Controlling locally produced acidity in miniaturized spaces is of high importance to manage simultaneous chemical reactions. Here, we present a platform that hosts miniaturized micro-reactors each one enabling electrochemical control of the acidity in ∼nL volumes. We demonstrated the local control of chemical reactions with the deprotection of strong acid labile groups in a region of 150 μm of diameter of upstanding glass using high proton concentrations (∼10⁻¹ M) and the acidity contrasts between the cell region and the outside. We demonstrated accurate control of the proton concentration in aqueous and organic solvents and the control of chemical reactions in organic electrolytes achieved with a sulfonated tetrafluoroethylene-based membrane, which isolates the acid generating electrodes from the reagents in the solution. The quantitative control of the acidity by faradaic currents was demonstrated by the calibration of carboxyfluorescein adjusted with external titrations and with a tautomer transition occurring at pH 4.2. To the best of our knowledge, this platform shows the best control of acidity in the smallest volume reported so far.

In this paper we report an acid-modulated strategy for novel peptide microarray production on biosensor interfaces. We initially selected a controlled pore glass (CPG) as a support for solid-phase peptide synthesis (SPPS) to implement a chemistry that can be performed at the interface of multiple field effect transistor (FET) sensors, eventually to generate label-free peptide microarrays for protein screening. Our chemistry uses a temporary protection of the N-terminal amino function of each amino acid building block with a tert-butyloxycarbonyl (Boc) group that can be removed after each SPPS cycle, in combination with semi-permanent protection of the side chains of trifunctional amino acid residues. Such a protection scheme with a well-proven record of application in conventional, batchwise SPPS has been fine-tuned for optimal performance on CPG and, from there, translated to SPR chips that allow layer-by-layer monitoring of amino acid coupling. Our results validate this acid-modulated synthesis as a feasible approach for producing peptides in high yields and purity on flat glass surfaces, such as those in bio-FETs.

In this work, we present the introductory methodology for a graphene field-effect transistor (GFET)-based platform for probing the electrochemical fingerprints of amino acids, designed to enable stable and controlled surface chemistry and electrochemical measurements toward peptide and protein sequencing. We begin with a focused conceptual review that motivates electrochemical fingerprinting as a strategy for amino acid and peptide identification and contextualizes this approach within recent advances in protein manipulation relevant to sequencing. We then describe a graphene functionalization protocol that facilitates the directional attachment of amino acids onto the graphene surface. This surface chemistry is quantitatively characterized through surface plasmon resonance (SPR), yielding surface densities in the order of 10¹² molecules/cm². The same functionalization protocol enables in situ peptide synthesis directly on graphene, as demonstrated by the successful synthesis of a model tripeptide. To support electrochemical interrogation, we developed three complementary platforms for sensor preconditioning, surface functionalization, and titration-based electrochemical measurements, compatible with both aqueous and organic solutions. Preliminary stability measurements indicate a Dirac point drift below 10 mV over 45 min. Altogether, this work establishes the experimental foundations for electrochemical amino acid and peptide fingerprinting using GFET sensors and provides a framework for the future development of electrochemically enabled protein sequencing technologies.

Controlling locally produced acidity in miniaturized spaces is of high importance yielding to manage simultaneous chemical reactions. Here we present a platform that hosts miniaturized micro reactors, each one enabling electrochemical control of the acidity in ~nL volumes. We demonstrated the local control of chemical reactions with the deprotection of strong acid labile groups in a region of 150 μm of diameter of an upstanding glass using high proton concentrations (~10-1M) and the acidity contrasts between the cell region and the outside. We demonstrated an accurate control of the proton concentration in aqueous and organic solvents and the control of chemical reactions in organic electrolytes achieved with a sulfonated tetrafluoroethylene-based membrane, that isolates the acid generating electrodes from the reagents in the solution. The quantitative control of the acidity by the Faradaic currents was demonstrated by the calibration of carboxyfluorescein adjusted with external titrations, and with a tautomer transition occurring at pH 4.2. To the best of our knowledge, this platform shows the best control of acidity in the smallest volume reported so far.

In the ElectroMed project, we are interested in screening certain peptide sequences for their ability to selectively interact with antibodies or MHC proteins. This poses a combinatorial challenge that requires a highly multiplexed setup of label-free immunosensors. Label-free FET-based immunosensors are good candidates due to their high multiplexing capability and fast response time. Nanowire-based FET sensors have shown high sensitivity but are unreliable for clinical applications due to drift and gate stability issues. To address this, a label-free immuno-FET architecture based on planar junctionless FET devices is proposed. This geometry can improve the signal-to-noise ratio due to its larger planar structure, which is less prone to defects that cause noise and is better suited to the functionalization of different receptor molecules.