Analysis Of Binding Kinetics Research Articles

Label-free evanescent imaging of cellular heterogeneity in membrane protein binding kinetics

Quantifying cellular heterogeneity of membrane protein binding kinetics is challenging but important for exploring drug resistance and screening drugs. Label-free analysis methods have emerged as promising tools for in situ binding kinetics analysis, but they have not been used for high throughput single cell analysis in live cells. Here we show that this is possible with Evanescent Scattering Microscopy (ESM). The ESM permits analyzing the kinetics of ligand binding onto membrane proteins in individual fixed and live cells, and provides a throughput of ∼200 cells in a single measurement with a period of ∼7 minutes. The statistical analysis further shows that the dissociation rate constant dominates the heterogeneity of cell responses to ligand binding, providing evidence for a long-standing hypothesis that the drug-target residence time may play a critical role in drug treatment. In addition, the ESM reveals that under some conditions the cells have responses to drug binding at the single cell level, whereas the ensemble measurements may average out the individual differences and present false negative results. We anticipate that the new evanescent imaging method will provide a powerful tool to quantify the functions of cellular proteins, especially their cell-to-cell heterogeneity that can provide fuel for drug resistance.

Efficient characterization of multiple binding sites of small molecule imaging ligands on amyloid-beta, tau and alpha-synuclein.

There is an unmet need for compounds to detect fibrillar forms of alpha-synuclein (αSyn) and 4-repeat tau, which are critical in many neurodegenerative diseases. Here, we aim to develop an efficient surface plasmon resonance (SPR)-based assay to facilitate the characterization of small molecules that can bind these fibrils. SPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds toward recombinant amyloid-beta (Aβ)42, K18-tau, full-length 2N4R-tau and αSyn fibrils. In silico modeling was performed to examine the binding pockets of ligands on αSyn fibrils. Immunofluorescence staining of postmortem brain tissue slices from Parkinson's disease patients and mouse models was performed with fluorescence ligands and specific antibodies. We optimized the protocol for the immobilization of Aβ42, K18-tau, full-length 2N4R-tau and αSyn fibrils in a controlled aggregation state on SPR-sensor chips and for assessing their binding to ligands. The SPR results from the analysis of binding kinetics suggested the presence of at least two binding sites for all fibrils, including luminescent conjugated oligothiophenes, benzothiazole derivatives, nonfluorescent methylene blue and lansoprazole. In silico modeling studies for αSyn (6H6B) revealed four binding sites with a preference for one site on the surface. Immunofluorescence staining validated the detection of pS129-αSyn positivity in the brains of Parkinson's disease patients and αSyn preformed-fibril injected mice, 6E10-positive Aβ in arcAβ mice, and AT-8/AT-100-positivity in pR5 mice. SPR measurements of small molecules binding to Aβ42, K18/full-length 2N4R-tau and αSyn fibrils suggested the existence of multiple binding sites. This approach may provide efficient characterization of compounds for neurodegenerative disease-relevant proteinopathies.

Structure-Activity Studies of 1,2,4-Oxadiazoles for the Inhibition of the NAD+-Dependent Lysine Deacylase Sirtuin 2.

The NAD+-dependent lysine deacylase sirtuin 2 (Sirt2) is involved in multiple pathological conditions such as cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purposes. Furthermore, the orthologue from Schistosoma mansoni (SmSirt2) has been considered for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the "Kinetobox" library as a dual inhibitor of human Sirt2 (hSirt2) and SmSirt2. Herein, we describe the structure-activity studies on 1,2,4-oxadiazole-based analogues, which are potent inhibitors of human Sirt2 deacetylation. As proposed by docking studies, a substrate-competitive and cofactor-noncompetitive binding mode of inhibition could be determined in vitro via binding assays and kinetic analysis and further confirmed by a crystal structure of an oxadiazole inhibitor in complex with hSirt2. Optimized analogues reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.

Antitumor Activity of Tasurgratinib as an Orally Available FGFR1-3 Inhibitor in Cholangiocarcinoma Models With FGFR2-fusion.

Cholangiocarcinoma (CCA) is an aggressive tumor with limited treatment options especially in 2nd line or later treatments. Targeting fibroblast growth factor receptor (FGFR) 2 has recently emerged as a promising treatment option for patients with CCA harboring FGFR2-fusion. This study investigated the antitumor activities of tasurgratinib as an orally available FGFR1-3 inhibitor, in preclinical FGFR2-driven CCA models. Antitumor activities of tasurgratinib were examined in vitro and in vivo using NIH/3T3 cells expressing FGFR2-fusion as FGFR2-driven CCA models, and in vivo using a CCA patient-derived xenograft model. The molecular mechanism of action of tasurgratinib was elucidated through co-crystal structure analysis with FGFR1, manual complex model analysis with FGFR2, and binding kinetics analysis with FGFR2. Furthermore, the cell-based inhibitory activities against acquired resistant FGFR2 mutations in patients with CCA treated with FGFR inhibitors were evaluated. Tasurgratinib showed antitumor activity in preclinical FGFR2-driven CCA models by inhibiting the FGFR signaling pathway in vitro and in vivo. Furthermore, cell-based target engagement assays indicated that tasurgratinib had potent inhibitory activities against FGFR2 mutations, such as N549H/K, which are the major acquired mutations in CCA. We also confirmed that tasurgratinib exhibited fast association and slow dissociation kinetics with FGFR2, binding to the ATP-binding site and the neighboring region, and adopting an Asp-Phe-Gly (DFG)-"in" conformation. These data demonstrate the therapeutic potential of tasurgratinib in FGFR2-driven CCA and provide molecular mechanistic insights into its unique inhibitory profile against secondary FGFR2 resistance mutations in patients with CCA treated with FGFR inhibitors.

Antidote-controlled DNA aptamer modulates human factor IXa activity

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

Flubendazole suppresses VEGF-induced angiogenesis in HUVECs and exerts antitumor effects in PC-3 cells.

Flubendazole, an FDA-approved anthelmintic, has been predicted to show strong VEGFR2 inhibitory activity in silico screening combined with invitro experimental validation, and it has shown anti-cancer effects on some human cancer cell lines, but little is known about the anti-angiogenesis effects and anti-prostate cancer effects. In this study, we analyzed the binding modes and kinetic analysis of flubendazole with VEGFR2 and first demonstrated that flubendazole suppressed VEGF-stimulated cell proliferation, wound-healing migration, cell invasion and tube formation of HUVEC cells, and decreased the phosphorylation of extracellular signal-regulated kinase and serine/threonine kinase Akt, which are the downstream proteins of VEGFR2 that are important for cell growth. What's more, our results showed that flubendazole decreased PC-3 cell viability and proliferation ability, and suppressed PC-3 cell wound healing migration and invasion across a Matrigel-coated Transwell membrane in a concentration-dependent manner. The antiproliferative effects of flubendazole were due to induction of G2-M phase cell cycle arrest in PC-3 cells with decreasing expression of the Cyclin D1 and induction of cell apoptosis with the number of apoptotic cells increased after flubendazole treatment. These results indicated that flubendazole could exert anti-angiogenic and anticancer effects by inhibiting cell cycle and inducing cell apoptosis.

Shine-Dalgarno Accessibility Governs Ribosome Binding to the Adenine Riboswitch.

Translational riboswitches located in the 5' UTR of the messenger RNA (mRNA) regulate translation through variation of the accessibility of the ribosome binding site (RBS). These are the result of conformational changes in the riboswitch RNA governed by ligand binding. Here, we use a combination of single-molecule colocalization techniques (Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) and Single-Molecule Kinetic Analysis of Ribosome Binding (SiM-KARB)) and microscale thermophoresis (MST) to investigate the adenine-sensing riboswitch in Vibrio vulnificus, focusing on the changes of accessibility between the ligand-free and ligand-bound states. We show that both methods faithfully report on the accessibility of the RBS within the riboswitch and that both methods identify an increase in accessibility upon adenine binding. Expanding on the regulatory context, we show the impact of the ribosomal protein S1 on the unwinding of the RNA secondary structure, thereby favoring ribosome binding even for the apo state. The determined rate constants suggest that binding of the ribosome is faster than the time required to change from the ON state to the OFF state, a prerequisite for efficient regulation decision.

A two-step mechanism for the binding of the HIV-1 MPER epitope by the 10E8 antibody onto biosensor-supported lipid bilayers.

HIV-1 antibodies targeting the carboxy-terminal area of the membrane-proximal external region (ctMPER) are close to exerting viral pan-neutralization. Here, we reconstituted the ctMPER epitope as the N-terminal extremity of the Env glycoprotein transmembrane domain helix and immobilized it onto biosensor-supported lipid bilayers. We assessed the binding mechanism of anti-MPER antibody 10E8 through Surface Plasmon Resonance, and found, through equilibrium and kinetic binding analyses as a function of bilayer thickness, peptide length, and paratope mutations, that 10E8 engages first with the epitope peptide (encounter), limited by ctMPER helix accessibility at the membrane surface, and then inserts into the lipid bilayer assisted by favorable Fab-membrane interactions (docking). This mechanistic information may help in devising new strategies to develop more efficient MPER-targeting vaccines.

Real-time kinetic analysis and detection of glycated hemoglobin A1c using a quartz crystal microbalance-based aptasensor.

Glycated hemoglobin (HbA1c) has been an important biomarker for long-term diagnosis and monitoring of diabetes mellitus. The development of a rapid, reliable, and less sophisticated device to measure HbA1c is imperative to facilitate efficient early-care diabetes management. To date, no existing aptamer-based biosensor (aptasensor) for detecting HbA1c has been developed using a quartz crystal microbalance (QCM). In this study, the aptamer specific to HbA1c as a novel biosensing receptor was covalently functionalized onto a QCM substrate via mixed self-assembled monolayers (SAMs). A portable QCM equipped with a liquid-flow module was used to investigate the biospecificity, sensitivity, and interaction dynamics of the aptamer functionalized surfaces. The real-time kinetic analysis of HbA1c binding to the surface-functionalized aptamers revealed "on" and "off" binding rates of 4.19 × 104 M-1 s-1 and 2.43 × 10-3 s-1, respectively. These kinetic parameters imply that the QCM-based aptasensor specifically recognizes HbA1c with an equilibrium dissociation constant as low as 57.99 nM. The linear detection of HbA1c spanned from 13 to 108 nM, with a limit of detection (LOD) of 26.29 nM. Moreover, the spiked plasma sample analysis offered compelling evidence that this aptasensor is a promising technique for developing a point-of-care device for diabetes mellitus.

Evaluation of an Image-Derived Input Function for Kinetic Modeling of Nicotinic Acetylcholine Receptor-Binding PET Ligands in Mice.

Positron emission tomography (PET) radioligands that bind with high-affinity to α4β2-type nicotinic receptors (α4β2Rs) allow for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. Here, we investigate the use of an image-derived arterial input function and the cerebellum for kinetic analysis of radioligand binding in mice. Two radioligands were explored: 2-[18F]FA85380 (2-FA), displaying similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, displaying similar pKa and binding affinity to nicotine. Time-activity curves of the left ventricle of the heart displayed similar distribution across wild type mice, mice lacking the β2-subunit for ligand binding, and acute nicotine-treated mice, whereas reference tissue binding displayed high variation between groups. Binding potential estimated from a two-tissue compartment model fit of the data with the image-derived input function were higher than estimates from reference tissue-based estimations. Rate constants of radioligand dissociation were very slow for 2-FA and very fast for Nifene. We conclude that using an image-derived input function for kinetic modeling of nicotinic PET ligands provides suitable results compared to reference tissue-based methods and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.

Discovery of Selective Histone Deacetylase 1 and 2 Inhibitors: Screening of a Focused Library Constructed by Click Chemistry, Kinetic Binding Analysis, and Biological Evaluation.

Histone deacetylase 1 and 2 (HDAC1/2) inhibitors are potentially useful as tools for probing the biological functions of the isoforms and as therapeutic agents for cancer and neurodegenerative disorders. To discover potent and selective inhibitors, we screened a focused library synthesized by using click chemistry and obtained KPZ560 as an HDAC1/2-selective inhibitor. Kinetic binding analysis revealed that KPZ560 inhibits HDAC2 through a two-step slow-binding mechanism. In cellular assays, KPZ560 induced a dose- and time-dependent increase of histone acetylation and showed potent breast cancer cell growth-inhibitory activity. In addition, gene expression analyses suggested that the two-step slow-binding inhibition by KPZ560 regulated the expression of genes associated with cell proliferation and DNA damage. KPZ560 also induced neurite outgrowth of Neuro-2a cells and an increase in the spine density of granule neuron dendrites of mice. The unique two-step slow-binding character of o-aminoanilides such as KPZ560 makes them interesting candidates as therapeutic agents.

One-step production of fully biotinylated and glycosylated human Fc gamma receptors.

Initiating and regulating humoral immunity, Fc gamma receptors (FcγRs) have been identified both as therapeutics and as drug targets, and thus production of biologically active FcγRs is highly demanded for biopharmaceutical development. Focusing on low-affinity FcγRs IIA (131H/R allotypes), IIB, and IIIA (176F/V), this study used human 293-F cells to achieve correct post-translational modifications (PTMs) including biotinylation, N-glycosylation, and disulfides. Approaches involving co-expression of FcγR-AviTag and Escherichia coli biotin ligase BirA, endoplasmic reticulum retention, stable and transient transfections, and optimization of transgene ratio were investigated. Protein electrophoresis under reducing and non-reducing conditions, enzymatic deglycosylation, streptavidin pull-down assays, and binding kinetic analysis collectively indicated that the produced FcγR ectodomains were fully biotinylated, N-glycosylated, had formed disulfide bond, and exhibited expected binding affinities toward IgG1 trastuzumab and its Fc mutants. A clear trade-off between production yield and PTM quality was also observed. Achieving multiple types of PTMs completely by one-step cell culture should have applications for the production of a variety of complex proteins of biomedical importance.

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Kinetic analysis of fluorescent ligand binding to cell surface receptors: Insights into conformational changes and allosterism in living cells.

Equilibrium binding assays are one of the mainstays of current drug discovery efforts to evaluate the interaction of drugs with receptors in membranes and intact cells. However, in recent years, there has been increased focus on the kinetics of the drug-receptor interaction to gain insight into the lifetime of drug-receptor complexes and the rate of association of a ligand with its receptor. Furthermore, drugs that act on topically distinct sites (allosteric) from those occupied by the endogenous ligand (orthosteric site) can induce conformational changes in the orthosteric binding site leading to changes in the association and/or dissociation rate constants of orthosteric ligands. Conformational changes in the orthosteric ligand binding site can also be induced through interaction with neighbouring accessory proteins and receptor homodimerisation and heterodimerisation. In this review, we provide an overview of the use of fluorescent ligand technologies to interrogate ligand-receptor kinetics in living cells and the novel insights that they can provide into the conformational changes induced by drugs acting on a variety of cell surface receptors including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and cytokine receptors.

Spatially Resolved Protein Binding Kinetics Analysis in Microfluidic Photonic Crystal Sensors

Organ-on-a-Chip systems are emerging as an important in vitro analysis method for drug screening and medical research. For continuous biomolecular monitoring of the cell culture response, label-free detection within the microfluidic system or in the drainage tube is promising. We study photonic crystal slabs integrated with a microfluidic chip as an optical transducer for label-free biomarker detection with a non-contact readout of binding kinetics. This work analyzes the capability of same-channel reference for protein binding measurements by using a spectrometer and 1D spatially resolved data evaluation with a spatial resolution of 1.2 μm. A cross-correlation-based data-analysis procedure is implemented. First, an ethanol–water dilution series is used to obtain the limit of detection (LOD). The median of all row LODs is (2.3±0.4)×10−4 RIU with 10 s exposure time per image and (1.3±0.24)×10−4 RIU with 30 s exposure time. Next, we used a streptavidin–biotin binding process as a test system for binding kinetics. Time series of optical spectra were recorded while constantly injecting streptavidin in DPBS at concentrations of 1.6 nM, 3.3 nM, 16.6 nM and 33.3 nM into one channel half as well as the whole channel. The results show that localized binding within a microfluidic channel is achieved under laminar flow. Furthermore, binding kinetics are fading out at the microfluidic channel edge due to the velocity profile.

NanoBiT- and NanoBiT/BRET-based assays allow the analysis of binding kinetics of Wnt-3a to endogenous Frizzled 7 in a colorectal cancer model.

Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt-FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt-FZD7 binding should be performed using receptors expressed under endogenous promotion.

Anti-CD30 (Ber-H2) epitope requires structural elements as shown by mass spectroscopy and dual-site associated kinetics.

The Ber-H2 mouse monoclonal antibody has been in use for thirty-five years for detecting the CD-30 biomarker in a variety of lymphomas. Despite the wide use of this clone, we have not been successful in applying synthetic peptides derived from the published epitope sequence and affinity data towards the development of a new Ber-H2-based in-vitro diagnostic reagent (IVDR) assay. We found that synthetic peptides based on the published epitope sequence do not function to inhibit antibody-binding activity, thus indicating that the sequence is not the full epitope recognized by Ber-H2. In this report, we used mass spectroscopic analysis of proteolysed CD30 fragments capable of binding Ber-H2 to identify additional regions within the epitope that participate in binding. Using surface plasmon resonance binding kinetic analyses and immuno-histochemical peptide-inhibition assays, we also demonstrate that the epitope sequence as originally reported is missing two key elements necessary for binding the Ber-H2 antibody. This article is protected by copyright. All rights reserved.

Abstract 2759: Real-time cellular target engagement and protein quantification for drug discovery

Abstract Preclinical assessment of direct drug-target engagement and analysis of binding kinetics within the cellular environment is essential for development of safer and more effective therapeutics. Infusion of such critical data into early drug discovery would significantly reduce the failure rate of new drug candidates, accelerate drug discovery, and validate repurposing of existing drugs. Described here is a novel drug-target engagement technology that can sensitively interrogate direct binding of drugs to cellular, bacterial, or viral protein targets within the physiological environment. Micro-Tag cell target engagement technology is based on complementation of a small 15-amino acid subunit with a large subunit into an active RNA-processing enzyme. The small subunit can be cloned to any drug target for transient or stable expression using existing tools such as CRISPR. Upon complementation, the active enzyme cleaves a FRET-based oligonucleotide substrate resulting in rapid generation of fluorescent signal that can be quantified in real time. The Micro-Tag technology enables in-cell quantitation of drug target levels using qPCR systems. This is the next-generation of target engagement technology that allows for real-time monitoring of drug-target interaction in the cell. We show here data demonstrating direct engagement of several reference compounds and novel small molecules with high-profile cancer targets such as K-RAS, MTH1, EGFR, and UBE2N. Selectivity of these drugs to the targets in the cell is further delineated using their mutant counterparts as well as inactive stereoisomer compounds. The Micro-Tag cell target engagement technology provides the power of cell target engagement to a large family of target proteins. It can be employed for high-throughput screens directed at initial on-target ranking or med-chem optimization of drug candidates. It is amenable for interrogation of drug candidates across various modalities: small molecules, peptides, antibodies and PROTACs. Importantly, this next-generation target engagement technology seamlessly integrates with qPCR systems, fluorescence microscopy, live-cell microscopy, FACS analysis, and offers multiplexing capability, thus allowing for further mechanistic insight. Citation Format: Ivan Babic, Nikolas Bryan, Claire Cunningham, Avery Sampson, Daniel Starczynowski, Elmar Nurmemmedov. Real-time cellular target engagement and protein quantification for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2759.

Primer exchange reaction-amplified protein-nucleic acid interactions for ultrasensitive and specific microRNA detection

Protein-nucleic acid interactions are not only fundamental to genetic regulation and cellular metabolism, but molecular basis to artificial biosensors. However, such interactions are generally weak and dynamic, making their specific and sensitive quantitative detection challenging. By using primer exchange reaction (PER)-amplified protein-nucleic acid interactions, we here design a universal and ultrasensitive electrochemical sensor to quantify microRNAs (miRNAs) in blood. This PER-miR sensor leverages specific recognition between S9.6 antibodies and miRNA/DNA hybrids to couple with PER-derived multi-enzyme catalysis for ultrasensitive miRNA detection. Surface binding kinetic analysis shows a rational Kd (8.9 nM) between the miRNA/DNA heteroduplex and electrode-attached S9.6 antibody. Based on such a favorable affinity, the programmable PER amplification enables the sensor to detect target miRNAs with sensitivity up to 90.5 aM, three orders of magnitude higher than that without PER in routine design, and with specificity of single-base resolution. Furthermore, the PER-miR sensor allows detecting multiple miRNAs in parallel, measuring target miRNA in lysates across four types of cell lines, and differentiating tumor patients from healthy individuals by directly analyzing the human blood samples (n = 40). These advantages make the sensor a promising tool to enable quantitative sensing of biomolecular interactions and precision diagnostics.

Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule Counting.

Multiplexed protein detection is critical for improving the drug and biomarker screening efficiency. Here, we show that multiplexed protein detection and parallel protein interaction analysis can be realized by evanescent scattering microscopy (ESM). ESM enables binding kinetics measurement with label-free digital single-molecule counting. We implemented an automatic single-molecule counting strategy with high temporal resolution to precisely determine the binding time, which improves the counting efficiency and accuracy. We show that digital single-molecule counting can recognize proteins with different molecular weights, thus making it possible to monitor the protein binding processes in the solution by real-time tracking of the numbers of free and bound proteins landing on the sensor surface. Furthermore, we show that this strategy can simultaneously analyze the kinetics of two different protein interaction processes on the surface and in the solution. This work may pave a way to investigate complicated protein interactions, such as the competition of biomarker-antibody binding in biofluids with biomarker-protein binding on the cellular membrane.