Desorption Of Monolayers Research Articles

A duplexed high-throughput mass spectrometry assay for bifunctional POLB polymerase and lyase activity

Polymerase β (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.

Mechanistic and Kinetic Measurements of Elementary Surface Reactions Using Temperature-Programmed X-ray Photoelectron Spectroscopy.

This paper describes a method by which surface-reaction kinetics can be measured by slowly and precisely ramping up the surface temperature at a constant rate while simultaneously collecting X-ray photoelectron spectra (XPS). This approach results in the collection of a large amount of data over relatively small temperature steps to produce quasi-continuous kinetic data. The method is illustrated for the desorption and reaction of diethyl disulfide (DEDS) on a Au(111) substrate in ultrahigh vacuum, where the results can be compared with previous conventional temperature-programmed desorption (TPD) data from Au(111). Experiments were carried out using a double-pass cylindrical-mirror analyzer with a channeltron detector to demonstrate how this approach can be implemented in a routine, multitechnique vacuum chamber. The approach will be even more effective in a more modern, specialized XPS apparatus with high-transmission hemispherical analyzers with multichannel array detectors, which will enable the spectra of several elements to be measured simultaneously. The results yielded an activation energy for multilayer desorption of DEDS of 41 ± 1 kJ/mol, with a pre-exponential factor of 8 ± 7 × 1012 s-1, an activation energy of 53 ± 6 kJ/mol and pre-exponential factor of 9 ± 8 × 1013 s-1 for monolayer desorption and an activation energy of 90 ± 6 kJ/mol with a prefactor of 1.0 ± 0.3 × 1015 s-1 for the reaction of adsorbed ethyl thiolate species to adsorbed DEDS. While these results were collected for a system for which the kinetic data could have been obtained using conventional TPD, this method can be more usefully applied to those surface reaction processes that do not rely on the formation of desorption products. This system, having been previously studied by TPD, facilitates a comparison with results obtained by conventional methods.

Surface properties of an InGaZnO4 layer with a monolayer formed using a phosphonic acid: wettability, work function, and thermal stability

The surface properties of an InGaZnO4 (IGZO) layer with a monolayer formed on the surface using octadecyl phosphonic acid (ODPA) or (1H, 1H, 2H, 2H-heptadecafluorodecyl) phosphonic acid (FDPA) were investigated. Surface roughness, wettability, and work function were investigated using atomic force microscopy, water contact angle measurement, and photoelectron yield spectroscopy, respectively. The reaction time of monolayer formation on an IGZO layer was discussed based on the measured contact angle. An ODPA-monolayer formed at a slightly higher rate than an FDPA-monolayer. The work function measurement provided an estimate of the density of the molecule in the monolayer. Furthermore, the measured contact angle was used to evaluate the thermal stability of a monolayer. The evaluation suggested that annealing above 500 K causes monolayer desorption for both ODPA- and FDPA-monolayers.

Structure Formation in an Ionic Liquid Wetting Layer: A Combined STM, IRAS, DFT and MD Study of [C2C1Im][OTf] on Au(111).

In a solid catalyst with ionic liquid layer (SCILL), ionic liquid (IL) coatings are used to improve the selectivity of noble metal catalysts. To understand the origins of this selectivity control, we performed model studies by surface science methods in ultrahigh vacuum (UHV). We investigated the growth and thermal stability of ultrathin IL films by infrared reflection absorption spectroscopy (IRAS). We combined these experiments with scanning tunneling microscopy (STM) to obtain information on the orientation of the ions, the interactions with the surface, the intermolecular interactions, and the structure formation. Additionally, we performed density-functional theory (DFT) calculations and molecular dynamics (MD) simulations to interpret the experimental data. We studied the IL 1-ethyl-3-methylimidazolium trifluoromethanesulfonate [C2C1Im][OTf] on Au(111) surfaces. We observe a weakly bound multilayer of [C2C1Im][OTf], which is stable up to 390 K, while the monolayer desorbs at ~450 K. [C2C1Im][OTf] preferentially adsorbs at the step edges and elbows of the herringbone reconstruction of Au(111). The anion adsorbs via the SO3 group with the molecular axis perpendicular to the surface. At low coverage, the [C2C1Im][OTf] crystallizes in a glass-like 2D phase with short-range order. At higher coverage, we observe a phase transition to a 6-membered ring structure with long-range order.

Cu-Cu Thermocompression Bonding with a Self-Assembled Monolayer as Oxidation Protection for 3D/2.5D System Integration.

Cu-Cu direct interconnects are highly desirable for the microelectronic industry as they allow for significant reductions in the size and spacing of microcontacts. The main challenge associated with using Cu is its tendency to rapidly oxidize in air. This research paper describes a method of Cu passivation using a self-assembled monolayer (SAM) to protect the surface against oxidation. However, this approach faces two main challenges: the degradation of the SAM at room temperature in the ambient atmosphere and the monolayer desorption technique prior to Cu-Cu bonding. In this paper, the systematic investigation of these challenges and their possible solutions are presented. The methods used in this study include thermocompression (TC) bonding, X-ray photoelectron spectroscopy (XPS), shear strength testing, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX). The results indicate nearly no Cu oxidation (4 at.%) for samples with SAM passivation in contrast to the bare Cu surface (27 at.%) after the storage at -18 °C in a conventional freezer for three weeks. Significant improvement was observed in the TC bonding with SAM after storage. The mean shear strength of the passivated samples reached 65.5 MPa without storage. The average shear strength values before and after the storage tests were 43% greater for samples with SAM than for the bare Cu surface. In conclusion, this study shows that Cu-Cu bonding technology can be improved by using SAM as an oxidation inhibitor, leading to a higher interconnect quality.

Multiple equilibria description of type H1 hysteresis in gas sorption isotherms of mesoporous materials

We report argon adsorption/desorption isotherms of MCM-41 and of SBA-15. The shape of all hysteresis loops we have observed corresponds to type H1. The data have been analyzed quantitatively using the multiple equilibria description and applying the notion of metastable thermodynamic equilibrium. It is remarkable and important that for both mesoporous materials the desorption process can be understood according to this description but with a corresponding equilibrium constant. This procedure therefore allows determining the thermodynamic values for the enthalpy and the free enthalpy of cavity desorption and thus to obtain information not available so far. We observed that the MCM-41 adsorption isotherms show first an increase with the characteristic Langmuir shape, followed by the almost instantaneous filling of cavities that ends as soon as all cavities are completely filled. The SBA-15 isotherms show a characteristic Langmuir shape at low relative pressure. The pressure slightly below the inflection point at prel, infl = 0.312 marks the beginning of the growth of a second layer, before an almost instantaneous filling of cavities takes place. It is interesting to observe that the values of the enthalpy and the free enthalpy for cavity filling and cavity desorption differ by about 0.1 kJ/mol for both, MCM-41 and SBA-15. This means that the driving force for developing a hysteresis is small. The hysteresis loop is therefore driven by delicate changes occurring within the cavities, partially or completely filled by the adsorbate. The monolayer coverage and monolayer desorption processes are decoupled from the cavity filling and cavity emptying processes. Knowing thermodynamic parameters for the hysteresis loop ultimately helps to better characterize and understand the experimental observations.

Dual-frequency comb spectroscopy studies of ionic strength effects in time-resolved ATR-SEIRAS

• DFCS allows sub-millisecond resolution of temporal processes occurring at an electrode surface. • IR transient time constants are directly proportional to the RC cell constant. • Absorption spectra line shape depends on coverage. Attenuated total reflection surface enhanced infrared absorption spectroscopy (ATR-SEIRAS) studies of the potential induced desorption/adsorption of a model organic monolayer is described using a paradigm-challenging dual frequency comb spectrometer. Experimentally measured ATR-SEIRAS transients reveal that both the rates of adsorption and desorption of 4-dimethylaminopyridine (DMAP) are dependent on the ionic strength of the supporting electrolyte. The characteristic time scales of monolayer desorption are found to be directly proportional to the spectroelectrochemical cell time constant indicating that the rate of film dissolution is much faster than the RC charging of the interface. The slow response of the interfacial charging process relative to the kinetics of the film re-organization/desorption processes means that the transient is parametrically linked to the potential dependent DMAP adsorption isotherm. The transients also reveal that the line shape of the molecular IR absorption feature exhibits a temporal dependence. A Lorentzian band is seen at early stages of the desorption transient but increasingly exhibit an anomalous bimodal line-shape at longer times. The development of the anomalous line shape is more pronounced at equivalent times in higher ionic strength electrolytes. Such an effect has been modelled using effective medium theory and is explained by the fact that the fraction of organic layer in the SEIRAS active film influences the observed apparent absorption features. The observation of a coverage dependence on SEIRAS line shapes adds new complexity to previous reports that linked anomalous absorption features exclusively to the metal film morphology and metal volume fraction.

Characterization of the Reductive Desorption of Self-Assembled Monolayers on Platinum Surfaces

Thiol-based self-assembled monolayers (SAMs) have been widely characterized on gold surfaces, with the desorption potentials of these SAMs being of special interest for nanotechnology and biological applications. However, the study and determination of desorption potentials of thiol SAMs on platinum surfaces has been limited by the hydrogen evolution reaction (HER) catalyzed by the metal, which dominates response signals when studying the system through conventional electrochemical techniques.Through the coupling of electrochemical techniques with fluorescence microscopy, the problem of the HER signal can be avoided in the study of desorption potentials for the platinum-thiol monolayer systems. By using fluorescent thiols, as well as monocrystalline platinum spheres, the desorption potential for different crystallographic orientations and thiol length combinations can be determined. The results obtained show that the desorption of Bodipy C10SH thiols from platinum surfaces occur at more positive potentials in [111] crystallographic orientations, and at more negative potentials at [100] ones. On the other hand, no notable difference was found in the desorption order of Bodipy C16SH from these two orientations.However, the use of fluorescence microscopy for the solution of this problem poses other challenges: the time the thiols need to separate enough from the metallic substrate to not be affected by quenching, as well as the facts that thiols fluoresce less as they dilute and the existing spectral overlap for the molecules, adds difficulties to the establishment of the desorption potentials.Through COMSOL Multiphysics simulations, we try to simulate the systems hereby proposed of platinum with thiol-based SAMs. Using them, the desorption potentials, as well as the monolayer coverage under specific conditions, can be determined. We expect that, with further development of the model, more specific values for these variables can be determined. Figure 1

Using Surface Science Techniques to Investigate the Interaction of Acetonitrile with Dust Grain Analogue Surfaces

Surface science methodologies, such as reflection-absorption infrared spectroscopy (RAIRS) and temperature programmed desorption (TPD), are ideally suited to studying the interaction of molecules with model astrophysical surfaces. Here we describe the use of RAIRS and TPD to investigate the adsorption, interactions and thermal processing of acetonitrile and water containing model ices grown under astrophysical conditions on a graphitic dust grain analogue surface. Experiments show that acetonitrile physisorbs on the graphitic surface at all exposures. At the lowest coverages, repulsions between the molecules lead to a decreasing desorption energy with increasing coverage. Analysis of TPD data gives monolayer desorption energies ranging from 28.8–39.2 kJ mol−1 and an average multilayer desorption energy of 43.8 kJ mol−1. When acetonitrile is adsorbed in the presence of water ice, the desorption energy of monolayer acetonitrile shows evidence of desorption with a wide range of energies. An estimate of the desorption energy of acetonitrile from crystalline ice (CI) shows that it is increased to ~37 kJ mol−1 at the lowest exposures of acetonitrile. Amorphous water ice also traps acetonitrile on the graphite surface past its natural desorption temperature, leading to volcano and co-desorption. RAIRS data show that the C≡N vibration shifts, indicative of an interaction between the acetonitrile and the water ice surface.

High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease.

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)-ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose-response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid-metabolizing enzymes.

Interrogation of Electrochemical Aptamer-Based Sensors via Peak-to-Peak Separation in Cyclic Voltammetry Improves the Temporal Stability and Batch-to-Batch Variability in Biological Fluids.

Electrochemical, aptamer-based (E-AB) sensors support continuous, real-time measurements of specific molecular targets in complex fluids such as undiluted serum. They achieve these measurements by using redox-reporter-modified, electrode-attached aptamers that undergo target binding-induced conformational changes which, in turn, change electron transfer between the reporter and the sensor surface. Traditionally, E-AB sensors are interrogated via pulse voltammetry to monitor binding-induced changes in transfer kinetics. While these pulse techniques are sensitive to changes in electron transfer, they also respond to progressive changes in the sensor surface driven by biofouling or monolayer desorption and, consequently, present a significant drift. Moreover, we have empirically observed that differential voltage pulsing can accelerate monolayer desorption from the sensor surface, presumably via field-induced actuation of aptamers. Here, in contrast, we demonstrate the potential advantages of employing cyclic voltammetry to measure electron-transfer changes directly. In our approach, the target concentration is reported via changes in the peak-to-peak separation, ΔEP, of cyclic voltammograms. Because the magnitude of ΔEP is insensitive to variations in the number of aptamer probes on the electrode, ΔEP-interrogated E-AB sensors are resistant to drift and show decreased batch-to-batch and day-to-day variability in sensor performance. Moreover, ΔEP-based measurements can also be performed in a few hundred milliseconds and are, thus, competitive with other subsecond interrogation strategies such as chronoamperometry but with the added benefit of retaining sensor capacitance information that can report on monolayer stability over time.

Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.

Estimating the Electrochemically Active Area: Revisiting a Basic Concept in Electrochemistry

The well-known electrochemical probe Fe(CN)63-/Fe(CN)64- is widely used for estimating the electrochemically active area of electrodes modified with carbon nanotubes, conductive polymers, enzymes, etc. In this study, we used the platinum electrode, smooth or platinized with different roughness factors, to demonstrate that such a redox couple fails to respond to a surface roughness variation. We determined the roughness factors of the Pt surfaces by atomic force microscopy (AFM) images, which yielded values between 2.72 and 25.91. Almost the same values were found by using the charge of the hydrogen monolayer desorption obtained from steady-state cyclic voltammetry experiments performed in an acid medium. They were then compared with those provided by peak current in voltammetry or chronoamperometry with Fe(CN)6 3-/Fe(CN)64- which all yielded values nearly to one. Such comparison demonstrates that the electrochemical behavior of the redox probe is an outer sphere reaction with a quite small interaction with the electrode surface, thus not being suitable to be related with active areas.

London Dispersion Corrections to Density Functional Theory for Transition Metals Based on Fitting to Experimental Temperature-Programmed Desorption of Benzene Monolayers.

Standard implementations of generalized gradient approximation (GGA)-based density functional theory (DFT) describe well strongly bound molecules and solids but fail to describe long-range London dispersion or van der Waals (vdW) attraction interactions that are important in molecular crystals and two-dimensional solids. To provide accurate values for the vdW distance and energies for the metals Cu, Ag, Au, Ni, Pd, and Pt, we determined empirical vdW corrections to Perdew, Burke, and Ernzerhof (PBE) DFT by fitting the experimental adsorption enthalpies measured by temperature-programmed desorption (TPD) from benzene monolayers by Campbell and co-workers ( J. Phys. Chem. C 2016, 120, 25161-25172). Benzene physisorbed to these metals without chemical reaction; therefore, we consider the bonding to be vdW. We use the low gradient form for the vdW corrections, EvdW-LG = -C6LG/[R6 + RvdwLG6] with just two parameters per atom (C6LG and RvdwLG). This LG form leads to negligible changes in bond distances and angles, so adjusting the parameters should not sacrifice accuracy for the bonding interactions. We demonstrate that the parameters fitted to benzene also describe well the physisorption enthalpies for other hydrocarbons (naphthalene, cyclohexane, methane, ethane, and propane) on Pt. We also report low gradient vdW correction parameters for the noble gases that fit the equilibrium lattice parameter and heat of vaporization of the crystals.

(Invited) Extending the Stability of Electrochemical, Aptamer-Based Sensors In Vivo

Electrochemical aptamer-based (E-AB) sensors are a technology that enables precise measurements of specific molecular targets in the body. They have been demonstrated to support real-time therapeutic drug monitoring and feedback-controlled drug dosing in living animals. However, one drawback of E-AB sensors is the rapid degradation of their sensing interface upon continuous interrogation via, for example, voltammetry. Over time, the faradaic current from their aptamer-attached redox reporter decreases, and the currents from charging the electrical double-layer increase beyond the faradaic current, thus preventing signaling. The latter effect is due to progressive desorption of the electrode-passivating alkyl thiol monolayers employed in these sensors. This progressive degradation limits their in-vivo operational life to hours, a period too short for monitoring the vast majority of drugs in humans. Attempting to address this problem, my laboratory is investigating novel monolayer chemistries that extend the operational life of E-AB sensors without compromising their sensing performance or biocompatibility. In this presentation, I will discuss the effects that different monolayer chemistries have on E-AB sensing performance and how they extend the in-vitro operational life of E-AB sensors for days in undiluted biological fluids.

Evaporation and Fragmentation of Organic Molecules in Strong Electric Fields Simulated with DFT

Atom probe tomography allows us to measure the three-dimensional composition of materials with up to atomic resolution by evaporating the material using high electric fields. Initially developed for metals, it is increasingly used for covalently bound structures. To aid the interpretation of the obtained fragmentation pattern, we modeled the fragmentation and desorption of self-assembled monolayers of thiolate molecules on a gold surface in strong electrostatic fields using density functional theory. We used a cluster model and a periodic model of amino-undecanethiolate, NH2(CH2)11S, and fluoro-decanethiolate, CF3(CF2)7(CH2)2S. In the former molecule, the fragment CH2NH2+ was found to evaporate at fields of 5.4-7.7 V/nm. It was followed by different hydrocarbon fragments. Fluoro-decanethiolate evaporates CF3+ at fields of 5.7-6.7 V/nm in the cluster model and at 15.4-23.1 V/nm in the periodic model, followed by CF2+ and C2F42+. Detailed analysis of the electronic structure during the evaporation process revealed a stepwise accumulation of the charge in the head groups exposed to the strongest fields, followed by dissociation of covalent bonds. These observations will facilitate the analysis of atom probe experiments of covalently bound structures.

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 μM), calpain inhibitors II and XII (IC50 ~20–25 μM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human β-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2.

Improving Monolayer Chemistries to Achieve Multiday Aptamer-Based Sensing

Electrochemical aptamer-based (E-AB) sensors are a technology that enables precise measurements of specific molecular targets in the body. They have been demonstrated to support real-time therapeutic drug monitoring and feedback-controlled drug dosing in living animals. However, one drawback of E-AB sensors is the rapid degradation of their sensing interface upon continuous interrogation in biological fluids by, for example, voltammetry. Over time, the faradaic current from their aptamer-attached redox reporter decreases, and the currents from charging the electrical double-layer increase beyond the faradaic current, thus preventing signaling. The latter effect is due to progressive desorption of electrode-passivating alkyl thiol monolayers employed in these sensors. This progressive degradation limits their in-vivo operational life to hours, a period too short for monitoring the vast majority of drugs in humans, which generally have half-lives on the order of days. In response, our laboratory is investigating novel monolayer chemistries that extend the operational life of E-AB sensors without compromising their sensing performance or biocompatibility. In this presentation, I will discuss the effects that different monolayer chemistries have on E-AB sensing performance and how they extend the in-vitro operational life of E-AB sensors for days in undiluted biological fluids.

Adsorption and desorption of self-assembled L-cysteine monolayers on nanoporous gold monitored by in situ resistometry.

Surface modifications of nanoporous metals have become a highly attractive research field as they exhibit great potential for various applications, especially in biotechnology. Using self-assembled monolayers is one of the most promising approaches to modify a gold surface. However, only few techniques are capable of characterizing the formation of these monolayers on porous substrates. Here, we present a method to in situ monitor the adsorption and desorption of self-assembled monolayers on nanoporous gold by resistometry, using cysteine as example. During the adsorption an overall relative change in resistance of 18% is detected, which occurs in three distinct stages. First, the cysteine molecules are adsorbed on the outer surface. In the second stage, they are adsorbed on the internal surfaces and in the last stage the reordering accompanied by additional adsorption takes place. The successful binding of cysteine on the Au surface was confirmed by cyclic voltammetry, which showed a significant decrease of the double-layer capacitance. Also, the electrochemically controlled desorption of cysteine was monitored by concomitant in situ resistometry. From the desorption peak related to the (111) surface of the structure, which is associated with a resistance change of 4.8%, an initial surface coverage of 0.48 monolayers of cysteine could be estimated.