBI-OFET devices have been compared for the three bio-recognition elements.
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