Correction: Universal control of proton concentration using an electrochemically generated acid compatible with miniaturization.

[This corrects the article DOI: 10.1039/D2NA00275B.].

Acid-Modulated Peptide Synthesis for Application on Oxide Biosensor Interfaces.

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.

Miniaturized Control of Acidity in Multiplexed Microreactors.

The control of acidity drives the assembly of biopolymers that are essential for a wide range of applications. Its miniaturization can increase the speed and the possibilities of combinatorial throughput for their manipulation, similar to the way that the miniaturization of transistors allows logical operations in microelectronics with a high throughput. Here, we present a device containing multiplexed microreactors, each one enabling independent electrochemical control of acidity in ∼2.5 nL volumes, with a large acidity range from pH 3 to 7 and an accuracy of at least 0.4 pH units. The attained pH within each microreactor (with footprints of ∼0.3 mm2 for each spot) was kept constant for long retention times (∼10 min) and over repeated cycles of >100. The acidity is driven by redox proton exchange reactions, which can be driven at different rates influencing the efficiency of the device in order to achieve more charge exchange (larger acidity range) or better reversibility. The achieved performance in acidity control, miniaturization, and the possibility to multiplex paves the way for the control of combinatorial chemistry through pH- and acidity-controlled reactions.

Planar Junctionless Field-Effect Transistor for Detecting Biomolecular Interactions.

Label-free field-effect transistor-based immunosensors are promising candidates for proteomics and peptidomics-based diagnostics and therapeutics due to their high multiplexing capability, fast response time, and ability to increase the sensor sensitivity due to the short length of peptides. In this work, planar junctionless field-effect transistor sensors (FETs) were fabricated and characterized for pH sensing. The device with SiO2 gate oxide has shown voltage sensitivity of 41.8 ± 1.4, 39.9 ± 1.4, 39.0 ± 1.1, and 37.6 ± 1.0 mV/pH for constant drain currents of 5, 10, 20, and 50 nA, respectively, with a drain to source voltage of 0.05 V. The drift analysis shows a stability over time of -18 nA/h (pH 7.75), -3.5 nA/h (pH 6.84), -0.5 nA/h (pH 4.91), 0.5 nA/h (pH 3.43), corresponding to a pH drift of -0.45, -0.09, -0.01, and 0.01 per h. Theoretical modeling and simulation resulted in a mean value of the surface states of 3.8 × 1015/cm2 with a standard deviation of 3.6 × 1015/cm2. We have experimentally verified the number of surface sites due to APTES, peptide, and protein immobilization, which is in line with the theoretical calculations for FETs to be used for detecting peptide-protein interactions for future applications.

Analytical techniques for multiplex analysis of protein biomarkers.

INTRODUCTION: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. AREAS COVERED: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. EXPERT COMMENTARY: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.

The influence of geometry and other fundamental challenges for bio-sensing with field effect transistors.

We present a review of field effect transistors (FET) from the point of view of their applications to label-free sensing in the era of genomics and proteomics. Here, rather than a collection of Bio-FET achievements, we propose an analysis of the different issues hampering the use of these devices into clinical applications. We make a particular emphasis on the influence of the sensor geometry in the phenomena of mass transport of analytes, which is a topic that has been traditionally overlooked in the analysis and design of biosensors, but that plays a central role in the achievement of low limits of detection. Other issues like the screening of charges by the ions in liquids with physiological ionic strength and the non-specific binding are also reviewed. In conclusion, we give an overview of different solutions that have been proposed to address all these challenges, demonstrating the potential of field effect transistors owing to their ease of integration with other semiconductor components for developing cost-effective, highly multiplexed sensors for next-generation medicines.

High Aspect Ratio Fin-Ion Sensitive Field Effect Transistor: Compromises toward Better Electrochemical Biosensing.

The development of next generation medicines demands more sensitive and reliable label-free sensing able to cope with increasing needs of multiplexing and shorter times to results. Field effect transistor-based biosensors emerge as one of the main possible technologies to cover the existing gap. The general trend for the sensors has been miniaturization with the expectation of improving sensitivity and response time but presenting issues with reproducibility and noise level. Here we propose a Fin-Field Effect Transistor (FinFET) with a high height to width aspect ratio for electrochemical biosensing solving the issue of nanosensors in terms of reproducibility and noise, while keeping the fast response time. We fabricated different devices and characterized their performance with their response to the pH changes that fitted to a Nernst-Poisson model. The experimental data were compared with simulations of devices with different aspect ratio, establishing an advantage in linearity and lower device resistance to provide higher current signals for the FinFETs with higher aspect ratio. In addition, these FinFETs promise the optimization of reliability and efficiency in terms of limits of detection for which the interplay of the size and geometry of the sensor with the diffusion of the analytes plays a pivotal role.

Single step fabrication of Silicon resistors on SOI substrate used as Thermistors.

Temperature sensing is one of the important features of Micro Electro Mechanical Systems and a monolithic integration provides advantages for both fabrication simplicity and performance. The use of Silicon On Insulator substrates allows simple fabrication of integrated wires that can be used as thermistors. We fabricated rectangular and triangular silicon wires with different dimensions in a single step fabrication process based on the wet etching of a <110> Silicon On Insulator substrate. We determined the experimental resistivity of the two kinds of devices and tested their performance as thermistors in a temperature range between 24 and 100 °C. The accuracy and normalized sensitivities of our devices were 0.4 °C and 0.3-0.5%/°C, respectively. The potential of the proposed method resides in the possibility of having devices with different shapes in a single straightforward process.

Electrochemical Control of pH in Nanoliter Volumes.

The electrochemical management of the proton concentration in miniaturized dimensions opens the way to control and parallelize multistep chemical reactions, but still it faces many challenges linked to the efficient proton generation and control of their diffusion. Here we present a device operated electrochemically that demonstrates the control of the pH in a cell of ∼140 nL. The device comprises a microfluidic reactor integrated with a pneumatic mechanism that allows the exchange of reagents and the isolation of protons to decrease the effect of their diffusion. We monitored the pH with a fluorescence marker and calculated the final value from the redox currents. We demonstrate a large pH amplitude control from neutral pH values beyond the fluorescence marker range at pH 5. On the basis of the calculations from the Faradaic currents, the minimum pH reached should undergo pH ∼ 0.9. The pH contrast between neutral and acid pH cells can be maintained during periods longer than 15 min with an appropriate design of a diffusion barrier.

Influence of polymerisation on the reversibility of low-energy proton exchange reactions by Para-Aminothiolphenol.

The reversibility of redox processes is an important function for sensing and molecular electronic devices such as pH reporters or molecular switches. Here we report the electrochemical behaviour and redox reversibility of para-aminothiolphenol (PATP) after different polymerisation methods. We used electrochemical and photo-polymerisation in neutral buffers and plasma polymerisation in air to induce reversible redox states. The chemical stoichiometry and surface coverage of PATP in the polymerized layers were characterized by X-ray photoelectron spectroscopy (XPS), while cyclic voltammetry (CV) was used to measure the charge transfer, double layer capacitance and electrochemical rate of the layers during successive potential cycles. Our results show that the surface coverage of the redox active species is higher on electro-polymerised samples, however, after consecutive cycles all the methods converge to the same charge transfer, while the plasma polymerised samples achieve higher efficiency per molecule and UV polymerised samples have a higher electron transfer rate.

Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.

In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity.