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Abstract Protein-ligand binding site prediction is a useful tool for understanding the functional behaviour and potential drug-target interactions of a novel protein of interest. However, most binding site prediction methods are tested by providing crystallised ligand-bound (holo) structures as input. This testing regime is insufficient to understand the performance on novel protein targets where experimental structures are not available. An alternative option is to provide computationally predicted protein structures, but this is not commonly tested. However, due to the training data used, computationally-predicted protein structures tend to be extremely accurate, and are often biased toward a holo conformation. In this study we describe and benchmark IF-SitePred, a protein-ligand binding site prediction method which is based on the labelling of ESM-IF1 protein language model embeddings combined with point cloud annotation and clustering. We show that not only is IF-SitePred competitive with state-of-the-art methods when predicting binding sites on experimental structures, but it performs better on proxies for novel proteins where low accuracy has been simulated by molecular dynamics. Finally, IF-SitePred outperforms other methods if ensembles of predicted protein structures are generated.

Gallium-68-labeled FAPI-46 has recently been proposed as a novel positron emission tomography imaging probe to diagnose and monitor a wide variety of cancers. Promising results from several ongoing clinical trials have led to a soaring demand for this radiotracer. Typical [68Ga]Ga-FAPI-46 labeling protocols do not cope with multiple generator elutions, leaving radiopharmacies unable to scale-up the production and meet the demand. Here, we propose a robust and efficient automated radiosynthesis of [68Ga]Ga-FAPI-46 on the Trasis miniAllinOne synthesizer, featuring a prepurification step which allows multiple generator elutions and ensures compatibility with a wide range of gallium-68 generators. Our approach was to optimize the prepurification step by first testing five different cationic cartridge chemistries. Only the strong cationic exchange (SCX) cartridges tested had sufficient affinities for quantitative trapping of >99.9%, while the weak cationics did not exceed 50%. Packaging, rinsing, or flowing of the selected SCX cartridges was not noticeable, but improvements in fluidics managed to save time. Based on our previous development experience of [68Ga]Ga-FAPI-46, radiolabeling optimization was also carried out at different temperatures during 10 min. At temperatures above 100°C, radiochemical yield (RCY) > 80% was achieved without significantly increasing the chemical impurities (88%). A comparison with radiosyntheses carried out without prepurification steps was also conclusive in terms of RCY, radiochemical yield, and chemical purity. Finally, high-activity tests using elutions from three generators were also successful for these parameters. [68Ga]Ga-FAPI-46 was consistently obtained in good radiochemical yields (>89%, n = 3), and the final product quality was compliant with internal specifications based on European Pharmacopoeia. This process is suitable for GMP production and allows scaling-up of routine productions, higher throughput, and, ultimately, better patient care.

We report on a quartz pin that can be interfaced easily to existing pin printers. The new pin surface can be reversibly derivatized using silanization chemistry, allowing one to reliably print a wide variety of liquid solutions. Feature sizes as small as 9 [micro]m can be produced with the new pin, allowing one to readily create microarrays with a feature density approaching [10.sup.6] spots/[cm.sup.2].

We report on a new sensor strategy that we have termed site selectively templated and tagged xerogels (SSTTX). The SSTTX platform is completely self-contained, and it achieves analyte recognition without the use of biomolecules. To illustrate the SSTTX scheme's potential, we present results for the selective detection and quantification of a model compound, 9-anthrol. The first-generation SSTTX shows the following: (i) exhibits an apparent dissociation constant of (1.8 [+ or -] 0.3) x [10.sup.-4] M for 9-anthrol; (ii) provides 0.3 [micro]M detection limits for 9-anthrol; (iii) yields a 45-s response time (2-[micro]m-thick film); (iv) is completely reversible (6% relative standard deviation after 25 cycles); (v) yields a selectivity factor for 9-anthrol over several structurally similar analogues/ interferences (e.g., anthracene, 9,10-anthracenediol, benzophenone, 2-naphthol, phenol, and pyrene) of between 290 and 520; and (vi) is stable (

We report on the analytical figures of merit for [O.sub.2]-responsive sensor arrays and films formed by sequestering tris(4,7-diphenyl- 1,10-phenanthroline)ruthenium(II) within class II organically modified silicates that are composed of tetramethoxysilane or tetraaethoxysilane and monoalkylsiloxanes of the form ([C.sub.n][H.sub.2n+1])-Si-[(OR).sub.3] (n = 1-12, R = Me or Et). These sensors exhibit a reasonably linear response to gaseous and dissolved [O.sub.2] ([r.sup.2] > 0.99), and the sensor responses are stable for over 2 years. Sensor sensitivity can be tuned continuously by adjusting n. For gas-phase [O.sub.2] detection, changes in the sensor sensitivity depend primarily on the [O.sub.2] diffusion coefficient within the xerogel phase. The [O.sub.2] solubility coefficient within the xerogel phase is also a factor but to a lesser degree. For dissolved [O.sub.2] detection, changes in the sensor sensitivity depend on the [O.sub.2] diffusion coefficient and the [O.sub.2] solubility coefficient within the xerogel phase. A linear correlation also exists between the sensor sensitivity and the polarity within these xerogels. Finally, the feature size of pin-printed sensor elements was found to depend linearly on pin velocity. The results of these experiments demonstrate a new strategy for creating xerogel-based sensor arrays consisting of diversified sensor elements for the same target analyte.

A radioluminescent (RL) light source is evaluated for the development of photonically based chemical-responsive sensor arrays (CRSAs). The RL light source is comprised of a strontium-90 ([sup.90]Sr) radionuclide and a plastic scintillator. The [beta] particles emitted from the [sup.90]Sr generate blue light ([lambda]max = 435 nm) from the plastic scintillator, and the blue light excites the analyte-responsive luminophores within the CRSA. To assess the RL light source utility, we have determined the analytical figures of merit from two tris(4,7'-diphenyl- 1,10'-phenathroline)ruthenium(II)-doped xerogel-based sensor platforms: (i) a planar 5 x 5 multielement array and (ii) a discrete sensor element formed on the proximal face of poly(styrene) pillars that have a frustrated cone (frustum) geometry. We compare the performance from each platform when it is excited by a He-Cd laser (442 nm), a blue light-emitting diode (460-470 nm), and the RL light source. The RL light source yields results that are statistically equivalent to results from either electrically powered light source. The RL light source consumes no electrical power, is compact and simple, and has an extremely stable time-averaged signal. The primary trade-offs for these advantages are the RL light source's lower radiant power and the corresponding longer data acquisition times.

Novel [O.sub.2]-sensing materials based on spin-coated noctyltriethoxysilane (Octyl-triEOS)/tetraethylorthosilane (TEOS) composite xerogel films have been synthesized and investigated. These sensors are based on the [O.sub.2] quenching of tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) ([[Ru[(dpp).sub.3].sup.2+]) sequestered within the xerogels. Scanning electron microscopy and luminescence measurements (steady state and time resolved) have been used to investigate the structure of these films and their analytical figures of merit and determine the underlying reasons for their observed performance. The results show that certain [[Ru[(dpp).sub.3]].sup.2+]-doped Octyl-triEOS/TEOS composites form uniform, crack-free xerogel films that can be used to construct high-sensitivity [O.sub.2] sensors that have linear calibration curves and excellent long-term stability. For example, an 11-month-old sensor based on 50 mol % Octyl-triEOS exhibits more than 4-fold greater sensitivity in comparison to an equivalent sensor based on pure TEOS. Over an 11-month time period, the sensitivity of a pure TEOS-based sensor drops by more than 400% whereas a sensor based on 50 mol % Octyl-triEOS remains stable (RSD = 4%).

We report the first biosensor arrays based on pin printing protein-doped xerogels. The individual biosensor elements are on the order of 100 [micro]m in diameter. Arrays are formed (1) onto a planar substrate that is excited by an external source (laser) or (2) directly on the face of a light-emitting diode. We illustrate the potential of our approach by fabricating, testing, and characterizing four types of pin-printed biosensor arrays (PPBSA) for the simultaneous detection of glucose and [O.sub.2]. The analytically reliable operating ranges for the PPBSAs are 0.1-10 mM for glucose and 0.1-100% for [O.sub.2]. The PPBSAs exhibit short- and long-term reproducibilities of no worse than 4 and 8%, respectively. The overall array-to-array response reproducibilities are [less than or equal to] 12%. These results demonstrate for the first time the combination sol--gel processing and pin printing methods as a way to rapidly form ensembles of integrated, reusable, and stable biosensor arrays for simultaneous multianalyte detection.

A new approach to rapidly produce micrometer-scale sensor elements into reusable multianalyte chemical sensor arrays is demonstrated. By using pin printing technology in concert with sol-gel processing methods, we form discrete xerogel-based microsensors on a planar substrate. We illustrate the new approach by forming discrete [O.sub.2]- and pH-responsive sensing elements into arrays that allow one to simultaneously determine [O.sub.2] and pH in aqueous samples. The pin printing method allows one to prepare sensor elements that are on the order of 100 [micro]m in diameter, 1-2 [micro]m thick, at a rate of approximately one sensor element per second with a single pin. Within a given calibrated array, the sensor element-to-sensor element response is reproducible to within 5%, the sensor element short- and long-term reproducibilities are 3 and 6%, respectively, and the array-to-array response reproducibility is 11%. These results demonstrate the potential of this methodology for rapidly forming ensembles of reusable sensor arrays for simultaneous multianalyte detection.

Research is presented concerning the characterization and synthesis of three fluorescent dendrimers which contain carboxylic acid groups and include a sole dansyl group which is focally located.

We report the effects of EtOH volume percent (0-70%) on spinach apo- and holocalmodulin that have been site-selectively labeled with fluorescein (F). In these experiments, calmodulin (CaM) has one F reporter group attached to Cys-26, and this site is located immediately adjacent to one of the four primary Ca(super 2+)-binding sites (EF hands). The optimum analytical CaM-F sensitivity to Ca(super 2+) occurs between approximately 10 and 30% EtOH. Our results also show that added EtOH causes changes in CaM and these changes are surprisingly different for apo- and holo-CaM. Apo-CaM-F appears to lose one of its two waters of hydration at approximately 20% EtOH and retains one water of hydration between approximately 20 and 70% EtOH. In apo-CaM-F, the semiangle that describes the range over which the fluorescein reporter group can precess remains essentially constant (42 +/- 2 degrees) between 0 and 70% EtOH. This shows that the fluorescein reporter group precessional freedom in apo-CaM-F is not affected significantly by EtOH. Holo-CaM-F also appears to lose one water of hydration at approximately 20-30% EtOH but then appears to denature as the EtOH volume percent increases. The fluorescein reporter group semiangle within holo-CaM-F decreases from 43 +/- 1 degree in neat aqueous buffer to 36 +/- 1 degree at 70% EtOH. This shows that holo-CaM-F is less nativelike and the EF hand 'closes down' about the fluorescein reporter group in holo-CaM-F as the EtOH volume percent increases.

We report on the performance of a new, multiharmonic frequency-domain instrument that uses the high harmonic content of a passively mode-locked, pulse-picked femto-second Ti-sapphire laser as the excitation source for the determination of one-, two-, or three-photon excited time-resolved fluorescence anisotropy and intensity decay kinetics. In operation, the new instrument can provide a complete frequency-domain data set at 100 modulation frequencies in less than 1 min. The new instrument exhibits 5-10-ps measurement precision and it can rapidly and accurately recover complex excited-state fluorescence anisotropy and intensity decay kinetics under one-, two-, or three-photon excitation for dilute or optically dense samples that exhibit single or multiexponential decay kinetics. This latter aspect of the instrument is demonstrated by successfully determining the excited-state intensity decay kinetics for a dilute aqueous solution of rhodamine 6G dissolved in a high concentration of bromocresol green. This approach is extended by determining the excited-state fluorescence intensity decay kinetics of dilute fluorescein directly in undiluted, whole blood as a function of pH under two-photon excitation conditions. The high-speed capabilities of the new instrument are exploited by performing two-photon excited fluorescence anisotropy decay experiments on the fly for site-selectively labeled bovine serum albumin as it undergoes enzymatic digestion by trypsin.

Biosensor interfacial dynamics in a monoclonal anti-fluorescein system were analyzed to determine the characteristics of surface-immobilized biorecognition elements. Multifrequency phase and modulation total internal reflection fluorescence analysis of the complex demonstrated the ability of the method used to immobilize the biorecognition element in affecting interfacial homogeneity. Furthermore, the G intermediate protein layer in the fluorescein-anti-fluorescein system led to a surface-immobilized biorecognition element that behaved like native monoclonal immunoglobulins.

Fluorescence quenching techniques are used to investigate the accessibility of a model biorecognition element-reporter group system when in buffer, surface-adsorbed, and covalently attached to a silica surface. The site-selective fluorescent reporter group, 6-acryloyl(dimethyl-amino)naphthalene (acrylodan, Ac), is attached covalently (at cysteine-34) to bovine and human serum albumin (BSA and HSA, respectively) and serves as a surrogate recognition element-reporter group system. Molecular oxygen is used to quench the Ac fluorescence and the accessibility, in the form of bimolecular rate constants ([k.sub.q]), in each model system is quantified. Although one might expect these systems to exhibit similar behavior, differences in quenching characteristics are observed, such as wavelength dependency of the Stern-Volmer quenching constant ([K.sub.SV]) for the native proteins in buffer. BSA-Ac exhibits wavelength dependent [K.sub.SV] values as well as a blue-shifted emission spectrum on [O.sub.2] addition. Physisorption of BSA-Ac onto a fused-silica optical fiber lowers the accessibility of Ac to [O.sub.2], whereas covalent attachment of BSA-Ac to APTES/glutaraldehyde-modified silica enhances the accessibility of the Ac reporter group to [O.sub.2].

The interaction of carbon dioxide with polymers containing basic groups was investigated by Fourier transform infrared (IR) spectroscopy. IR transmission and attenuated total reflectance spectra suggested that polymers bearing electron-donating functional groups display specific interactions with carbon dioxide (CO2). Use of the CO2 bending mode (v2) arising from the CO2-polymer interaction revealed splitting of the band corresponding to the CO2 v2 mode, suggesting that changes in the v2 mode of CO2 when CO2 was incorporated into polymers bearing basic functional groups.

Poly[2[prime]-O-(2,4-dinitrophenyl)lpoly(A) [DNP-poly(A)] has been found to be a potent inhibitor in solution for RNases A, B, S, [T.sub.1], [T.sub.2], and H as well as phosphodiesterases I and II. Kinetic measurements with RNase B and RNase [T.sub.1] showed DNP-poly(A) to be a reversible competitive inhibitor with [K.sub.I] equal to 1.03 and 1.05 [[micro]molar], respectively. Data on the quenching of fluorescence of RNase [T.sub.1] by DNP-poly(A) indicate the existence of more than one RNase-binding site in each DNP-poly(A) molecule. By attaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column was prepared for selective removal of RNases from aqueous solutions by simple filtration. It was found that a 1000-fold reduction in RNase concentration can be obtained by passing either 7.0 [[micro]molar] or 7.0 nM RNase A solution through a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solution and subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedly with no significant decrease in RNase-binding affinity and capacity. By titration of the derivatized beads with RNase, the first dissociation constant ([K.sub.d]) and binding capacity for the bound enzyme can be determined. The [K.sub.d] was found to be 0.66 [[micro]molar] for RNase B and 0.48 [[micro]molar] for RNase [T.sub.1]; the corresponding binding capacities were found to be 21.0 x [10.sup.-8] and 9.6 x [10.sup.-8] mol/g, respectively.

Optimization of supercritical fluid extraction conditions requires accurate information on solute solubility under a given set of experimental conditions. In situ electronic absorbance spectroscopy is used commonly to determine solute solubilities, and one assumes that the solute molar absorptivity ([Epsilon]) is constant over a broad density range when monitoring at a particular wavelength. Using UV-vis spectroscopy, we have found that [Epsilon] for anthracene and pyrene in supercritical [CO.sub.2] is density dependent. Systematic increases in [Epsilon] of 1.3-2.7-fold (30-170%) are observed as [CO.sub.2] density increases from 0.3 to 0.9 g/[cm.sup.3]. We account for the observed density dependence in terms of solute - solvent dielectric interactions. These results illustrate the pitfalls associated with using in situ spectroscopic techniques for the determination of solute solubilities in supercritical fluids.

We investigate acrylodan-labeled bovine and human serum albumin (BSA-Ac and HSA-Ac) entrapped within a tetramethylorthosilane-derived biogel composite. The effects of biogel aging and drying were studied by following the acrylodan steady-state and time-resolved emission, the decay of anisotropy, and the dipolar relaxation kinetics as a function of ambient storage time. The results indicate that there is a substantial amount of nanosecond and subnanosecond dipolar relaxation within the local environment surrounding cysteine-34 in both proteins, even when they are fully encapsulated in a dry biogel. Time-resolved anisotropy experiments show that the acrylodan residue and the protein are able to undergo nanosecond motion within the biogel. The semiangle through which the acrylodan can precess is the same for a freshly formed biogel and the native protein in buffer. However, once the biogel begins to dry, the semiangle increases ([approximately]20 [degrees] and 10 [degrees] for BSA-Ac and HSA-Ac, respectively). This suggests that the 'pocket' hosting the acrylodan reporter group opens as the biogel dries.

Currently, there are no simple one-step methods available to remove ribonucleases (RNases) from solution; however, such is imperative for researchers working with ribonucleic acids (RNAs) because RNases can destroy RNA. We report a simple means to remove RNase from solution. The scheme is based on the encapsulation of a new RNase inhibitor, poly[2[prime]-O-(2,4-dinitrophenyl)]poly(adenyiic acid) (DNP-poly(A)), within porous tetramethoxy-silane- (TMOS) or tetraethoxysilane- (TEOS) based sol-gel-derived silica particles that are used to form a small bioaffinity column. We investigate the activity, binding capacity, and stability of the hybrid DNP-poly(A)/sol-gel-derived bioaffinity material. Radioactivity and fluorometric assays are used to determine the response of the sol-gel entrapped DNP-poly(A) to a variety of RNases. The results clearly demonstrate that DNP-poly(A) entrapped within the sol-gel matrix is active and binds to all RNases tested. There is minimal (

Experiments were performed to characterize solute-fluid and solute-solute interactions in dilute supercritical solutions. The experiments were carried out using the fluorescence probe pyrene. Results indicate that the excimer-like emission from pyrene is not due to soluble pyrene aggregates or dissolved ground-state pyrene species by are caused by the pyrene molecules' tendency to solvate individually.

We report the steady-state and time-resolved fluorescence of 6-acryloyl(dimethylamino)naphthalene (acrylodan) covalently attached to Cys-34 in bovine serum albumin (BSA). For this conceptually simple system, complicated fluorescence intensity and anisotropy decay kinetics are observed. The steady-state and time-resolved results demonstrate the presence of an excited-state reaction for the BSA-acrylodan system. Additional analysis shows that dipolar relaxation of the environment surrounding acrylodan within BSA is responsible for most of the observed time-dependent evolution of the emission spectrum. The effects of temperature, chemical denaturation, and protein adsorption to a bare silica substrate are also investigated. These results demonstrate the complexity of the changes within a protein/biorecognition element that affect the signal from a single fluorescent reporter group.

Sol-gel-derived glasses have emerged as a new class of materials well suited for the immobilization of biomolecules. As a consequence, they are also finding new applications as platforms for chemical sensors. Room temperature (or lower) processing conditions, chemical inertness, negligible swelling effects, tunable porosity, and the high purity of sol-gel-derived glasses make them ideal for many types of sensor applications. We report here on the characterization of tetraethyl orthosilicate-(TEOS-) derived thin sol-gel films, doped with glucose oxidase (GOx), as a sensing platform for a prototypical biosensor. GOx was immobilized in/on a thin TEOS sol-gel film using physisorption, microencapsulation, and a new sol-gel:GOx: sol-gel sandwich configuration. Amperometric and photometric detection modes are used to study the response profiles and in turn quantify glucose. The results clearly demonstrate that the sandwich configuration exhibits a fast response and high enzyme loading. This particular scheme is stable for at least 2 months under ambient storage conditions.

Abstract Transition metal dichalcogenides (TMDs) have attracted much attention in the fields of valley- and spintronics due to their property of forming valley-polarized excitons when illuminated by circularly polarized light. In TMD-heterostructures it was shown that these electron-hole pairs can scatter into valley-polarized interlayer exciton states, which exhibit long lifetimes and a twist-angle dependence. However, the question how to create a valley polarization of free charge carriers in these heterostructures after a valley selective optical excitation is unexplored, despite its relevance for opto-electronic devices. Here, we identify an interlayer transfer mechanism in twisted WSe2/MoSe2 heterobilayers that transfers the valley polarization from excitons in WSe2 to free charge carriers in MoSe2 with valley lifetimes of up to 12 ns. This mechanism is most efficient at large twist angles, whereas the valley lifetimes of free charge carriers are surprisingly short for small twist angles, despite the occurrence of interlayer excitons.

Low temperature processed, porous sol-gel glasses represent a new class of materials for immobilization of biomolecules. The ability to form these materials into films, monoliths, and fibers to produce high-purity, porous glasses and the fact that they are optically transparent and chemically inert make them an intriguing platform for the development of chemical biosensors. In this paper, we report on the first attempt to encapsulate an intact antibody in a sol-gel glass matrix. Specifically, we present results on the affinity of sol-gel-encapsulated polyclonal antifluorescein. The results demonstrate that the sol-gel-encapsulated antibody retains an affinity for fluorescein; the affinity constant (K(sub f)) for antibody-hapten complex is on the order of 10 to the seventh power M to the -1. The encapsulation process decreases K(sub f) by about 2 orders of magnitude compared to the native system in buffer solution. The effect of aging and drying on the K(sub f) for sol-gel-encapsulated antifluorescein is reported. Finally, we demonstrate that the intact antibody affinity can be maintained using simple storage protocols.

The solute-entrainer interactions in matrix-modified supercritical fluids. The fluids, which consist of CO2 and small amounts of acetonitrile or methanol are taken as a function of fluid density, with pyrene acting as the solute probe. Fluorescence spectroscopy is then employed to probe the ground and excited states of pyrene. Results reveal that entrainers enhance solute-solvent clustering and slow the excimer formation reaction.