Angewandte Chemie

Interactions between proteins and glycosphingolipids (GSLs) regulate various cellular processes and altered GSL metabolism contributes to numerous diseases. The diverse glycan headgroups and ceramide backbones of GSLs shape membrane organisation, fluidity, curvature, and tension. As protein recognition frequently depends on both glycan specificity and the organisation of GSLs within the membrane, these interactions remain challenging to characterise in vitro. Here, we introduce FLiPA (Fluorescent Liposome Plate Assay), a versatile method that utilises fluorescent agarose-embedded giant liposomes for the quantitative analysis of protein-GSL interactions. By enabling systematic control of membrane and buffer composition, FLiPA provides an accessible and robust platform for dissecting the molecular determinants of protein-GSL interactions, including the roles of cholesterol, membrane order, protein oligomerisation and ionic strength.

Controlling protein glycosylation as a critical quality attribute of biopharmaceuticals remains challenging when glycosylation is coupled to cellular production systems. Here, we present a proof-of-concept glycosyltransferase immobilised enzyme reactor (IMER) housed within a 3D-printed column that enables directed post-production glycan modification of purified glycoproteins. Using {beta}-1,4-galactosyltransferase ({beta}4GalT1-IMER) and -2,6-sialyltransferase (ST6Gal1-IMER) immobilised on Ni-NTA resin, the IMER achieved near-complete galactosylation and substantial sialylation of partially deglycosylated bovine fetuin N-glycans with their respective substrates with a maximum substrate-enzyme contact time of four minutes. Isomeric-level analysis revealed arm-specific addition preferences for both enzymes, consistent with known specificities. The modular IMER design permits sequential connection of individual enzyme chambers, potentially offering a scalable, plug-and-play platform for constructing defined glycan structures on recombinant glycoprotein therapeutics.

The function of a protein depends not only on its sequence but on post-translational modifications and folding that produce functionally distinct proteoforms. Single-molecule methods for protein identification, such as nanopore sequencing, typically require denaturation or proteolysis, sacrificing conformational information that contributes to proteoform diversity. Here, we identify intact, folded proteins by recording an optical fingerprint of their local surface chemistry using a single covalent label. The signal is produced by a spontaneously blinking fluorophore attached to the protein through established bioconjugation reactions. The thermodynamics and kinetics of its switching between a fluorescent and a dark state are influenced by the immediate protein environment in a chemically interpretable manner. Further discriminative information can be extracted using deep learning to achieve excellent identification accuracy. Using this approach, we distinguish different proteins, different pockets of the same protein, and the presence of a single post-translational modification, in each case tracing the classification back to a distinct physicochemical mechanism. These results establish single-molecule fluorescence blinking as both a protein fingerprinting method and a probe of local chemistry on the surface of folded proteins.

The multivalent display of surface glycoprotein hemagglutinin (HA) on Influenza A viruses (IAVs) enhances the overall binding avidity to sialylated glycans on host cell surfaces. While precomplexing HA trimers with antibodies increases multivalency and avidity, this method does not replicate the virions geometry and limits insights into the multivalent binding process. Here, we use perfectly controllable icosahedral protein nanoparticles to examine the multivalent HA receptor-binding properties. We compare three HA presentation systems with varying degrees of multivalency: single HA trimers, antibody-precomplexed HA trimers, and HA trimers on nanoparticles. Our results indicate that increasing HA valency enhances binding avidity across various glycan surfaces, including erythrocytes, cells, and lipid bilayers with varying glycan densities, while maintaining receptor specificity. By combining functional and non-functional HA trimers during nanoparticle formation, we create statistical mixtures of nanoparticles with varying valencies. At high receptor densities, nanoparticles with few functional trimers still bind strongly, whereas at low receptor densities, a patch of five HA trimers appears necessary for binding. As a key finding, we observe that such a statistical mixture of nanoparticles with functional and nonfunctional HAs binds to glycan surfaces in a stronger density-dependent manner than fully functional particles. We also observe differences in binding modes that correlate with the number of functional trimers, the glycan structure (linear vs branched), and the densities achievable with these glycans. Overall, our findings demonstrate that the presentation of multivalent HA plays an enormous role in the response to glycan receptor type and density, with implications for the future design of virus monitoring, viral inhibitors, and targeting vectors.

Protein cages provide useful scaffolds for nanoscale engineering due to their highly ordered structures and in vivo self-assembly. These scaffolds are amendable to late-stage conjugation, enabling expansion in functionality. However, many conjugation techniques either lack site-selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Salmonella Typhimurium bacteriophage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions, via a minimal extension to the P22 coat protein C-terminus, is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction whereby two therapeutically relevant receptor targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for site-selective functionalization of large molecular assemblies like VLPs.

Cell penetrating thyclotides (CPTs) are synthetic molecules that promote highly efficient cellular uptake and endosomal escape of bioactive peptides. While peptides are valuable as medicinal agents, their translation to therapies is often limited by their inability to cross cell membranes. CPTs have a unique combination of chiral tetrahydrofurans and polar sidechains within a molecular scaffold that can be optimized to efficiently deliver peptide cargo into cells. The cellular uptake and endosomal escape of two peptides with anticancer biological activities but low bioavailabilities were remarkably improved after conjugation to a CPT. Using CPTs to overcome barriers to cellular uptake represents a new direction for the intracellular delivery of bioactive molecules, and will accelerate drug development for new medical therapies.

Cell-free biosensors leverage in vitro gene expression reactions to detect chemicals. While inexpensive, modular, and distributable, these platforms are constrained by slow readouts at ambient temperatures, precluding practical field operation. In cells, phase separation accelerates biochemical reactions; however, recapitulating these gains in vitro has remained challenging for complex biochemistries. Here, we report the first self-assembling coacervate system that accelerates in vitro transcription. Prepared by simple mixing, coacervation with spermine and polyacrylic acid occurs dynamically in response to NTP consumption and co-localizes DNA templates and RNA polymerase to accelerate transcription, mimicking intracellular phenomena. We exploit this discovery to accelerate the cell-free biosensing of six ligands, demonstrating that coacervation can preserve platform modularity, improve sensitivity, retain lyophilization compatibility, function in field matrices, and reduce ambient-temperature time-to-signal by hours. This work contributes to a growing understanding of phase separation in biology and advances the use of membrane-less organization for real-world applications.

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionite's ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied this method to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

Proximity labeling has transformed spatial proteomics by enabling compartment-resolved mapping of protein environments in living cells, yet its extension to small-molecule metabolites has not been demonstrated, probably due to limitations in labeling chemistry and identification of labeled metabolites. Here, we introduce DESTNI, an engineered desthiobiotin (DTB) ligase derived from TurboID through directed evolution, and establish a platform for spatially resolved profiling of amine-containing metabolites. A directed evolution strategy based on a yeast display system yielded DESTNI with an efficient DTB-dependent reactivity, enabling robust and compartment-specific proximity labeling across diverse subcellular environments. To identify the DTB-modified amino metabolome, we developed an integrated analytical framework combining DTB-modified amino metabolite standards, in vitro DESTNI profiling, and in silico MS/MS prediction, enabling systematic annotation of DTB-modified amino metabolites. To extend this chemistry to metabolites, we combined synthetic DTB-conjugated metabolite reference standards, in vitro DESTNI-reactive metabolite discovery, and machine-learning prediction of DTB-derivatized metabolites and oligopeptides. Organelle-targeted DESTNI recovered reproducible compartment-enriched amino metabolite signatures, including mitochondrial matrix-enriched glycine, 5-aminolevulinic acid, ornithine and spermidine adducts, as well as nuclear-enriched {gamma}-aminobutyric acid and 5-aminovaleric acid adducts. Together, this work establishes DESTNI as a proximity labeling platform that bridges spatial proteomics and metabolomics and provides a general strategy for mapping subcellular biochemical environments in living cells.

Establishing tools to couple biological processes to a DNA sequence has transformed our ability to monitor life at the molecular scale due to the scalability, flexibility, and low cost of DNA sequencing. Key examples include DNA-protein (ChIP-seq1), RNA-protein (CLIP-seq2), protein-protein (proximity ligation assay3), and Cas-based recording of cellular events4. In contrast, this paradigm has not yet significantly enhanced studies of glycans, which are mostly limited to non-DNA based chemical and biochemical assays. While classical asparagine-linked and serine/threonine-linked glycans can be directly sequenced using mass spectrometry, glycosaminoglycans - notable players in the extracellular matrix - cannot be easily analyzed in their full-length form. Here we introduce HS-nano-seq, a generalized framework to selectively label, process, and detect features of heparan sulfate on a nanopore sequencing platform. Recognizing that heparan sulfate is biochemically analogous to a nucleic acid, we report purification techniques using rapid nucleic acid strategies and conjugation methods to couple DNA adapters, generating HS-DNA chimeras resolved as discrete species by capillary electrophoresis (CE). The CE assay can distinguish features of chain length and sulfation patterns. At the single-molecule level enabled by nanopore sensing, we classify a library of synthetic heparan sulfate standards and demonstrate that nanopore ionic current fingerprints encode sulfation-dependent structural features of individual HS chains. Analysis of intact, cell-derived HS could discriminate features of individual chains with different sulfation patterns, defining the heterogeneity of binding motifs across cell types and how cells organize and program the tethered extracellular matrix. More broadly, HS-nano-seq establishes a framework for achieving full-length readouts of ECM glycopolymers that are amenable to the same biological interrogation as nucleic acids.

mNeonGreen (mNG) is among the brightest and most photostable monomeric green fluorescent proteins and is widely used for protein tagging. Here, we present sdAb(mNG), a high-affinity single-domain antibody (sdAb) that enables biochemical capture, imaging, and manipulation of mNG-tagged proteins. A 1.26 [A] crystal structure reveals an extensive interaction surface between mNG and sdAb(mNG), accounting for its high affinity (KD = 0.39 nM) and robust target recognition across diverse experimental conditions. This allows a single sdAb to support applications that typically require multiple specialized tools. We demonstrate the utility of sdAb(mNG) in several example applications including highly specific immunoprecipitation, direct immunofluorescence, and super-resolution imaging. Importantly, sdAb(mNG) retains high-performance target recognition even in intracellular environments. When expressed as an intrabody in living mammalian cells, sdAb(mNG) enables relocalization of mNG-tagged proteins to defined compartments or visualization of synaptic vesicle transport in primary neurons. In zebrafish, fusion of sdAb(mNG) to an F-box degradation domain induces cell-autonomous depletion of an endogenous mNG-tagged transcription factor and produces a clear developmental phenotype. These findings establish sdAb(mNG) as a versatile and robust affinity reagent that converts mNG from a passive fluorescent reporter into a multifunctional handle for imaging, proteomics, and programmable manipulation of endogenous and engineered proteins.

Serotonin is a key neurotransmitter, and aptamer-based tools using the 44 nt Apt44 have been successfully developed for its in vitro and in vivo detection. Nevertheless, the structural basis of recognition by this aptamer remains unclear. Here we report high resolution NMR structures of Apt38, a 6-nt truncated variant in the third loop of Apt44, in free and serotonin-bound states. Both structures reveal a two layered antiparallel chair type G quadruplex core with three edgewise loops and a terminal duplex, forming a G quadruplex duplex hybrid structure. Serotonin binds at the G quadruplex duplex junction, stabilized by stacking, electrostatic attraction, hydrogen bonding, and hydrophobic contacts. Apt38 is preorganized for binding, whereas the longer third loop of Apt44 introduces conformational dynamics into the G quadruplex scaffold, which enables a pronounced binding triggered conformational switch in PBS buffer, explaining its sensing mechanism. Our work reveals the recognition and sensing mechanism of the serotonin aptamer and provides a framework for aptamer design in serotonin biosensing.

Single-cell metabolomics remains analytically challenging due to the low abundance and chemical diversity of metabolites in individual cells. We have developed complementary microflow HILIC and ion pair LC-MS methods to expand metabolite coverage in single macrophages. Ion pair LC-MS was applied to single cells for the first time, enabling retention of highly polar and ionic metabolites that elute early under conventional reversed-phase conditions. Across Mycobacterium bovis BCG infected, uninfected bystander, and control unexposed THP-1 macrophages, both microflow methods detected significantly more features than a previously reported analytical-flow HILIC method. The two microflow methods provided complementary chemical space, together yielding 633 unique named metabolites with MS2 spectra. This depth enabled pathway-level interpretation at single-cell resolution, revealing infection-associated changes in purine-, arginine-, glutathione-, and one-carbon folate-associated metabolism. Metabolite-level interrogation indicated shared purine and amino acid changes in both infected and neighbouring macrophages, while revealing a distinct bystander phenotype characterised by elevated glycine and heterogeneous ATP levels. Finally, we demonstrate sequential IP and HILIC analysis of the same single cell, establishing a route toward maximal coverage from individual cells. These results position microflow HILIC and IP LC-MS as powerful, orthogonal strategies for advancing single-cell metabolomics and unveiling heterogeneity within complex biological microenvironments. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/733771v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d0d02dorg.highwire.dtl.DTLVardef@11364d5org.highwire.dtl.DTLVardef@40e1a1org.highwire.dtl.DTLVardef@19d24a5_HPS_FORMAT_FIGEXP M_FIG Figure made in BioRender. C_FIG

Fluorescence correlation spectroscopy (FCS) provides valuable information about molecular dynamics, however, experimental setup typically requires labour-intensive passivation to prevent non-specific binding of molecules to sample containers. Furthermore, precious samples can be wasted by having to use relatively high sample volumes in existing sample containers. We overcome these major issues using a simple method of sample encapsulation into water-in-oil droplets, using purified proteins and cell lysates as proof-of-concept. FCS of fluorescently labelled protein samples in the nanomolar (nM) range confirmed that water-in-oil droplets yield more accurate measurements than conventional open-chamber methods. We first optimized the droplet composition to prevent protein coating at the water-oil interface using pegylated-lipids. We then utilized FCS to accurately measure protein concentrations and diffusion speeds in nanolitre volumes. Additionally, we used fluorescence cross-correlation spectroscopy (FCCS) to measure enzymatic cleavage of substrate inside our droplet system, demonstrating the capacity of this platform to measure biological processes at the nanoscale. Overall, conducting FCS in droplets offers a cost-effective, robust, and accessible alternative for measuring molecular dynamics, with promising potential for high-throughput and resource-limited applications. TOC Image + Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/734730v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e0023dorg.highwire.dtl.DTLVardef@b32cb0org.highwire.dtl.DTLVardef@13ad832org.highwire.dtl.DTLVardef@47dc12_HPS_FORMAT_FIGEXP M_FIG C_FIG Conventional single-molecule fluorescence requires slow, expensive glass passivation procedures to prevent proteins adsorbing to surfaces. By encapsulating proteins in lipid-coated nanolitre water droplets, the passivation requirement is removed, enabling accurate measurement of protein dynamics in low nanolitre volumes. Water-in-oil droplets thus provide a passivation-free platform for fluorescence correlation spectroscopy.

The chorioallantoic membrane (CAM) model represents a promising three-dimensional in vivo platform for preclinical drug testing in human tissues. In this study, we investigated whether the tissue penetration and distribution of benzbromarone, a known inhibitor of the Ca2+ activated chloride channel TMEM16A and potential therapeutic agent for autosomal dominant polycystic kidney disease (ADPKD), can be successfully visualized in human renal cyst tissue cultured on the CAM. To this end, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) combined with an ultrahigh-resolution time-of-flight mass spectrometer was employed. We achieved spatially resolved molecular mapping of endogenous metabolites and lipids as well as the applied compound. MSI enabled clear differentiation between CAM and cystic tissue based on their distinct lipid profiles. Benzbromarone was reproducibly detected in the cyst specimens and exhibited selective accumulation along the cyst epithelium, which is considered the principal site of action. These observations were complemented by multivariate analyses including Uniform Manifold Approximation and Projection (UMAP), and sparse multinomial logistic zero-sum classification. The data-driven approach confirmed molecular differences between tissue types and allowed accurate classification of drug-treated and untreated regions. This study demonstrates that topically applied benzbromarone penetrates human renal cyst tissue in the CAM model and localizes to pharmacologically relevant tissue regions, notably the location of the Ca2+ activated chloride channel TMEM16A in the epithelial lining. The integration of high-resolution DESI-MSI with advanced statistical analysis provides a robust and label-free method to study drug distribution in human tissue grafts. Our findings contribute to the advancement of translational research in analytical chemistry and pharmacology.

Poor aqueous solubility remains a major obstacle to the translational development of targeted anticancer compounds. VS1, a first-in-class inhibitor of the cholesterol-transfer protein STARD3, has emerged as a promising chemosensitizing agent in colorectal cancer (CRC), but its clinical applicability is limited by its poor water solubility. Here, we combine structural biology, nanotechnology, and functional pharmacology to establish STARD3 inhibition as a delivery-enabled strategy to potentiate fluoropyrimidine therapy. To define the molecular basis of STARD3 inhibition, we solved the crystal structure of VS1 bound to the STARD3 ligand-binding domain at 2.1 [A] resolution, revealing direct occupation of the sterol-binding cavity. Molecular dynamics simulations confirmed a stable binding mode and identified the {Omega}1 loop as a dynamic gate regulating ligand binding and dissociation. To overcome the formulation barrier of VS1, we engineered carrier-free, albumin-coated nanocrystals through sonication-assisted nanocrystallization followed by surfactant exchange with human serum albumin. The resulting rod-shaped nanocrystals displayed nanometric size, narrow size distribution, sustained release, and improved aqueous dispersibility, increasing the apparent solubility of VS1 by more than 14-fold while preserving its molecular integrity and crystallinity. Biologically, VS1 selectively potentiated 5-fluorouracil (5-FU) in CRC cells, with synergistic effects restricted to 5-FU-sensitive models and associated with enhanced reactive oxygen species accumulation. Albumin-coated formulation retained the chemosensitizing activity of the free compound. In HCT-116 xenografts, combined treatment with albumin-coated VS1 nanocrystals and 5-FU significantly reduced tumor growth, prolonged tumor doubling time, and increased intratumoral necrosis without exacerbating systemic toxicity. Together, these findings establish that albumin-coated nanocrystals can overcome the delivery limitations of an insoluble STARD3 inhibitor and provide a formulation-enabled strategy to enhance fluoropyrimidine therapy in colorectal cancer.

Peptidoglycan is an essential component of the bacterial cell wall, providing mechanical strength and maintaining cell shape. It consists of glycan chains crosslinked by short peptide stems, resulting in a chemically heterogeneous macromolecule that remains challenging to study in a well-defined form. Access to discrete peptidoglycan fragments has therefore been critical for advancing biochemical and structural studies of cell wall-active enzymes. However, current synthetic, semi-synthetic, and cell wall extraction approaches remain limited by the complexity of carbohydrate chemistry and the difficulty of isolating pure, well-defined material. Here, we report a facile enzymatic approach for generating defined, denuded peptidoglycan oligosaccharides from the cell walls of two Staphylococcus species. These oligosaccharides, which terminate in N-acetylglucosamine and range from two to five disaccharide units in length, serve as substrates for a diverse panel of peptidoglycan-active enzymes that cleave or chemically modify the glycan backbone. We further show that these denuded oligosaccharides can be used in lysozyme-catalyzed transglycosylation reactions to generate p-nitrophenyl derivatives, enabling continuous colorimetric monitoring of peptidoglycan-cleaving enzymes. This method provides a practical route to defined peptidoglycan glycans and establishes a platform for further structural diversification, including stem peptide reattachment, quantitative enzyme assays, and structural characterization of peptidoglycan-binding proteins.

Targeting the overwhelming inflammation driven by neutrophil extracellular traps (NETs) during infection provides an opportunity to manage severe sepsis. This potential needs to be realized by exploring selective NET-neutralization materials, which remains a challenge. Herein, we report a multivalent macromolecular strategy that targets NET-associated DNA-histone chromatin complexes while preserving antibacterial activity of aminoglycoside. We identify 8-arm PEG-conjugated netilmicin (8-arm Netil) as a lead NETs-neutralizer from a library of multivalent aminoglycoside-displayed materials. When compared with 2- and 4-arm counterparts, 8-arm Netil exhibits potent antibacterial activity and high-affinity binding to DNA-histone chromatin complexes through stable multivalent noncovalent interactions, thereby suppressing NET-induced TLR4/TLR9 activation and macrophage inflammatory responses. In severe septic mice, intravenously administered 8-arm Netil preferentially accumulates in inflamed tissues, leading to improved survival protection, owing to the reduction of bacterial dissemination, NET accumulation, systemic cytokine production, and multiple-organ injury. These findings establish NET-associated DNA-histone chromatin complexes as actionable extracellular targets and demonstrate multivalent chromatin targeting as a rational material design strategy for selective NET neutralization and inflammation control in severe sepsis.

Single-particle tracking (SPT) over time enables direct observation of molecular transport and interactions in living cells. Fluorescence-based SPT has provided insights into intracellular processes such as endocytosis, receptor signaling, and drug delivery. Extending the observation window to several hours and beyond is critical for capturing slow intracellular dynamics, including the full course of endosomal trafficking, the long-term accumulation of particles within subcellular compartments, and transitions between transport modes that occur on hour-scale timescales. However, long-term intracellular SPT under visible-wavelength excitation remains challenging because fluorescence probes generally suffer from photobleaching and phototoxicity. While near-infrared (NIR) excitation can simultaneously mitigate these issues, generally weak emission of NIR-emitting dyes has hindered its wide application in long-term SPT. Here, we demonstrate long-term NIR SPT using atomically precise gold quantum needles, Au42(PET)32 (PET = 2-phenylethanethiolate). Continuous tracking of intracellular particles in living HEK293 cells was achieved for up to 12 h. Trajectory analysis revealed temporal transitions between directional and diffusive transport, as well as the accumulation of multiple particles within localized intracellular domains over several-hour timescales. The high photostability of Au42, combined with low phototoxicity of NIR excitation, enables visualization of intracellular transport dynamics over timescales difficult to access using conventional visible fluorescent probes. These results establish Au42-based NIR imaging as a platform for long-term, low-phototoxicity intracellular SPT and provide a framework for investigating slow intracellular dynamics in living systems.

Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.