Label-free Format Research Articles

Hierarchical Au@Pt nanoparticle/amino benzoic acid polymer-based hybrid material for labeled and label-free detection of interleukin-6: a comparative assessment.

Interleukin-6 (IL6) is a cytokine mainly involved in inflammatory processes associated with various diseases, from rheumatoid arthritis and pathogen-caused infections to cancer, where malignant cells exhibit high proliferation and overexpression of cytokines, including IL6. Furthermore, IL6 plays a fundamental role in detecting and differentiating tumor cells, including colorectal cancer (CRC) cells. Therefore, given its range of biological activities and pathological role, IL6 determination has been claimed for the diagnosis/prognosis of immune-mediated diseases. Herein, a comparative study is presentedof labeled and label-free electrochemical immunosensors involving a hierarchical Au@Pt nanoparticle/polymer hybrid material for detecting IL6. The electrochemical immunosensors were independently coupled to the surface of screen-printed carbon electrodes (SPCEs) previously modified with polymeric layers. While in the label-free immunosensor, an anti-IL6 antibody (IL6-Ab) was covalently bound to the modified SPCE surface, in the sandwich-like amperometric immunosensor, an anti-biotinylated-IL6 antibody (B-IL6-Ab) was attached to the electrode through biotin-avidin affinity interactions. The label-free format employed a straightforward detection of IL6 by differential pulse voltammetry (DPV). The resulting electrochemical immunosensors exhibited a linear dynamic range from 50 to 750pg/mL IL6, with detection limits (LOD) of 14.4 and 6.0pg/mL for label-free and sandwich-like immunosensors, respectively. This outstanding performance makes them versatile platforms for clinical analysis of a panel of biomarkers for early diagnosis/prognosis of inflammatory processes associated with oncological diseases, among other pathologies.

Electron transmittance by means of quantum capacitive states as a signal amplification mechanism for biosensing applications

The use of quantum capacitive signals within redox switch interfaces is a sensitive and specific method for detecting biomolecules in a label-free format. Presently, an electrochemical signal amplification mechanism is demonstrated based on the adjustment of the energy level of quantum redox capacitive probes assembled at the interface to that of free redox probes added to the biological sample. This adjustment of the energy levels permits electron transmittance (in diffusionless mode) and communication with the electrode through the quantum redox capacitive states chemically assembled at the interface. This electron transport mode is owing to a quantum transmittance mechanism that promotes an enhancement of the electrochemical current of the interface. As quantum capacitive moieties can be chemically designed within neighboring biological receptors, an increase is observed in the electric signal sensitivity of the interface upon the binding of a biological analyte of interest by about a thousand times. The detection of the NS1 dengue virus biomarker was performed as a proof-of-concept of the above-described capacitive signal amplification methodology for biosensing, enabling the diagnosis of dengue in a point-of-care format within a limit-of-detection of 0.67 pg mL−1 in undiluted human serum samples.

Label-Free SERS for Rapid Differentiation of SARS-CoV-2-Induced Serum Metabolic Profiles in Non-Hospitalized Adults.

COVID-19 represents a multi-system infectious disease with broad-spectrum manifestations, including changes in host metabolic processes connected to the disease pathogenesis. Understanding biochemical dysregulation patterns as a consequence of COVID-19 illness promises to be crucial for tracking disease course and clinical outcomes. Surface-enhanced Raman scattering (SERS) has attracted considerable interest in biomedical diagnostics for the sensitive detection of intrinsic profiles of unique fingerprints of serum biomolecules indicative of SARS-CoV-2 infection in a label-free format. Here, we applied label-free SERS and chemometrics for rapid interrogation of temporal metabolic dynamics in longitudinal sera of mildly infected non-hospitalized patients (n = 22), at 4 and 16 weeks post PCR-positive diagnosis, and compared them with negative controls (n = 8). SERS spectral markers revealed distinct metabolic profiles in patient sera that significantly deviated from the healthy metabolic state at the two sampling time intervals. Multivariate and univariate analyses of the spectral data identified abundance dynamics in amino acids, lipids, and protein vibrations as the key spectral features underlying the metabolic differences detected in convalescent samples and perhaps associated with patient recovery progression. A validation study performed using spontaneous Raman spectroscopy yielded spectral data results that corroborated SERS spectral findings and confirmed the detected disease-specific molecular phenotypes in clinical samples. Label-free SERS promises to be a valuable analytical technique for rapid screening of the metabolic phenotype induced by SARS-CoV-2 infection to allow appropriate healthcare intervention.

One-Step and Real-Time Detection of microRNA-21 in Human Samples for Lung Cancer Biosensing Diagnosis.

The rapid diagnosis of cancer, especially in its early stages, is crucial for on-time medical treatment and for increasing the patient survival rate. Lung cancer shows the highest mortality rate and the lowest 5-year survival rate due to the late diagnosis in advanced cancer stages. Providing rapid and reliable diagnostic tools is a top priority to address the problem of a delayed cancer diagnosis. We introduce a nanophotonic biosensor for the direct and real-time detection in human plasma of the microRNA-21-5p biomarker related to lung cancer. The biosensor employs a silicon photonic bimodal interferometric waveguide that provides a highly sensitive detection in a label-free format. We demonstrate a very competitive detectability for direct microRNA-21-5p biomarker assays in human plasma samples (estimated LOD: 25 pM). The diagnostic capability of our biosensor was validated by analyzing 40 clinical samples from healthy individuals and lung cancer patients, previously analyzed by reverse-transcription quantitative polymerase chain reaction (qRT-PCR). We could successfully identify and quantify the levels of microRNA in a one-step assay, without the need for DNA extraction or amplification steps. The study confirmed the significance of implementing this biosensor technique compared to the benchmarking molecular analysis and showed excellent agreement with previous results employing the traditional qRT-PCR. This work opens new possibilities for the true implementation of point-of-care biosensors that enable fast, simple, and efficient early diagnosis of cancer diseases.

An alternative label-free DNA sensor based on the alternating-current electroluminescent device for simultaneous detection of human immunodeficiency virus and hepatitis C co-infection

Coinfection of HIV/HCV is a significant public health issue globally, as it increases the risk of liver cancer in co-infected individuals. The point-of-care testing (POCT) device for HIV/HCV DNA detection is promptly needed for diagnosis and monitoring of the disease progression. Here, the alternating-current electroluminescence (ACEL) technique is proposed as a sensitive POCT sensing platform for HIV/HCV cDNA detection. A conductance-based light emission modulated by the hybridization between a pyrrolidinyl PNA probe and the DNA target enabled the DNA detection in a label-free format. Enhanced electroluminescence was observed in the presence of the target DNA due to the increased proton conductivity. Under the optimal conditions, the linearity range from 1 nM to 1 μM was achieved for HIV and HCV cDNA with LODs of 1.86 pM (HIV cDNA) and 1.96 pM (HCV cDNA). The spiked HIV/HCV cDNA in healthy human serum was successfully detected, demonstrating the feasibility of the developed device for the detection of cDNA in real biological samples. Additionally, simultaneous HIV/HCV cDNA detection on a single ACEL device employing a 2x2-array detection zone design. The cross-reactivity with other viral DNA was shown to be minimal due to the high specificity of the PNA probes used. Finally, the negative and positive samples from the patient's serum were tested and the results were in 100% agreement with the commercial kit based-on real-time PCR method, thus illustrating the high sensitivity and specificity of the developed sensor.

Split light up aptamers as a probing tool for nucleic acids.

Aptamers that bind non-fluorescent dyes and increase their fluorescence can be converted to fluorescent sensors. Here, we discuss and provide guidance for the design of split (binary) light up aptameric sensors (SLAS) for nucleic acid analysis. SLAS consist of two RNA or DNA strands and a fluorogenic organic dye added as a buffer component. The two strands hybridize to the analyzed DNA or RNA sequence and form a dye-binding pocket, followed by dye binding, and increase in its fluorescence. SLAS can detect nucleic acids in a cost-efficient label-free format since it does not require conjugation of organic dyes with nucleic acids. SLAS design is preferable over monolith fluorescent sensors due to simpler assay optimization and improved selectivity. RNA-based SLAS can be expressed in cells and used for intracellular monitoring and imaging biological molecules.

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.

A Regenerable Biosensing Platform for Bacterial Toxins.

Waterborne diarrheal diseases such as travelers' diarrhea and cholera remain a threat to public health in many countries. Rapid diagnosis of an infectious disease is critical in preventing the escalation of a disease outbreak into an epidemic. Many of the diagnostic tools for infectious diseases employed today are time-consuming and require specialized laboratory settings and trained personnel. There is hence a pressing need for fit-for-purpose point-of-care diagnostic tools with emphasis in sensitivity, specificity, portability, and low cost. We report work toward thermally reversible biosensors for detection of the carbohydrate-binding domain of the Escherichia coli heat-labile enterotoxin (LTB), a toxin produced by enterotoxigenic E. coli strains, which causes travelers' diarrhea. The biosensing platform is a hybrid of two materials, combining the optical properties of porous silicon (pSi) interferometric transducers and a thermoresponsive multivalent glycopolymer, to enable recognition of LTB. Analytical performance of our biosensors allows us to detect, using a label-free format, sub-micromolar concentrations of LTB in solution as low as 0.135 μM. Furthermore, our platform shows a temperature-mediated "catch-and-release" behavior, an exciting feature with potential for selective protein capture, multiple readouts, and regeneration of the sensor over consecutive cycles of use.

“Signal-On” electrochemical biosensor based on a competitive immunoassay format for the sensitive determination of oxytetracycline

In this work, we describe a new “signal-on” electrochemical biosensor based on a competitive immunoassay with a label-free format for the facile, sensitive and cost-effective determination of oxytetracycline (OTC). The device pattern was designed and printed on low-cost filter paper to construct a disposable paper-based analytical device (PAD) using a wax printing technique. The sensor consisted of a capture anti-OTC antibody (anti-OTC) and OTC-conjugated bovine serum albumin (OTC-BSA). Anti-OTC was first anchored onto the functionalized PAD, followed by the competitive binding of the OTC target and OTC-BSA. The amount of the captured OTC was examined by observing the signal response of the redox [Fe(CN)6]3−/4−. In the presence of OTC, the current response was significantly increased with increasing OTC concentration, whereas the signal response was negligible in the absence thereof. The detected OTC concentration was linear over a range of 1−200 ng mL-1, and the corresponding values of 0.33 and 1.1 ng mL-1 were obtained for the limit of detection (LOD) and limit of quantitation (LOQ), respectively. This introduced sensor displayed high sensitivity and specificity, and the amounts of OTC were successfully quantified in agricultural products. This developed signal-on sensor may be an alternative tool for determining OTC residues and broader antibiotic targets.

Laser-induced graphene interdigitated electrodes for label-free or nanolabel-enhanced highly sensitive capacitive aptamer-based biosensors

Highly porous laser-induced graphene (LIG) is easily generated in complex electrode configurations such as interdigitated electrodes (IDEs). Here, we demonstrate that their superior capacitive response at low frequencies can be exploited in affinity biosensors using thrombin aptamers as model biorecognition elements. Of specific interest was the effect of electrode surface area on capacitance detection, and the comparison between a label-free format and enhancement strategies afforded by carboxy group bearing polymeric nanoparticles or liposomes. Electrochemical impedance spectroscopy (EIS) was used to investigate the LIG performance and optimize the biosensor design. Interestingly, the label-free strategy performed extremely well and additional labels decreased the limit of detection or increased the sensitivity only minimally. It is assumed that the highly porous nature of the LIG structures dominates the capacitive response so that labels removed from the surface have only limited influence Also, while slight performance changes can be observed for smaller vs. larger electrode structures, the performance of a LIG IDE is reasonably independent of its size. In the end, a dynamic range of 5 orders of magnitude was obtained (0.01 nM–1000 nM) with a limit of detection as low as 0.12 pM. When measured in serum, this increased to 1.3 pM. The good reproducibility (relative standard deviation (RSD), 4.90%) and repeatability (RSD, 2.59%) and good long-term stability (>7 weeks at 4 °C) prove that a LIG-based capacitance sensor is an excellent choice for affinity-based biosensor. The ease-of-production, the simplicity of modification and the superior performance even in a label-free format indicate that LIG-based biosensors should be considered in point-of-care diagnostics in the future.

Ultrasensitive and Label-Free Detection of Cell Surface Glycan Using Nanochannel-Ionchannel Hybrid Coupled with Electrochemical Detector.

In this work, asymmetric nanochannel-ionchannel of porous anodic alumina (PAA) coupled with electrochemical detector was used for sensitive and label-free detection of cell surface glycan. The amplified ionic current caused by array nanochannels as well as the ionic current rectification (ICR) caused by asymmetric geometry endows PAA with sensitive ionic current response. Functionalized with the special molecular probe, the constructed nanofluidic device can be used for selective recognition and detection of glycan in a real-time and label-free format. In addition, due to the subnanosize of ionchannels, the probe immobilization and glycan recognition is carried out on the outer surface of PAA, avoiding the blockage of PAA nanochannel by samples, which promises the reproducibility and accuracy of the present method toward bioanalysis. Results show that the glycan concentration ranging from 10 fM to 10 nM can be successfully detected with a detection limit of ∼10 aM, which is substantially lower than most previous works. The designed strategy provides a valuable platform for sensitive and label-free detection of cell surface glycan, which acts as a promising candidate in pathological research and cancer diagnosis.

Aptamers vs. antibodies as capture probes in optical porous silicon biosensors.

Over the past decade aptamers have emerged as a promising class of bioreceptors for biosensing applications with significant advantages over conventional antibodies. However, experimental studies comparing aptasensors and immunosensors, under equivalent conditions, are limited and the results are inconclusive, in terms of benefits and limitations of each bioreceptor type. In the present work, the performance of aptamer and antibody bioreceptors for the detection of a his-tagged protein, used as a model target, is compared. The bioreceptors are immobilized onto a nanostructured porous silicon (PSi) thin film, used as the optical transducer, and the target protein is detected in a real-time and label-free format by reflective interferometric Fourier transform spectroscopy. For the antibodies, random-oriented immobilization onto the PSi nanostructure results in a poor biosensing performance. Contrary, Fc-oriented immobilization of the antibodies shows a similar biosensing performance to that exhibited by the aptamer-based biosensor, in terms of binding rate, dynamic detection range, limit of detection and selectivity. The aptasensor outperforms in terms of its reusability and storability, while the immunosensor could not be regenerated for subsequent experiments.

A sensitive and selective approach for detection of IL 1α cancer biomarker using disposable ITO electrode modified with epoxy-substituted polythiophene polymer

A highly sensitive and label-free electrochemical immunosensor for sensitive detection of interleukin 1α cancer biomarker was described by using epoxy-substituted polythiophene polymer modified disposable indium tin oxide electrode. This conjugated polymer was synthesized to fabricate this immunosensor for the first time and it offered a good biosensing platform for anti-IL 1α antibody immobilization due to epoxy groups present on the side groups of the polymer. Furthermore, the epoxy-substituted polythiophene polymer coated indium tin oxide electrode was used for the determination of IL 1α antigen for the first time. Electrode fabrication stages were characterized by using electrochemical (electrochemical impedance spectroscopy and cyclic voltammetry) and morphological (scanning electron microscopy and atomic force microscopy) techniques. Under optimum experimental conditions, impedimetric responses of the immunosensor increased with the increasing of IL 1α antigen concentration, and the proposed immunosensor displayed a wide linear detection range from 0.01 pg/mL to 5.5 pg/mL with a detection limit of 3.4 fg/mL. The proposed immunosensor exhibited outstanding performance including excellent reproducibility, good repeatability, high selectivity and long storage stability. With the advantages of simple operation, low-cost fabrication and label-free format, the suggested immunosensor was expected to have potential applications for IL 1α cancer biomarker detection.

A wash-free and label-free colorimetric biosensor for naked-eye detection of aflatoxin B1 using G-quadruplex as the signal reporter

A wash-free and label-free colorimetric biosensor for the amplified detection of aflatoxin B1 (AFB1) has been constructed by the integration of an ingenious hairpin DNA probe with exonuclease III (Exo III)-assisted signal amplification. The presence of the AFB1 activates the continuous cleavage reactions by Exo III toward a hairpin probe, resulting in the autonomous accumulation of numerous free G-quadruplex sequences, which can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to produce a colorimetric response. The naked-eye biosensor is ultrasensitive, enabling the visual detection of trace amounts of AFB1 as low as 1 pM without instrumentation. The sensor is robust and can work even when challenged with complex sample matrices such as peanut samples. With the advantages of simple operation, wash-free and label-free format, visible and intuitive output, and low cost, the naked-eye based colorimetric biosensor is expected to have potential applications for in-field detection of AFB1.

Nanoparticle Bragg reflectors: A smart analytical tool for biosensing

In this work, we present a novel optical biosensor based on periodically alternated layers of SiO2 and TiO2 nanoparticles grown by wet methods and working as a Bragg mirror in a label free format. The sensing principle relies on the change of the optical response generated by variations in the dielectric constant of the voids due to a biorecognition probe-target event. This change is reflected in a modification in the photonic band gap frequencies of the Bragg reflector. As a proof of concept, biotin and streptavidin biorecognition on the biosensor surface is monitored in label free format, achieving a LOD of 3.09 mg L−1 (54 nM) for the determination of streptavidin. Our proposed biosensor also shows selective determination against a variety of targets (horseradish peroxidase and bovine serum albumin antibody) working in physiological conditions (diluted human serum). To get a picture of the potential of our development and its ease of integration, we incorporated the immunosensor in an electronic consumable technology, such as compact disk, using the optical disk as analytical platform and a CD player detector for the determination of streptavidin. Thus, our findings point out the feasibility of nanoparticle based Bragg reflectors in the biosensing field and open new avenues towards the rational design of a new generation of versatile, easy to fabricate in mass scale and cost-competitive biosensors.

High-Throughput Density Measurement Using Magnetic Levitation.

This work describes the development of an integrated analytical system that enables high-throughput density measurements of diamagnetic particles (including cells) using magnetic levitation (MagLev), 96-well plates, and a flatbed scanner. MagLev is a simple and useful technique with which to carry out density-based analysis and separation of a broad range of diamagnetic materials with different physical forms (e.g., liquids, solids, gels, pastes, gums, etc.); one major limitation, however, is the capacity to perform high-throughput density measurements. This work addresses this limitation by (i) re-engineering the shape of the magnetic fields so that the MagLev system is compatible with 96-well plates, and (ii) integrating a flatbed scanner (and simple optical components) to carry out imaging of the samples that levitate in the system. The resulting system is compatible with both biological samples (human erythrocytes) and nonbiological samples (simple liquids and solids, such as 3-chlorotoluene, cholesterol crystals, glass beads, copper powder, and polymer beads). The high-throughput capacity of this integrated MagLev system will enable new applications in chemistry (e.g., analysis and separation of materials) and biochemistry (e.g., cellular responses under environmental stresses) in a simple and label-free format on the basis of a universal property of all matter, i.e., density.

Development of Impedimetric Immunosensors for the Diagnosis of DOCK8 and STAT3 Related Hyper-Immunoglobulin E Syndrome

Hyper Immunoglobulin E syndrome (HIES) is a rare inherited inborn error of primary immunodeficiency. HIES is characterized by high levels of serum immunoglobulin E (IgE), severe eczema, which overlap with atopic dermatitis, as well as lung infections and even death. HIESs are genetically inherited by heterozygous or homozygous mutations in signal transducer and activator of transcription 3 (STAT3) or Dedicator of cytokinesis 8 (DOCK8) genes, in autosomal dominant and recessive forms, respectively. Therefore, the early detection of DOCK8 and STAT3 protein levels in humans would facilitate the early diagnosis of these disorders and thus, help in the disease management. Here, we present the development of immunosensors for the detection of DOCK8 and STAT3 using electrochemical impedance spectroscopy in a label-free format. The immunosensors were fabricated by the covalent attachment of specific antibodies for DOCK8 and STAT3 on gold electrodes via cysteamine/phenylene diisothiocyanate linkers. The detection was achieved by monitoring the change in the charge transfer resistance (R-ct) of ferro/ferricyanide redox couple upon binding of the proteins to the immunosensor surface. These biosensors enabled the detection of DOCK8 and STAT3 levels with low detection limits of 1.2 and 9.0pg/ml, respectively. The immunosensor was also applied for the detection of DOCK8 in human serum samples showing high recovery percentages which indicates great promise of this method for early diagnosis of HIES in newborn infants.

Longitudinal Monitoring of Biofilm Formation via Robust Surface-Enhanced Raman Scattering Quantification of Pseudomonas aeruginosa-Produced Metabolites.

Detection of bacterial metabolites at low concentrations in fluids with complex background allows for applications ranging from detecting biomarkers of respiratory infections to identifying contaminated medical instruments. Surface-enhanced Raman scattering (SERS) spectroscopy, when utilizing plasmonic nanogaps, has the relatively unique capacity to reach trace molecular detection limits in a label-free format, yet large-area device fabrication incorporating nanogaps with this level of performance has proven difficult. Here, we demonstrate the advantages of using chemical assembly to fabricate SERS surfaces with controlled nanometer gap spacings between plasmonic nanospheres. Control of nanogap spacings via the length of the chemical crosslinker provides uniform SERS signals, exhibiting detection of pyocyanin, a secondary metabolite of Pseudomonas aeruginosa, in aqueous media at concentration of 100 pg·mL-1. When using machine learning algorithms to analyze the SERS data of the conditioned medium from a bacterial culture, having a more complex background, we achieve 1 ng·mL-1 limit of detection of pyocyanin and robust quantification of concentration spanning 5 orders of magnitude. Nanogaps are also incorporated in an in-line microfluidic device, enabling longitudinal monitoring of P. aeruginosa biofilm formation via rapid pyocyanin detection in a medium effluent as early as 3 h after inoculation and quantification in under 9 h. Surface-attached bacteria exposed to a bactericidal antibiotic were differentially less susceptible after 10 h of growth, indicating that these devices may be useful for early intervention of bacterial infections.

Identification of Small-Molecule Noncovalent Binders Utilizing SAMDI Technology

In recent years, the ability to unambiguously identify complex mixtures of analytes with high accuracy and resolving power in a label-free format continues to expand the application of mass spectrometry (MS) in the drug discovery process. This advantage combined with improved instrumentation makes MS suitable for targets with limited alternative assays for high-throughput screening (HTS). We describe a novel screening format using Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization (SAMDI) technology. SAMDI enables affinity capture of a target protein for use in a small-molecule–binding assay format. Subsequent ionization enables the inferred identification of noncovalent compound interactions. SAMDI technology overcomes shot-to-shot variability by uniformly saturating the surface with captured protein, thereby minimizing matrix crystallization “hot spots.” Furthermore, the combination with high-resolution matrix-assisted laser desorption/ionization time of flight significantly reduces interference of small-molecule detection from salt, detergent, and matrix. By using a pooled library format, the SAMDI assay can significantly improve the throughput of MS-based screening irrespective of enzyme activity. Finally, we demonstrate binding affinity rank ordering from a pool of compounds that correlates with potency data from a biochemical assay.

Biomolecular Interaction Determination and Quantification by Microscale Thermophoresis

MicroScale Thermophoresis (MST), an immobilization-free technology, is used to rapidly quantify biomolecular interactions (pM-mM). MST, the directed movement of molecules in optically generated microscopic temperature gradients, is monitored by fluorescence. Thermophoretic movement is highly sensitive to binding reactions, which affect the size, charge, conformation, and/or hydration shell of target molecules. Here, we demonstrate that MST can be used to quantify interactions between biomolecules of interest, including proteins, nucleic acids, ions, and more. In one example, the binding site of a protein-protein interaction is investigated via protein engineering. As MST is suitable for probe interactions with membrane proteins in detergent or liposomes, we also demonstrate the quantification of interactions with GPCRs in a label free format using tryptophan fluorescence. Thus, MST is a critical addition to the biophysical toolbox allowing greater user-flexibility for analyzing molecular interactions.