Enhanced Electron Transfer Efficiency of Fructose Dehydrogenase onto Roll-to-Roll Thermal Stamped Laser-Patterned Reduced Graphene Oxide Films.

Herein, a strategy to stamp laser-produced reduced graphene oxide (rGO) onto flexible polymers using only office-grade tools, namely, roll-to-roll thermal stamping, is proposed, proving for the first time its effectiveness for direct bioelectrocatalysis. This straightforward, scalable, and low-cost approach allows us to overcome the limits of the integration of laser-induced rGO-films in bioanalytical devices. Laser-produced rGO has been thermally stamped (TS) onto different polymeric substrates (PET, PVC, and EVA) using a simple roll-laminator; the obtained TS-rGO films have been compared with the native rGO (untransferred) via morphochemical and electrochemical characterization. Particularly, the direct electron transfer (DET) reaction between fructose dehydrogenase (FDH) and TS-rGO transducers has been investigated, with respect to the influence of the amount of enzyme on the catalytic process. Remarkable differences have been observed among TS-rGO transducers; PET proved to be the elective substrate to support the transfer of the laser-induced rGO, allowing the preservation of the morphochemical features of the native material and returning a reduced capacitive current. Noteworthily, TS-rGOs ensure superior electrocatalysis using a very low amount of FDH units (15 mU). Eventually, TS-rGO-based third-generation complete enzymatic biosensors were fabricated via low-cost benchtop technologies. TS-rGOPET exhibited bioanalytical performances superior to the native rGO, allowing a sensitive (0.0289 µA cm-2 µM-1) and reproducible (RSD = 3%, n = 3) d-fructose determination at the nanomolar level (LOD = 0.2 µM). TS-rGO exploitability as a point-of-need device was proved via the monitoring of d-fructose during banana (Musa acuminata) postharvest ripening, returning accurate (recoveries 110-90%; relative error -13/+1%) and reproducible (RSD ≤ 7%; n = 3) data.

Spiers Memorial Lecture: Challenges and prospects in organic photonics and electronics.

While a substantial amount of research activity has been conducted in fields related to organic photonics and electronics, including the development of devices such as organic field-effect transistors, organic photovoltaics, and organic light-emitting diodes for applications encompassing organic thermoelectrics, organic batteries, excitonic organic materials for photochemical and optoelectronic applications, and organic thermoelectrics, this perspective review will primarily concentrate on the emerging and rapidly expanding domain of organic bioelectronics and neuromorphics. Here we present the most recent research findings on organic transistors capable of sensing biological biomarkers down at the single-molecule level (i.e., oncoproteins, genomes, etc.) for the early diagnosis of pathological states and to mimic biological synapses, paving the way to neuromorphic applications that surpass the limitations of the traditional von Neumann computing architecture. Both organic bioelectronics and neuromorphics exhibit several challenges but will revolutionize human life, considering the development of artificial synapses to counteract neurodegenerative disorders and the development of ultrasensitive biosensors for the early diagnosis of cancer to prevent its development. Moreover, organic bioelectronics for sensing applications have also triggered the development of several wearable, flexible and stretchable biodevices for continuous biomarker monitoring.

Bioelectrochemically triggered apoferritin-based bionanoreactors: synthesis of CdSe nanoparticles and monitoring with leaky waveguides.

Herein, we describe a novel method for producing cadmium-selenide nanoparticles (CdSe NPs) with controlled size using apoferritin as a bionanoreactor triggered by local pH change at the electrode/solution interface. Apoferritin is known for its reversible self-assembly at alkaline pH. The pH change is induced electrochemically by reducing O2 through the application of sufficiently negative voltages and bioelectrochemically through O2 reduction catalyzed by laccase, co-immobilized with apoferritin on the electrode surface. Specifically, a Ti electrode is modified with (3-aminopropyl)triethoxysilane, followed by glutaraldehyde cross-linking (1.5% v/v in H2O) of apoferritin (as the bionanoreactor) and laccase (as the local pH change triggering system). This proposed platform offers a universal approach for controlling the synthesis of semiconductor NPs within a bionanoreactor solely driven by (bio)electrochemical inputs. The CdSe NPs obtained through different synthetic approaches, namely electrochemical and bioelectrochemical, were characterized spectroscopically (UV-Vis, Raman, XRD) and morphologically (TEM). Finally, we conducted online monitoring of CdSe NPs formation within the apoferritin core by integrating the electrochemical system with LWs. The quantity of CdSe NPs produced through bioelectrochemical means was determined to be 2.08 ± 0.12 mg after 90 minutes of voltage application in the presence of O2. TEM measurements revealed that the bioelectrochemically synthesized CdSe NPs have a diameter of 4 ± 1 nm, accounting for 85% of the size distribution, a result corroborated by XRD data. Further research is needed to explore the synthesis of nanoparticles using different biological nanoreactors, as the process can be challenging due to the elevated buffer capacitance of biological media.

Analysis of Clinical Samples of Pancreatic Cyst's Lesions with A Multi-Analyte Bioelectronic Simot Array Benchmarked Against Ultrasensitive Chemiluminescent Immunoassay.

Pancreatic cancer, ranking as the third factor in cancer-related deaths, necessitates enhanced diagnostic measures through early detection. In response, SiMoT-Single-molecule with a large Transistor multiplexing array, achieving a Technology Readiness Level of 5, is proposed for a timely identification of pancreatic cancer precursor cysts and is benchmarked against the commercially available chemiluminescent immunoassay SIMOA (Single molecule array) SP-X System. A cohort of 39 samples, comprising 33 cyst fluids and 6 blood plasma specimens, undergoes detailed examination with both technologies. The SiMoT array targets oncoproteins MUC1 and CD55, and oncogene KRAS, while the SIMOA SP-X planar technology exclusively focuses on MUC1 and CD55. Employing Principal Component Analysis (PCA) for multivariate data processing, the SiMoT array demonstrates effective discrimination of malignant/pre-invasive high-grade or potentially malignant low-grade pancreatic cysts from benign non-mucinous cysts. Conversely, PCA analysis applied to SIMOA assay reveals less effective differentiation ability among the three cyst classes. Notably, SiMoT unique capability of concurrently analyzing protein and genetic markers with the threshold of one single molecule in 0.1 mL positions it as a comprehensive and reliable diagnostic tool. The electronic response generated by the SiMoT array facilitates direct digital data communication, suggesting potential applications in the development of field-deployable liquid biopsy.

Point-Of-Care Ultra-Portable Single-Molecule Bioassays for One-Health.

Screening asymptomatic organisms (humans, animals, plants) with a high-diagnostic accuracy using point-of-care-testing (POCT) technologies, though still visionary holds great potential. Convenient surveillance requires easy-to-use, cost-effective, ultra-portable but highly reliable, in-vitro-diagnostic devices that are ready for use wherever they are needed. Currently, there are not yet such devices available on the market, but there are a couple more promising technologies developed at readiness-level 5: the Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) lateral-flow-strip tests and the Single-Molecule-with-a-large-Transistor (SiMoT) bioelectronic palmar devices. They both hold key features delineated by the World-Health-Organization for POCT systems and an occurrence of false-positive and false-negative errors <1-5% resulting in diagnostic-selectivity and sensitivity >95-99%, while limit-of-detections are of few markers. CRISPR-strip is a molecular assay that, can detect down to few copies of DNA/RNA markers in blood while SiMoT immunometric and molecular test can detect down to a single oligonucleotide, protein marker, or pathogens in 0.1mL of blood, saliva, and olive-sap. These technologies can prospectively enable the systematic and reliable surveillance of asymptomatic ones prior to worsening/proliferation of illnesses allowing for timely diagnosis and swift prognosis. This could establish a proactive healthcare ecosystem that results in effective treatments for all living organisms generating diffuse and well-being at efficient costs.

Experimental design of stencil-printed high-performance organic electrochemical transistors.

Organic electrochemical transistors (OECTs) are widely employed in several bioelectronic applications such as biosensors, logic circuits, and neuromorphic engineering, providing a seamless link between the realm of biology and electronics. More specifically, OECTs are endowed with remarkable signal amplification, the ability to operate in an aqueous environment, and the effective transduction of ionic to electrical signals. One main limiting factor preventing OECTs' wide use is the need for microfabrication processes, typically requiring specialized equipment. From this perspective, a robust and cost-effective production protocol to achieve high-performing OECT would be desirable. Herein, a straightforward stencil-printed OECT fabrication procedure is proposed, where the electrical performance can be controlled by adjusting the electronic channel fabrication conditions. An experimental design approach is undertaken to optimize OECT figures of merit by varying key parameters such as the annealing temperature and time, as well as the transistor active channel length. The resulting OECT devices, fabricated through a high-yield, cost-effective, and fast stencil printing technique, feature large transconductance values at low operating voltages. The experimental design allowed for minimizing the threshold voltage (VT = 260 mV) while keeping a high on/off ratio (7 × 103). A signal-to-noise ratio as high as 40 dB was obtained, which is among the highest for OECTs, operating in an aqueous electrolyte operated in a DC mode. An atomic force microscopy (AFM) characterization has been undertaken to analyze the channel morphology in the OECTs, correlating the annealing conditions with the charge transport properties.

Kelvin probe force microscopy on patterned large-area biofunctionalized surfaces: a reliable ultrasensitive platform for biomarker detection.

Kelvin probe force microscopy (KPFM) allows the detection of single binding events between immunoglobulins (IgM, IgG) and their cognate antibodies (anti-IgM, anti-IgG). Here an insight into the reliability and robustness of the methodology is provided. Our method is based on imaging the surface potential shift occurring on a dense layer of ∼5 × 107 antibodies physisorbed on a 50 µm × 90 µm area when assayed with increasing concentrations of antigens in phosphate buffer saline (PBS) standard solutions, in air and at a fixed scanning location. A comprehensive investigation of the influence of the main experimental parameters that may interfere with the outcomes of KPFM immune-assay is provided, showing the robustness and reliability of our approach. The data are supported also by a thorough polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) analysis of the physisorbed biolayer, in the spectral region of the amide I, amide II and amide A bands. Our findings demonstrate that a 10 min incubation in 500 µL PBS encompassing ≈ 30 antigens (100 zM) triggers an extended surface potential shift that involves the whole investigated area. Such a shift quickly saturates at increasing ligand concentration, showing that the developed sensing platform works as an OFF/ON detector, capable of assessing the presence of a few specific biomarkers in a given assay volume. The reliability of the developed methodology KPFM is an important asset in single molecule detections at a wide electrode interface.

A Single-Molecule Bioelectronic Portable Array for Early Diagnosis of Pancreatic Cancer Precursors.

A cohort of 47 patients is screened for pancreatic cancer precursors with a portable 96-well bioelectronic sensing-array for single-molecule assay in cysts fluid and blood plasma, deployable at point-of-care (POC). Pancreatic cancer precursors are mucinous cysts diagnosed with a sensitivity of at most 80% by state-of-the-art cytopathological molecular analyses (e.g., KRASmut DNA). Adding the simultaneous assay of proteins related to malignant transformation (e.g., MUC1 and CD55) is deemed essential to enhance diagnostic accuracy. The bioelectronic array proposed here, based on single-molecule-with-a-large-transistor (SiMoT) technology, can assay both nucleic acids and proteins at the single-molecule limit-of-identification (LOI) (1% of false-positives and false-negatives). It comprises an enzyme-linked immunosorbent assay (ELISA)-like 8 × 12-array organic-electronics disposable cartridge with an electrolyte-gated organic transistor sensor array, and a reusable reader, integrating a custom Si-IC chip, operating via software installed on a USB-connected smart device. The cartridge is complemented by a 3D-printed sensing gate cover plate. KRASmut , MUC1, and CD55 biomarkers either in plasma or cysts-fluid from 5 to 6 patients at a time, are multiplexed at single-molecule LOI in 1.5 h. The pancreatic cancer precursors are classified via a machine-learning analysis resulting in at least 96% diagnostic-sensitivity and 100% diagnostic-specificity. This preliminary study opens the way to POC liquid-biopsy-based early diagnosis of pancreatic-cancer precursors in plasma.

A stable physisorbed layer of packed capture antibodies for high-performance sensing applications.

Antibody physisorption at a solid interface is a very interesting phenomenon that has important effects on applications such as the development of novel biomaterials and the rational design and fabrication of high-performance biosensors. The strategy selected to immobilize biorecognition elements can determine the performance level of a device and one of the simplest approaches is physical adsorption, which is cost-effective, fast, and compatible with printing techniques as well as with green-chemistry processes. Despite its huge advantages, physisorption is very seldom adopted, as there is an ingrained belief that it does not lead to high performance because of its lack of uniformity and long-term stability, which, however, have never been systematically investigated, particularly for bilayers of capture antibodies. Herein, the homogeneity and stability of an antibody layer against SARS-CoV-2-Spike1 (S1) protein physisorbed onto a gold surface have been investigated by means of multi-parametric surface plasmon resonance (MP-SPR). A surface coverage density of capture antibodies as high as (1.50 ± 0.06) × 1012 molecules per cm-2 is measured, corresponding to a thickness of 12 ± 1 nm. This value is compatible with a single monolayer of homogeneously deposited antibodies. The effect of the ionic strength (is) of the antibody solution in controlling physisorption of the protein was thoroughly investigated, demonstrating an enhancement in surface coverage at lower ionic strength. An atomic force microscopy (AFM) investigation shows a globular structure attributed to is-related aggregations of antibodies. The long-term stability over two weeks of the physisorbed proteins was also assessed. High-performance sensing was proven by evaluating figures of merit, such as the limit of detection (2 nM) and the selectivity ratio between a negative control and the sensing experiment (0.04), which is the best reported performance for an SPR S1 protein assay. These figures of merit outmatch those measured with more sophisticated biofunctionalization procedures involving chemical bonding of the capture antibodies to the gold surface. The present study opens up interesting new pathways toward the achievement of a cost-effective and scalable biofunctionalization protocol, which could guarantee the prolonged stability of the biolayer and easy handling of the biosensing system.

Lab-made flexible third-generation fructose biosensors based on 0D-nanostructured transducers.

Herein, we report a scalable benchtop electrode fabrication method to produce highly sensitive and flexible third-generation fructose dehydrogenase amperometric biosensors based on water-dispersed 0D-nanomaterials. The electrochemical platform was fabricated via Stencil-Printing (StPE) and insulated via xurography. Carbon black (CB) and mesoporous carbon (MS) were employed as 0D-nanomaterials promoting an efficient direct electron transfer (DET) between fructose dehydrogenase (FDH) and the transducer. Both nanomaterials were prepared in water-phase via a sonochemical approach. The nano-StPE exhibited enhanced electrocatalytic currents compared to conventional commercial electrodes. The enzymatic sensors were exploited for the determination of D-fructose in model solutions and various food and biological samples. StPE-CB and StPE-MS integrated biosensors showed appreciable sensitivity (∼150 µA cm-2 mM-1) with µmolar limit of detection (0.35 and 0.16 µM, respectively) and extended linear range (2-500 and 1-250 µM, respectively); the selectivity of the biosensors, ensured by the low working overpotential (+0.15 V), has been also demonstrated. Good accuracy (recoveries between 95 and 116%) and reproducibility (RSD ≤8.6%) were achieved for food and urine samples. The proposed approach because of manufacturing versatility and the electro-catalytic features of the water-nanostructured 0D-NMs opens new paths for affordable and customizable FDH-based bioelectronics.

Electropolymerized molecularly imprinted polypyrrole film for dimethoate sensing: investigation on template removal after the imprinting process.

The development of ultrasensitive analytical detection methods for organophosphorus pesticides such as dimethoate (DMT) plays a key role in healthy food production. DMT is an inhibitor of acetylcholinesterase (AChE), which can lead to the accumulation of acetylcholine and result in symptoms related to the autonomous and central nervous systems. Herein, we report the first spectroscopic and electrochemical study on template removal after an imprinting process from a polypyrrole-based molecularly imprinted polymer (PPy-MIP) film for the detection of DMT. Several template removal procedures were tested and evaluated using X-ray photoelectron spectroscopy. The most effective procedure was achieved in 100 mM NaOH. The proposed DMT PPy-MIP sensor exhibits a limit of detection of (8 ± 2) × 10-12 M.

Self-powered logically operated fluorescent detection of hepatitis B virus (HBV).

In this study, a novel sensing strategy based on double sensing/actuating pathway is demonstrated, being capable to trigger the DNA-based AND gate for the sensitive and selective detection of hepatitis B virus DNA (HBV-DNA). Such an approach encompasses an enzymatic machinery logically operated using the variation of physiologically relevant biomarkers for liver dysfunctions. Alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) are used as inputs of an AND gate generating an output signal, namely lactate. In particular, lactate is oxidized back to pyruvate at the anodic electrode by lactate oxidase connected in mediated electron transfer through ferrocene moieties (creating an amplifying recycling mechanism). The anodic electrode is further connected with a Myrothecium verrucaria bilirubin oxidase (MvBOx) based biocathode modified with SiO2 nanoparticles (SiO2NPs) functionalized with phenyl boronic acid and trigonelline, triggering the release of quenching DNA (qDNA) upon local pH change at the electrode surface (notably, modified SiONPs gets negatively recharged upon local pH gradient releasing negatively charged DNA). Next, the released qDNA labeled with BHQ2 and detecting DNA (dDNA, labeled with FAM) are detecting HBV-DNA. The proposed biosensor can discriminate between the absence and presence of HBV-DNA setting the threshold at 0.05 fM in model buffer solutions and 1 fM in human serum. This enzymatic/DNA logic network can be of particular interest for future biomedical applications (e.g., early detection of liver cancer disease etc.). In the future development this technology could be easily integrated with a smartphone camera, allowing more user-friendly applications.

Fast and Reliable Electronic Assay of a Xylella fastidiosa Single Bacterium in Infected Plants Sap.

Pathogens ultra-sensitive detection is vital for early diagnosis and provision of restraining actions and/or treatments. Among plant pathogens, Xylella fastidiosa is among the most threatening as it can infect hundreds of plant species worldwide with consequences on agriculture and the environment. An electrolyte-gated transistor is here demonstrated to detect X. fastidiosa at a limit-of-quantification (LOQ) of 2 ± 1 bacteria in 0.1 mL (20 colony-forming-unit per mL). The assay is carried out with a millimeter-wide gate functionalized with Xylella-capturing antibodies directly in saps recovered from naturally infected plants. The proposed platform is benchmarked against the quantitave polymerase chain reaction (qPCR) gold standard, whose LOQ turns out to be at least one order of magnitude higher. Furthermore, the assay selectivity is proven against the Paraburkholderia phytofirmans bacterium (negative-control experiment). The proposed label-free, fast (30 min), and precise (false-negatives, false-positives below 1%) electronic assay, lays the ground for an ultra-high performing immunometric point-of-care platform potentially enabling large-scale screening of asymptomatic plants.

A handheld intelligent single-molecule binary bioelectronic system for fast and reliable immunometric point-of-care testing.

Molecular tests are highly reliable and sensitive but lack portability and are not simple to use; conversely, easy-to-use antigenic tests still lack high performance. BioScreen combines single-molecule sensitivity and outstanding reliability with ultraportability and simplicity of use. This digital platform is capable of artificial intelligence-based binary classification at the limit of identification of a single marker/virus in 0.1 ml. The diagnostic sensitivity, specificity, and accuracy reach 99.2% as validated through 240 assays, including a pilot clinical trial. The versatile immunometric system can detect the SARS-CoV-2 virus, spike S1, and immunoglobulin G antigen proteins in saliva, blood serum, and swab. BioScreen has a small footprint comprising a disposable cartridge and a handheld electronic reader connected to a smart device. The sample handling is minimal, and the assay time to result is 21 min. Reliable and sensitive self-testing with an ultraportable and easy-to-use diagnostic system operated directly by a patient holds the potential to revolutionize point-of-care testing and early diagnosis.

Why a Diffusing Single-Molecule can be Detected in Few Minutes by a Large Capturing Bioelectronic Interface.

Single-molecule detection at a nanometric interface in a femtomolar solution, can take weeks as the encounter rate between the diffusing molecule to be detected and the transducing nanodevice is negligibly small. On the other hand, several experiments prove that macroscopic label-free sensors based on field-effect-transistors, engaging micrometric or millimetric detecting interfaces are capable to assay a single-molecule in a large volume within few minutes. The present work demonstrates why at least a single molecule out of a few diffusing in a 100 µL volume has a high probability to hit a large capturing and detecting electronic interface. To this end, sensing data, measured with an electrolyte-gated FET whose gate is functionalized with 1012 capturing anti-immunoglobulin G, are here provided along with a Brownian diffusion-based modeling. The EG-FET assays solutions down to some tens of zM in concentrations with volumes ranging from 25 µL to 1 mL in which the functionalized gates are incubated for times ranging from 30 s to 20 min. The high level of accordance between the experimental data and a model based on the Einstein's diffusion-theory proves how the single-molecule detection process at large-capturing interfaces is controlled by Brownian diffusion and yet is highly probable and fast.

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers.

Early diagnosis in a premalignant (or pre-invasive) state represents the only chance for cure in neoplastic diseases such as pancreatic-biliary cancer, which are otherwise detected at later stages and can only be treated using palliative approaches, with no hope for a cure. Screening methods for the purpose of secondary prevention are not yet available for these cancers. Current diagnostic methods mostly rely on imaging techniques and conventional cytopathology, but they do not display adequate sensitivity to allow valid early diagnosis. Next-generation sequencing can be used to detect DNA markers down to the physical limit; however, this assay requires labeling and is time-consuming. The additional determination of a protein marker that is a predictor of aggressive behavior is a promising innovative approach, which holds the potential to improve diagnostic accuracy. Moreover, the possibility to detect biomarkers in blood serum offers the advantage of a noninvasive diagnosis. In this study, both the DNA and protein markers of pancreatic mucinous cysts were analyzed in human blood serum down to the single-molecule limit using the SiMoT (single-molecule assay with a large transistor) platform. The SiMoT device proposed herein, which exploits an inkjet-printed organic semiconductor on plastic foil, comprises an innovative 3D-printed sensing gate module, consisting of a truncated cone that protrudes from a plastic substrate and is compatible with standard ELISA wells. This 3D gate concept adds tremendous control over the biosensing system stability, along with minimal consumption of the capturing molecules and body fluid samples. The 3D sensing gate modules were extensively characterized from both a material and electrical perspective, successfully proving their suitability as detection interfaces for biosensing applications. KRAS and MUC1 target molecules were successfully analyzed in diluted human blood serum with the 3D sensing gate functionalized with b-KRAS and anti-MUC1, achieving a limit of detection of 10 zM and 40 zM, respectively. These limits of detection correspond to (1 ± 1) KRAS and (2 ± 1) MUC1 molecules in the 100 µL serum sample volume. This study provides a promising application of the 3D SiMoT platform, potentially facilitating the timely, noninvasive, and reliable identification of pancreatic cancer precursor cysts.

Electrochemical and X-ray Photoelectron Spectroscopy Surface Characterization of Interchain-Driven Self-Assembled Monolayer (SAM) Reorganization.

Herein, we report a combined strategy encompassing electrochemical and X-ray photoelectron spectroscopy (XPS) experiments to investigate self-assembled monolayer (SAM) conformational reorganization onto an electrode surface due to the application of an electrical field. In particular, 3-mercaptopriopionic acid SAM (3MPA SAM) modified gold electrodes are activated with a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHSS) (EDC-NHSS) mixture by shortening the activation time, from 2 h to 15/20 min, labelled as Protocol-A, -B and -C, respectively. This step, later followed by a deactivation process with ethanolamine (EA), plays a key role in the reaction yields (formation of N-(2-hydroxyethyl)-3-mercaptopropanamide, NMPA) but also in the conformational rearrangement observed during the application of the electrical field. This study aims at explaining the high performance (i.e., single-molecule detection at a large electrode interface) of bioelectronic devices, where the 3MPA-based SAM structure is pivotal in achieving extremely high sensing performance levels due to its interchain interaction. Cyclic voltammetry (CV) experiments performed in K4Fe(CN)6:K3Fe(CN)6 for 3MPA SAMs that are activated/deactivated show similar trends of anodic peak current (IA) over time, mainly related to the presence of interchain hydrogen bonds, driving the conformational rearrangements (tightening of SAMs structure) while applying an electrical field. In addition, XPS analysis allows correlation of the deactivation yield with electrochemical data (conformational rearrangements), identifying the best protocol in terms of high reaction yield, mainly related to the shorter reaction time, and not triggering any side reactions. Finally, Protocol-C's SAM surface coverage, determined by CV in H2SO4 and differential pulse voltammetry (DPV) in NaOH, was 1.29 * 1013 molecules cm-2, being similar to the bioreceptor surface coverage in single-molecule detection at a large electrode interface.

Large-Area Interfaces for Single-Molecule Label-free Bioelectronic Detection.

Bioelectronic transducing surfaces that are nanometric in size have been the main route to detect single molecules. Though enabling the study of rarer events, such methodologies are not suited to assay at concentrations below the nanomolar level. Bioelectronic field-effect-transistors with a wide (µm2-mm2) transducing interface are also assumed to be not suited, because the molecule to be detected is orders of magnitude smaller than the transducing surface. Indeed, it is like seeing changes on the surface of a one-kilometer-wide pond when a droplet of water falls on it. However, it is a fact that a number of large-area transistors have been shown to detect at a limit of detection lower than femtomolar; they are also fast and hence innately suitable for point-of-care applications. This review critically discusses key elements, such as sensing materials, FET-structures, and target molecules that can be selectively assayed. The amplification effects enabling extremely sensitive large-area bioelectronic sensing are also addressed.

Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein.

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10-9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10-5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.