Real-time Surface Plasmon Resonance Research Articles

PET Foils Functionalized with Reactive Copolymers as Adaptable Microvolume ELISA Spot Array Platforms for Multiplex Serological Analysis of SARS-CoV-2 Infections.

Microvolume ELISA platforms have become vital in diagnostics for their high-throughput capabilities and minimal sample requirements. High-quality substrates with advanced surface properties are essential for these applications. They enable both efficient biomolecule immobilization and antifouling properties, which are critical for assay sensitivity and specificity. This study presents PET-based microvolume ELISA spot arrays coated with amine- and DBCO-reactive copolymers MCP-2 and Copoly Azide. The platforms were designed for the sensitive and specific detection of specific antibodies such as COVID-19 biomarkers. Supporting robust attachment of the SARS-CoV-2 nucleoprotein (NP), these arrays outperform traditional approaches. It was demonstrated that covalent attachment methods proved more efficient than passive adsorption, together with the reduction of non-specific binding. Analytical performance was verified with classical ELISA and real-time Surface Plasmon Resonance (SPR) analysis. It enables sensitive detection of IgG and IgA antibodies, including IgG subclasses, in human serum. Clinically, the platform achieved 100.0% sensitivity and 92.9% specificity for anti-NP antibody detection in COVID-19-positive and negative samples. Additionally, DNA-directed immobilization extended the platform's utility to multiplex serological measurements. These findings underscore the potential of PET-based microvolume ELISA arrays as scalable, high-throughput diagnostic tools suitable for detecting multiple biomarkers in a single assay and easily integrated into microfluidic devices.

Level of blood Tau and p‐Tau : a non‐invasive approach for the diagnosis of Alzheimer’s Disease(AD)

AbstractBackgroundProminent expression of Tau and phosphorylated Tau in cerebrospinal fluid (CSF) is one of the well‐known hallmarks of the Alzheimer’s disease (AD). High levels of p‐Tau can easily differentiate AD from other neurodegenerative disease. However, the serum levels of these proteins in AD patients are not well explored. This study provides an alternative non‐invasive approach to diagnose the disease by evaluating the level of Tau and p‐Tau181 in serum samples.MethodBlood samples were collected from 39 AD patients, 37 MCI patients and 37 elderly individuals as controls. Quantification of tau and pTau in serum samples was done with the help of a label free real time Surface Plasmon Resonance technology, and further validated by Western Blot. Statistical analysis, including Receiver Operating Characteristic (ROC), was done for further affirmation.ResultThe concentrations of serum Tau and p‐Tau181 were significantly higher (p<0.00001) in AD (Tau; 47.49±9.00ng/µL, p‐Tau181; 0.161±0.04 ng/µL) compared to MCI (Tau; 39.26±7.78 ng/µL, p‐Tau181; 0.135±0.02 ng/µL) and were further compared to elderly controls (Tau; 34.92±6.58 ng/µL, p‐Tau181; 0.122±0.01 ng/ µL). A significant (p<0.0001) downhill correlation was found between Tau as well as p‐Tau181 levels with HMSE and MoCA score.ConclusionThis study for the first time reports the concentration of Tau and p‐Tau181 in serum of AD and MCI patients. The cut‐off values of Tau and p‐Tau181 of AD and MCI patients with sensitivity and specificity reveal that serum level of these proteins can be used as a predictive marker for AD and MCI.

Real-time hybrid angular-interrogation surface plasmon resonance sensor in the near-infrared region for wide dynamic range refractive index sensing

Herein, we integrated angle-scanning surface plasmon resonance (SPR) and angle-fixed SPR as a hybrid angular-interrogation SPR to enhance the sensing performance. Galvanometer-mirror-based beam angle scanning achieves a 100-Hz acquisition rate of both the angular SPR reflectance spectrum and the angle-fixed SPR reflectance, whereas the use of near-infrared light enhances the refractive index (RI) sensitivity, range, and precision compared with visible light. Simultaneous measurement of the angular SPR reflectance spectrum and angle-fixed SPR reflectance boosts the RI change range, RI resolution, and RI accuracy to 10–1–10–6 RIU, 2.24 × 10−6 RIU, and 5.22 × 10−6 RIU, respectively. The proposed hybrid SPR is a powerful tool for wide-dynamic-range RI sensing with various applications.

2D MOF-enhanced SPR sensing platform: Facile and ultrasensitive detection of Sulfamethazine via supramolecular probe

Sulfamethazine (SMZ) is widely present in the environment and can cause severe allergic reactions and cancer in humans. Accurate and facile monitoring of SMZ is crucial for maintaining environmental safety, ecological balance, and human health. In this work, a real-time and label-free surface plasmon resonance (SPR) sensor was devised using a two-dimensional metal-organic framework with superior photoelectric performance as an SPR sensitizer. The supramolecular probe was incorporated at the sensing interface, allowing for the specific capture of SMZ from other analogous antibiotics through host-guest recognition. The intrinsic mechanism of the specific interaction of the supramolecular probe-SMZ was elucidated through the SPR selectivity test in combination with analysis by density functional theory, including p-π conjugation, size effect, electrostatic interaction, π-π stacking, and hydrophobic interaction. This method facilitates a facile and ultrasensitive detection of SMZ with a limit of detection of 75.54 pM. The accurate detection of SMZ in six environmental samples demonstrates the potential practical application of the sensor. Leveraging the specific recognition of supramolecular probes, this direct and simple approach offers a novel pathway for the development of novel SPR biosensors with outstanding sensitivity.

FerryCalin: an engineered lipocalin protein directed against the transferrin receptor with potential for brain drug delivery.

The transferrin receptor (TfR) mediates transcytosis across the blood-brain barrier (BBB), which offers a promising approach for the non-invasive delivery of therapeutics into the brain parenchyma. Employing the recombinant homodimeric murine TfR ectodomain, prepared in a biochemically functional state, we have selected a cognate Anticalin via phage display and bacterial cell surface display from a random library based on the human lipocalin 2 (Lcn2). After affinity maturation, several engineered lipocalin were identified that bind murine TfR in a non-competitive manner with the natural ligand (transferrin•Fe3+), among those an Anticalin - dubbed FerryCalin - exhibiting a dissociation constant (KD) of 3.8 nM. Epitope analysis using the SPOT technique revealed a sequential epitope in a surface region of TfR remote from the transferrin-binding site. Due to their fast konrate and short complex half-life, as evidenced by real-time surface plasmon resonance (SPR) measurements, FerryCalin, or one of its related mutants, shows characteristics as a potential vehicle for the brain delivery of biopharmaceuticals.

A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration.

Age-related macular degeneration (AMD) is linked to 2 main disparate genetic pathways: a chromosome 10 risk locus and the alternative pathway (AP) of complement. Rare genetic variants in complement factor H (CFH; FH) and factor I (CFI; FI) are associated with AMD. FH acts as a soluble cofactor to facilitate FI's cleavage and inactivation of the central molecule of the AP, C3b. For personalised treatment, sensitive assays are required to define the functional significance of individual AP genetic variants. Generation of recombinant FI for functional analysis has thus far been constrained by incomplete processing resulting in a preparation of active and inactive protein. Using an internal ribosomal entry site (IRES)-Furin-CFI expression vector, fully processed FI was generated with activity equivalent to serum purified FI. By generating FI with an inactivated serine protease domain (S525A FI), a real-time surface plasmon resonance assay of C3b:FH:FI complex formation for characterising variants in CFH and CFI was developed and correlated well with standard assays. Using these methods, we further demonstrate that patient-associated rare genetic variants lacking enzymatic activity (e.g. CFI I340T) may competitively inhibit the wild-type FI protein. The dominant negative effect identified in inactive factor I variants could impact on the pharmacological replacement of FI currently being investigated for the treatment of dry AMD.

Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis.

Early detection of metastatic breast cancer (MBC) is a serious issue for the healthcare system. It is essential to develop potential non-invasive, low-cost molecular biomarkers. The present study explored specific serum proteins of inflammatory, MAPK, and cytoskeletal signaling pathways involved in the progression of MBC to establish a panel of blood-based diagnostic and prognostic biomarkers. Healthy-control (HC), non-metastatic (NM), and metastatic (M) (pre- and post-therapy) breast cancer (BC) patients were recruited. LOX5, Rac1, Rac1b, p38α, phospho-p38α (Y182), LIMK1, phospho-LIMK1 (T508), cofilin1, and phospho-cofilin1 (S3) were quantified in the serum of the study group by real-time label-free surface plasmon resonance technology and verified by Western blot. Proteins were found to be significantly elevated in the serum of BC patients compared to HC and also higher in M compared to NM, which further downregulated in post-therapy M patients. Elevation of phospho-LIMK1 and phospho-cofilin1, which are critical for M, was also indicated in the serum level and can differentiate from NM. Receiver operating characteristics (ROC) derived area under the curve (AUC) (0.9) is very strong to differentiate between HC and BC. Moreover, the combined ROC of 3 molecules phospho-LIMK, p38α, and phospho-p38α were found to be a potent predictive panel of biomarkers between M and NM with AUC0.95. The panel of inflammatory cytoskeleton signaling regime proteins specified in this study can have significant clinical utility for diagnosis as well as prognosis of MBC at an early stage. The study may have a high translational value in a simple and cost-effective way by avoiding frequent CT/PET scans.

Revealing the mechanism of surfactant-promoted enzymatic hydrolysis of dilute acid pretreated bamboo

To improve the enzymatic digestibility of dilute acid pretreated bamboo residue (DABR), surfactants including PEG 4000 and Tween 80 were added to prevent the non-productive adsorption between residual lignin and enzyme. At the optimal loadings (e.g., 0.2 and 0.3 g surfactant/g lignin), the enzymatic digestibility of DABR improved from 29.4% to 64.6% and 61.6% for PEG 4000 and Tween 80, respectively. Furthermore, the promoting mechanism of these surfactants on enzymatic hydrolysis was investigated by real-time surface plasmon resonance (SPR) and fluorescence spectroscopy. Results from SPR analysis showed that Tween 80 outperformed PEG 4000 in terms of dissociating the irreversible cellulase adsorption onto lignin. Fluorescence quenching mechanism revealed that PEG 4000 and Tween 80 intervened the interaction between lignin and cellulase by hydrogen bonds/Van der Waals and hydrophobic action, respectively. This work provided an in-depth understanding of the mechanisms of PEG 4000 and Tween 80 on enhancing the enzymatic hydrolysis efficiency.

Refractive index and temperature dual parameter sensor based on a twin-core photonic crystal fiber

A twin-core photonic crystal fiber sensor is proposed for measuring liquid refractive index (RI) and temperature simultaneously. The air holes of the sensor are arranged in a hexagonal pattern, and two planes are introduced by polishing in the cladding. On one side of the plane, the gold film is deposited for RI measurement, and on the other side, the gold film and polydimethylsiloxane are deposited for temperature measurement. We analyzed its sensing characteristics by using the finite element method. The numerical results show that the two channels for measuring RI and temperature have no mutual interference and the arrangement reduces the complexity of the sensing measurement. The maximum spectral sensitivity of the sensor is 20 000 nm/RIU and 9.2 nm °C−1, respectively, when the liquid RI is in the range of 1.36–1.42 and the temperature is in the range of 0 °C–50 °C. The results also show the sensing accuracy was not very sensitive to the change of structural parameters, which makes the sensor very easy to fabricate. Our work is very helpful for implementation of a high sensitivity, easy fabrication and real-time multi-parameter surface plasmon resonance sensor.

Real Time SPR Assessment of the Structural Changes of Adaptive Dynamic Constitutional Frameworks as a New Route for Sensing.

Cross linked gold-dynamic constitutional frameworks (DCFs) are functional materials of potential relevance for biosensing applications, given their adaptivity and high responsivity against various external stimuli (such as pH, temperature) or specific interactions with biomolecules (enzymes or DNA) via internal constitutional dynamics. However, characterization and assessment of their dynamic conformational changes in response to external stimuli has never been reported. This study proves the capability of Surface Plasmon Resonance (SPR) assays to analyse the adaptive structural modulation of a functional matrix encompassing 3D gold-dynamic constitutional frameworks (Au-DCFs) when exposed to pH variations, as external stimuli. We analyse Au-DCFs formed from Au nanoparticles, (AuNP) connected through constitutionally dynamic polymers, dynamers, with multiple functionalities. For increased generality of this proof-of-concept assay, Au-DCFs, involving DCFs designed from 1,3,5-benzene-tricarbaldehyde (BTA) connecting centres and polyethylene glycol (PEG) connectors, are covalently attached to standard SPR sensing chips (Au nanolayers, carboxyl terminated or with carboxymethyl dextran, CMD top-layer) and analysed using state-of-the art SPR instrumentation. The SPR effects of the distance from the Au-DCFs matrix to the Au nanolayer of the sensing chip, as well as of Au-DCFs thickness were investigated. This study reveals the SPR response, augmented by the AuNP, to the conformational change, i.e., shrinkage, of the dynamer and AuNP matrix when decreasing the pH, and provides an unexplored insight into the sensing applicability of SPR real-time analysis of adaptive functional materials.

Low-Cost Cell-Surface-Mimic Analysis of Ligand Interactions of Biotinylated Immune Receptors Using Surface Plasmon Resonance.

On the immune cell surface, many immune receptors are expressed and modulate the inhibitory or activating signals to control the immune responses. Recently, some of these receptors have been categorized as immune checkpoint receptors and targeted for cancer immunity or autoimmune diseases. To analyze the weak and fast binding typical for immune receptor-ligand interactions, a real-time surface plasmon resonance (SPR) technique is useful. However, it sometimes becomes difficult to optimize the immobilization conditions and appropriate controls. Considering that receptor orientation is relevant for achieving function on the cell surface, it is important to immobilize ligand proteins using specific tags at the membrane proximal end to avoid steric hindrance and structural changes in specific binding regions. Here we introduce a sensor chip, Sensor Chip CAP (Cytiva), which enables reversible and orientation-controlled immobilization of biotinylated ligands, resulting in a significant cost-effective method. We further show preparation methods of several biotinylated immune receptor proteins for SPR analysis, which are also useful for structural and other functional analyses.

The effect of N-glycosylation of SARS-CoV-2 spike protein on the virus interaction with the host cell ACE2 receptor

SummaryThe densely glycosylated spike (S) protein highly exposed on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) surface mediates host cell entry by binding to the receptor angiotensin-converting enzyme 2 (ACE2). However, the role of glycosylation has not been fully understood. In this study, we investigated the effect of different N-glycosylation of S1 protein on its binding to ACE2. Using real-time surface plasmon resonance assay the negative effects were demonstrated by the considerable increase of binding affinities of de-N-glycosylated S1 proteins produced from three different expression systems including baculovirus-insect, Chinese hamster ovarian and two variants of human embryonic kidney 293 cells. Molecular dynamic simulations of the S1 protein-ACE2 receptor complex revealed the steric hindrance and Coulombic repulsion effects of different types of N-glycans on the S1 protein interaction with ACE2. The results should contribute to future pathological studies of SARS-CoV-2 and therapeutic development of Covid-19, particularly using recombinant S1 proteins as models.

Fabrication and measurement of a fiber optic localized surface plasmon resonance sensor chip for molecular diagnostics

For several decades, the field of molecular diagnostics has been dominated by polymerase chain reaction technology, which usually entails relatively expensive equipment, trained laboratory staff, and problems in time management. Advances in molecular diagnostics aim to provide rapid tests, miniaturized devices, increased user convenience, and high sensitivity. The real-time label-free localized surface plasmon resonance (LSPR) biosensing technique merges with a small optical fiber and is combined with a microfluidic channel. This fiber optic LSPR sensor chip has the advantages of easy handling, isolation from the external environment, and continuous transport of sample fluid. The microfluidic channel can also accelerate hybridization by supplying the target to the sensor surface in succession. The conditions for the binding of probe deoxyribonucleic acids (DNAs) on gold nanoparticles are modified. Hybridization of the immobilized probe DNA and target DNA was analyzed using the proposed sensor and the results showed a good response to DNA hybridization with a limit of detection (LOD) level up to 12.3 ng/mL (1.5 nM). The LOD was compared with other LSPR-based DNA sensors and improvement was found. The proposed sensor responded with high linearity (R2 of 0.94) as a function of change in the concentration of the target DNA. Suitable reproducibility for biosensor applications was confirmed with an average coefficient of variation of 12.5 %.

A multiplex and regenerable surface plasmon resonance (MR-SPR) biosensor for DNA detection of genetically modified organisms

The continuous advancement of analytical technology has provided methods with increasing sensitivity and precision to detect genetically modified organisms (GMOs). Novel analytical strategy-based detection methods are alternatives to conventional polymerase chain reaction (PCR)-mediated assays, which are still the gold standard in this field. However, PCR primers and probes cannot be reused, which makes the technique uneconomical. Surface plasmon resonance (SPR) is an optical and label-free technique for studying ligand-analyte interactions, especially for DNA hybridization, and several SPR biosensors have been described for the detection of nucleic acids. Here, a multiplexed, regenerable and real-time SPR biosensor for the detection of GMOs is described. A biosensor was constructed for qualitative detection of T-nos, CaMV35S and cry1A and had good specificity and sensitivity. The limit of detection (LOD) of this biosensor was 0.1 nM without any signal amplification. Furthermore, our biosensor could be stably regenerated more than 100 times over at least 20 days and showed good reproducibility. This nucleic acid SPR biosensor has potential for application in other types of biological detection.

Electrosynthesis of polydopamine-ethanolamine films for the development of immunosensing interfaces

We report a straightforward and reproducible electrochemical approach to develop polydopamine-ethanolamine (ePDA-ETA) films to be used as immunosensing interfaces. ETA is strongly attached to polydopamine films during the potentiodynamic electropolymerization of dopamine. The great advantage of the electrochemical methods is to generate the oxidized species (quinones), which can readily react with ETA amine groups present in solution, with the subsequent incorporation of this molecule in the polymer. The presence of ETA and its effect on the electrosynthesis of polydopamine was accessed by cyclic voltammetry, ellipsometry, atomic force microscopy, FTIR and X-ray photoelectron spectroscopy. The adhesive and biocompatible films enable a facile protein linkage, are resilient to flow assays, and display intrinsic anti-fouling properties to block non-specific protein interactions, as monitored by real-time surface plasmon resonance, and confirmed by ellipsometry. Immunoglobulin G (IgG) and Anti-IgG were used in this work as model proteins for the affinity sensor. By using the one-step methodology (ePDA-ETA), the lower amount of immobilized biorecognition element, IgG, compared to that deposited on ePDA or on ETA post-modified film (ePDA/ETA), allied to the presence of ETA, improved the antibody-antigen affinity interaction. The great potential of the developed platform is its versatility to be used with any target biorecognition molecules, allowing both optical and electrochemical detection.

The Effect of  <i>N</i>-Glycosylation of SARS COV-2 Spike Protein on the Virus Interaction With the Host Cell ACE2 Receptor

The densely glycosylated spike (S) protein highly exposed on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) surface mediates host cell entry by binding to the receptor angiotensin-converting enzyme 2 (ACE2). However, the role of glycosylation has not been fully understood. In the present study, we investigated the effect of different N-glycosylation of S1 protein on its binding to ACE2. Using real-time surface plasmon resonance assay the negative effects were demonstrated by the considerable increase of binding affinities of de-N-glycosylated S1 proteins produced from three different expression systems including baculovirus-insect, Chinese hamster ovarian and two variants of human embryonic kidney 293 cells. Molecular dynamic simulations of the S1 protein-ACE2 receptor complex revealed the steric hinderance and Coulombic repulsion effects of the different types of N-glycans on the S1 protein interaction with ACE2. The results presented here should contribute to future pathological studies of SARS-CoV-2 and therapeutic development of Covid-19, particularly using recombinant S1 proteins as models.Funding Information: The authors declare that they have no conflict of interest.Declaration of Interests: The work was supported in part by the National Natural Science Foundation of China (31600650, 31671369, and 62072435), the March of Dimes Prematurity Research Center grant (22-FY18-82), and Wellcome Trust Biomedical Resource grant (218304/Z/19/Z).

Real-time nanoplasmonic sensing of three-dimensional morphological changes in a supported lipid bilayer and antimicrobial testing applications

We report the development of a real-time localized surface plasmon resonance (LSPR) biosensing strategy to detect three-dimensional morphological changes in a supported lipid bilayer (SLB) on a plasmonic substrate. The sensing concept advances on past efforts to detect subtle conformational changes in adsorbed biomacromolecules by demonstrating the capability to track large-scale, complex adsorbate shape changes and to classify different types of shape changes based on specific, multi-step measurement signatures. To validate this concept, we tested the addition of antimicrobial fatty acids, monoglycerides, and surfactants in micellar form to the SLB platform, which triggered specific three-dimensional membrane morphological changes such as tubule or bud formation along with solubilization. Experimentally, the different remodeling events were detected by distinct measurement signatures related to the shape and size of lipid protrusions that formed and evolved over time, which agreed well with a newly developed theoretical model. Our conceptual approach and formalism are applicable to various biosensing techniques, including not only LSPR but also surface plasmon resonance (SPR) and total internal reflection fluorescence (TIRF) microscopy. These sensing capabilities are advantageous for evaluating the mechanisms of antimicrobial drug candidates and other membrane-active compounds, and the measurement strategy is extendable to a wide range of biomimetic lipid compositions.

Comparative study of block copolymer-templated localized surface plasmon resonance optical fiber biosensors: CTAB or citrate-stabilized gold nanorods

Block copolymer (BCP) brush-like layer absorbed on optical fibers are shown to address the longstanding challenge of forming dense and well-dispersed gold nanorods (AuNRs) films on highly curved surface, allowing the development of real-time localized surface plasmon resonance (LSPR) biosensors for antigen-antibody interaction. This method requires only solution-based manufacturing processes and a plasma cleaning step to self-assemble AuNRs with various aspect ratios on the optical fibers. Because AuNRs capped with cetyltrimethylammonium bromide (CTAB) have been problematic for biosensing applications due to their cytotoxicity and displacement difficulty in the construction of biosensors, citrate-stabilized AuNRs were prepared from CTAB-stabilized AuNRs via surfactant exchange. For comparison, both a poly(styrene)-b-poly(acrylic acid) (PS-b-PAA) BCP template with CTAB-stabilized AuNRs and a poly(styrene)-b-poly(4-vinyl pyridine) (PS-b-P4VP) BCP template with citrate-stabilized AuNRs were employed for the fabrication of the optical fiber biosensor. The LSPR spectral analysis results demonstrate that the sensitivity of the sensor produced by the PS-b-P4VP templating method was 1.5 times better than that of the sensor produced by the PS-b-PAA templating method, which was due to the improved surface coverage and reduced aggregation of nanoparticles. The detection limit of the PS-b-P4VP-templated sensor for human IgG was 0.6 nM. Furthermore, for citrate-stabilized AuNRs, the PS-b-P4VP templating method also led to a better surface morphology and higher sensitivity as compared to the standard particle absorption methods using alkoxysilanes or polyelectrolytes on identical fibers. The simplicity of the fabrication process and the far superior optical performance of the PS-b-P4VP-templated LSPR optical fiber sensor illustrate the ability to regulate the detection range by tuning the aspect ratios of the AuNRs, and demonstrate its application prospects as a real-time biosensor without cytotoxicity.

SmartSPR sensor: Machine learning approaches to create intelligent surface plasmon based sensors

Surface plasmon resonance (SPR) based sensors allow the evaluation of aqueous and gaseous solutions from real-time measurements of molecular interactions. The reliability of the response generated by a SPR sensor must be guaranteed, especially in substance detection, diagnoses, and other routine applications since poorly handled samples, instrumentation noise features, or even molecular tampering manipulations can lead to wrong interpretations. This work investigates the use of different machine learning (ML) techniques to deal with these issues, and aim to improve and attest to the quality of the real-time SPR responses so-called sensorgrams. A new strategy to describe a SPR-sensorgram is shown. The results of the proposed ML-approach allow the creation of intelligent SPR sensors to give a safe, reliable, and auditable analysis of sensorgram responses. Our arrangement can be embedded in an Intelligence Module that can classify sensorgrams and identify the substances presents in it. Also made it possible to order and analyze interest areas of sensorgrams, standardizing data, and supporting eventual audit procedures. With those intelligence features, the new generation of SPR-intelligent biosensors is qualifying to perform automated testing. A properly protocol for Leishmaniasis diagnosis with SPR was used to verify this new feature.

Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways.

Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for Gs, Gq, and Gi signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds.