Use Of Surface Plasmon Resonance Research Articles

Operando UV-vis spectroscopy for real-time monitoring of nanoparticle size in reaction conditions: a case study on rWGS over Au nanoparticles.

We propose the use of surface plasmon resonance (SPR) as a distinctive marker for real-time monitoring in reaction conditions of gold nanoparticles supported on α-Al2O3. The study leverages the SPR shape-and-size dependency to monitor metal nanoparticles in reaction conditions, evidencing an influence of both dimensions and agglomerations on the SPR peak position. Operando measurements, coupling UV-vis spectroscopy and catalytic testing, allows to follow the dynamics during nanoparticle formation (Au3+ to Au0 reduction) and during the reverse water gas shift reaction (CO2 + H2 → CO + H2O). The catalyst structure and stability in reaction conditions was further confirmed by operando X-ray spectroscopy and PXRD data. Overall, this approach enables the direct acquisition of information on the structure-activity relationship of metal-based supported catalysts under actual reaction conditions.

Abstract B121: CPL976, an innovative bispecific antibody targeting AXL and PD-L1 axis as a potential new anticancer therapeutic

Abstract Background: Bispecific antibodies rapidly emerge as a powerful therapeutic tool in cancer treatment. Usage of the bispecific antibodies in oncology therapies targeting more than one antigen on cancer cells enhances the specificity of the therapy, increasing its effectiveness and decreasing the resistance rate. This approach may be used to modulate the mechanism of the defense and development of cancer cells, such as high-level expression on the cell surface the ligands of the immune checkpoints like PD-L1, and growth factor receptors like EGFR, VEGFR, or AXL. The latest reports reveal the correlation between the expression level of PD-L1 and AXL/EGFR1 in cancer. Notably, the disruption of the signaling pathway of AXL leads to a reduction of the PD-L1 on the cell surface. Adoption of this mechanism into oncology therapies may enhance the immune response against cancer cells. Moreover, the use of the bispecific antibodies simultaneously targeting PD-L1 and AXL may provoke internalization which maximizes the biological effect of the inhibition of the receptor/ligand complexes (AXL/GAS6, PD-1/PD-L1) formation. Materials and Methods: Bispecific antibody CPL976 was developed as a VHH-Fc-VHH format, with two VHHs raised against PD-L1 and AXL, selected from immune libraries generated after llamas’ immunization. Antibodies' biological activity and selectivity were studied by the Western blot technique on breast cancer cell line (MDA-MB-231) and functional assays constructed on Jurkat E6.1 and Raji cells with stable expression of the PD-1 and PD-L1, respectively. The immunomodulatory functions were also verified in cancer cell lines and PBMC co-cultures. Bispecific binding potential to AXL and PD-L1 and its effect on receptor internalization were evaluated with the use of surface plasmon resonance, Western blot technique, flow cytometry, and pH-dependent dye assays. Results: CPL976 (PD-L1 × AXL biAb), presents excellent binding kinetics with subnanomolar KDs and an innovative mechanism of action based on simultaneous internalization and degradation of both therapeutic targets at 150 nM. The compound also modulates the cell viability and migration in the breast cancer line stronger than the single agents. This data is supported by the observed decrease of the EMT (Epithelial-Mesenchymal Transition) markers. The immunomodulatory effect was confirmed in the co-culture, where the T-cell activity is blocked. The efficacy of the antibody in the tumor was checked in a murine cancer model. Conclusions: We present CPL976, an innovative bispecific antibody, with excellent binding parameters, strong biological effect on receptor presentation on the cell surface, and promising in vitro and in vivo results. These combined properties may establish a new generation of anticancer antibodies, that effectively block cancer development and break the PD-1/PD-L1 axis in patients with primary and secondary resistance to PD-1/PD-L1 targeted therapies. Such unique properties might also be later used as an effective carrier of toxic payloads in antibody-drug conjugate format. Citation Format: Agnieszka Bojko-Matuszek, Damian Kołakowski, Magdalena Bojko, Aleksandra Sowińska, Filip Mituła, Krzysztof Lacek, Delfina Popiel, Sebastian Kwiatkowski, Tomasz Kornatowski, Beata Kliszcz, Anna Jabłońska, Krzysztof Flis, Bartosz Wiernicki, Maciej Wieczorek, Jerzy Pieczykolan, Olga Abramczyk. CPL976, an innovative bispecific antibody targeting AXL and PD-L1 axis as a potential new anticancer therapeutic [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B121.

Study on Saccharide-Glucose Receptor Interactions with the Use of Surface Plasmon Resonance.

The aim of this study was to investigate the process of attachment of saccharide particles differing in degree of complexity to cell receptors responsible for transport of glucose across the cell membrane (GLUT proteins). This phenomenon is currently considered when designing modern medicines, e.g., peptide drugs to which glucose residues are attached, enabling drugs to cross the barrier of cell membranes and act inside cells. This study aims to help us understand the process of assimilation of polysaccharide nanoparticles by tumour cells. In this study, the interactions between simple saccharides (glucose and sucrose) and dextran nanoparticles with two species of GLUT proteins (GLUT1 and GLUT4) were measured using the surface plasmon resonance technique. We managed to observe the interactions of glucose and sucrose with both applied proteins. The lowest concentration that resulted in the detection of interaction was 4 mM of glucose on GLUT1. Nanoparticles were measured using the same proteins with a detection limit of 40 mM. These results indicate that polysaccharide nanoparticles interact with GLUT proteins. The measured strengths of interactions differ between proteins; thus, this study can suggest which protein is preferable when considering it as a mean of nanoparticle carrier transport.

Abstract LB180: SPOC, A highly multiplexed platform to study the kinetics of hundreds of biomolecular interactions: applications for early cancer detection

Abstract Tissue specific tumor neo-antigens and tumor associated antigens (TAAs) play a pivotal role in immune system interaction with cancer cells. Mutated, alternately spliced, over-expressed, mis-folded and abnormally post-translationally modified proteins may elicit immune responses that suppress tumor development. As these cancer specific proteins are distinct from normal self-proteins they offer the possibility of serving as highly-specific cancer biomarkers for early detection. TAAs are increasingly being targeted for priming the immune response to immunotherapies and cancer vaccines. Consequently, there has been an exponential increase in research on neo-antigen profiling using sequencing and multi-omic technologies. However, proteomic tools capable of studying and profiling the cancer proteome are limited. To contribute to this, we are developing a novel proteomic screening platform termed Sensor-integrated Proteome on Chip (SPOC) that enables high-throughput (HTP) screening of cancer proteins for loss or gain of function analysis, and humoral immune response profiling. SPOC platform enables use of surface plasmon resonance (SPR) biosensing for kinetic interaction profiling of 100s to 1000s of proteins at once, which can be applied to discovery and characterization of autoantibodies and neoantigens. SPOC uses human cell-free in vitro transcription and translation to express fusion capture-tagged proteins of interest from DNA plasmids printed in nanowells etched into a silicon slide. As proteins are expressed, they are captured covalently onto an overlaid gold-coated surface in individual spots via the capture ligand. The capture ligand requires the tag to be properly folded for binding, thus proteins on the surface are presumed to be fully folded when captured. When SPOC chips are analyzed using SPR, kinetic measurements of protein interactions can be obtained for each protein spot over time (RMax, Kon, Koff, etc.). SPOC arrays may be probed with a variety of biological molecules including small molecule drugs, antibodies, and other proteins. Data can be analyzed using bioinformatics and machine learning for neo-antigen discovery and to generate distinct signatures of disease. To demonstrate SPOC utility for neo-antigen discovery and characterization, a serum sample with known anti-p53 autoantibodies was analyzed on a SPOC SPR chip containing expressed p53 protein spots distributed throughout the array. Increases in SPR signals were observed for each p53 spot on the array as anticipated. Citation Format: Rebecca Cook, Chidozie Victor Agu, William Martelly, Peter A. Bell, Mukilan Mohan, Bharath Takulapalli. SPOC, A highly multiplexed platform to study the kinetics of hundreds of biomolecular interactions: applications for early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB180.

ПЕРСПЕКТИВИ ТА ТЕНДЕНЦІЇ РОЗВИТКУ ПРИЛАДІВ ТА МЕТОДІВ НА ОСНОВІ ЯВИЩА ПОВЕРХНЕВОГО ПЛАЗМОННОГО РЕЗОНАНСУ В ІНФРАЧЕРВОНОМУ ДІАПАЗОНІ (ОГЛЯД)

On the phenomenon of surface plasmon resonance (SPR) and its use for the study of gaseous and liquid substances, more and more papers are published in professional journals. These studies are used in clinical diagnostics, food industry, environmental monitoring. The advantages of SPR sensors include quick results, a small amount of analyte, the possibility of real-time measurement of such reactions as antigen-antibody, protein-protein, enzyme-substrate or inhibitor, protein-DNA, receptor-drug, protein-polysaccharide , virus protein and reactions of external stimulation of a living cell. The high sensitivity of the method is due to the properties of its resonant nature of plasmon-polariton oscillation. SPR devices are used in laboratory conditions and there is a potential possibility of further reduction of overall dimensions for use in field conditions. The use of SPR in the IR region of the spectrum has a number of advantages, namely: the laser light penetrates deeper into the sample, it becomes possible to study substances that are not transparent in the visible region, but transparent in the infrared, which allows working with larger research objects, provides a narrower characteristic of the SPR, which allows to more accurately determine the angle of the minimum of the SPR, and also requires the application of a thinner layer of gold on the glass chip, which reduces the cost of the analysis. The review analyzed literary sources and determined prospects for the development of devices and methods based on IR SPR, considered prospective materials of the sensitive element and prisms of the total internal reflection device according to the Kretchman scheme. It was determined that, in addition to precious metals (gold and silver), conductive metal oxides ITO, ZnO, CdO:Dy have the prospect of being used as materials for manufacturing the sensitive element of the sensor. Their compliance with the requirements for materials for maintaining the SPR was analyzed. It is shown that CdO:Dy has the greatest potential due to sufficient mobility and density of charge carriers. But in practice, the most common use is gold and silver, for which it is necessary to determine the optimal thicknesses for the IR range of SPR. Total internal reflection prisms (hereafter prisms) in SPR devices according to the Kretchman scheme are usually made of glass, but for the IR range it is advisable to consider other materials that are less sensitive to the temperature effect of the IR laser. The paper analyzes the dependence of the plasmon resonance parameters on the prism material. The possibility of using not only different brands of traditional glass, but also glass ceramics with practically zero coefficient of thermal expansion, semiconductor silicon and chalcogenide glass for the manufacture of prisms is shown.

Multivalent Interactions with Intrinsically Disordered Proteins Probed by Surface Plasmon Resonance.

Multivalent interactions underpin associations between intrinsically disordered proteins (IDPs) and their binding partners. This is a subject of considerable interest and governs how nuclear transport receptors (NTRs) orchestrate the nucleocytoplasmic transport (NCT) of signal-specific cargoes through nuclear pore complexes (NPCs) in eukaryotic cells. Specifically, IDPs termed phenylalanine-glycine nucleoporins (FG Nups) exert multivalent interactions with NTRs to facilitate their transport selectivity and speed through the NPC. Here, we document the use of surface plasmon resonance (SPR) to quantify the affinity and kinetics of NTR-FG Nup binding as a function of FG Nup surface density. Moreover, we describe an in situ method that measures conformational height changes that occur in a FG Nup layer following NTR-binding. Protocols by which the as-obtained SPR results are treated with respect to mass transport limitations are further described. Overall, the SPR methodology described here can be applied to studying multivalent interactions and the role of avidity in diverse biological and biointerfacial systems.

(Invited) New Advancements in Biosensor Technologies with Surface Plasmonic Resonance Microscopy

Since the first use of surface plasmon resonance (SPR) technology for biosensing more than four decades ago, SPR has become a powerful tool for characterizing and quantifying biomolecular interactions, especially in the pharmaceutical industry for drug discovery. It has made great strides both in instrumentation developments and applications. One of the most significant developments is SPR microscopy, which integrates SPR technology with optical microscopy. SPR microscopy has enabled many new studies, such as direct measurements of small drug molecules interacting with GPCR proteins of a single cell in its native state, as well as direct measurement of drug responses on over hundreds of cells simultaneously. In this way, a heterogenous population of cells with different phenotypes, growth stages, and receptor expression levels may be directly studied to reveal deeper insight on receptor functionalities while in their native cellular state.

Signal Demodulation for Surface Plasmon Resonance Tilted Fiber Bragg Grating Based on Root Sum Squared Method

There is an increasing interest in the use of surface plasmon resonance tilted fiber Bragg grating (SPR-TFBG) as a sensor for various chemical applications. Signal demodulation is an important process for extracting useful parameters from the unique optical spectra for an accurate measurement. In this work, we proposed a global-based estimator for SPR-TFBG based on a spectral subtraction approach. The estimator is generated based on the root sum squared (RSS) error of the spectral intensity difference between the tested spectrum and the reference spectrum. In the comparison with the other established methods, the proposed method has the lowest standard deviation, the maximum absolute deviation, the finest measurement resolution, and the fastest processing speed. Besides, the proposed method is immune to the interference of white noise in the transmission spectra and capable of producing a high-quality signal with the lowest noise level.

Development of a Novel SPR Assay to Study CXCR4-Ligand Interactions.

G protein-coupled receptors (GPCRs) are involved in a plethora of different diseases. Consequently, these proteins are considered as an important class of drug targets. Measuring detailed kinetic information on these types of proteins has been challenging. Surface plasmon resonance (SPR) can provide this information, however, the use of SPR on GPCRs remains a complex issue. Here, we report an SPR assay to investigate the interactions between the full-length chemokine receptor CXCR4 and nanobody-Fc (Nb-Fc) ligands. Nb-Fcs consist of two monovalent VHH domains fused with an Fc domain of a human IgG molecule. The CXCR4 protein used in this assay was produced with a C-terminal 10x-histidine tag and was immobilized on a nitrilotriacetic acid chip. In order to verify the sensitivity and effectiveness of this assay, the results were compared to data obtained from cellular assays as well as from another SPR assay using CXCR4 virus-like particles (VLPs). CXCR4 remained intact and stable for at least 12 h, and the kinetic results correlated well with both the cellular assays and the VLP SPR assay results. Apart from determining the binding kinetics of Nb-Fc with CXCR4, our results contributed to understanding CXCR4 interaction dynamics. In conclusion, this assay provides a viable experimental platform that has high potential to be expanded for studying other molecules as well as other histidine-tagged GPCRs.

Laboratory Diagnosis of Paratyphoid Fever: Opportunity of Surface Plasmon Resonance.

Paratyphoid fever is caused by the bacterium Salmonella enterica serovar Paratyphi (A, B and C), and contributes significantly to global disease burden. One of the major challenges in the diagnosis of paratyphoid fever is the lack of a proper gold standard. Given the absence of a licensed vaccine against S. Paratyphi, this diagnostic gap leads to inappropriate antibiotics use, thus, enhancing antimicrobial resistance. In addition, the symptoms of paratyphoid overlap with other infections, including the closely related typhoid fever. Since the development and utilization of a standard, sensitive, and accurate diagnostic method is essential in controlling any disease, this review discusses a new promising approach to aid the diagnosis of paratyphoid fever. This advocated approach is based on the use of surface plasmon resonance (SPR) biosensor and DNA probes to detect specific nucleic acid sequences of S. Paratyphi. We believe that this SPR-based genoassay can be a potent alternative to the current conventional diagnostic methods, and could become a rapid diagnostic tool for paratyphoid fever.

Non-Invasive Plasmonic-Based Real-Time Characterization of Cardiac Drugs on Cardiomyocytes Functional Behavior.

In the fabrication of cardiac tissue, an important factor is continuous measurement of its contraction features. A module that allows for a dynamic system capable of noninvasive and label-free monitoring of the contraction profile under administering chemicals and drugs is highly valuable for understanding accurate tissue mechanobiology. In this research, we have successfully demonstrated the use of surface plasmon resonance (SPR) technology for the first time to characterize the contractility of cardiac cells in response to Blebbistatin and ATP drug exposure in real tme. An optimal flow rate of 10 μL/min was selected for a continuous flow of warm media,and 10 μM drug administration effect was detected with high spatiotemporal sensitivity on contracting cardiomyocytes. Our drug screening has identified the source of the SPR periodic signal to be direct cell contraction rather than action potentials or calcium signaling. Per our results, SPR has high potential in applications in least-interference real-time and label-free tissue characterizations and cellular properties analysis from a functional and structural point of view.

Abstract 925: Insulin Inhibits β-adrenergic Receptor Resensitization Through PI3KγInsulin Inhibits β-adrenergic Receptor Resensitization Through PI3Kγ

Insulin regulates cardiac metabolism, myocyte survival, and cardiac growth. In addition, insulin is also known to modulate beta adrenergic receptors (βARs) function in the heart regulating cardiac function. βAR function in response to its agonist epinephrine/norepinephrine is regulated by G-protein coupled receptor kinase (GRKs) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization. Although insulin is known to modulate βAR function through GRKs, less is known about role in insulin in regulating resensitization mechanisms. Phosphoinositide 3-kinase γ (PI3Kγ) is a key regulator of resensitization at it inhibits PP2A activity and therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Insulin stimulation of HEK 293 cells stably expressing β 2 ARs resulted in significant β 2 AR phosphorylation. This β 2 AR phosphorylation was completely abolished with use of pharmacologic inhibitors of PI3K wortmannin or LY 294002 and knock-down of PI3Kγ showing an essential role for PI3Kγ in regulation of βAR function by insulin. Activation of insulin receptor induces downstream signaling via recruitment of the adaptor insulin receptor substrates (IRS). Interestingly, our data suggests that cross talk between the IRs and B 2 ARs are dependent on interaction between PI3Kγ, GRK2 and IRS as identified by co-immunoprecipitation studies and use of surface plasmon resonance using purified proteins. Since cardiomyocytes and fibroblasts represent major cell-types in the heart, primary adult cardiomyocytes and cardiac fibroblasts (CFs) were isolated from WT and PI3Kγ KO hearts for insulin stimulation. While β 2 AR phosphorylation was completely abolished in primary CFs from PI3Kγ KO compared to WT CFs, cardiomyocytes did not show any differences in β 2 AR phosphorylation in response to insulin. These data suggest that PI3Kγ may differentially regulate pathways in cardiomyocytes and CFs in the heart indicating the existence of cell-specific signaling mechanisms that may underlie response to insulin and thereby the cardio-metabolic process.

A brief review on the strategy of developing SPR-based biosensors for application to the diagnosis of neglected tropical diseases

The phenomenon of surface plasmon resonance (SPR) through optical sensors was developed from initial studies involving excitation of surface plasmons on metallic substrates. From the beginning, these optical systems have attracted increasing interest for application in different areas, ranging from physics, chemistry, and materials science to biology. Although numerous applications have been explored, the use of SPR in the development of biosensors is by far the most prominent. This review provides a brief account of fundamental aspects related to the recent applications of SPR as a tool for the development of new clinical diagnosis methods. The applications of SPR biosensors were illustrated through recent studies published in the field of neglected tropical diseases, with an emphasis on the contributions achieved in visceral leishmaniasis. It was possible to demonstrate the real benefits and the difficulties that the SPR biosensors have encountered in this important and complex system. Finally, future trends in the use of nanomaterials for the development of SPR-based portable devices for application to neglected tropical diseases have been demonstrated.

Monolithically integrated InGaAsP multiple quantum well tunable laser diode for integrated optic surface plasmon resonance sensing

We demonstrate an InGaAsP multiple quantum well tunable laser diode consisting of two gain sections with different bandgap energy levels. This tunable laser is used as a probing source for an optical sensor that makes use of surface plasmon resonance (SPR). Lasers fabricated on a single monolithic substrate can have widely differing output wavelengths. Therefore, by controlling the extent of the intermixing, the bandgap energy of the material can be accurately tuned over a broad spectral range. The regions of different bandgap energies are achieved using selective area intermixing of the multiple quantum wells, through impurity-free vacancy-induced disordering. By varying the current combination that was injected to each section, the laser wavelength can be effortlessly tuned from 1538 to 1578 nm with relatively constant output power. The free spectral range of the tunable laser is found to be 0.25 nm. This tunable laser was launched into an optical SPR sensor head that was fabricated on an inverted rib dielectric waveguide to provide an input light source for the SPR sensor. The average sensitivity of the SPR sensor devices was determined to be S  =  334  nm RIU  −  1.

SPR Biosensors in Direct Molecular Fishing: Implications for Protein Interactomics.

We have developed an original experimental approach based on the use of surface plasmon resonance (SPR) biosensors, applicable for investigation of potential partners involved in protein–protein interactions (PPI) as well as protein–peptide or protein–small molecule interactions. It is based on combining a SPR biosensor, size exclusion chromatography (SEC), mass spectrometric identification of proteins (LC-MS/MS) and direct molecular fishing employing principles of affinity chromatography for isolation of potential partner proteins from the total lysate of biological samples using immobilized target proteins (or small non-peptide compounds) as ligands. Applicability of this approach has been demonstrated within the frame of the Human Proteome Project (HPP) and PPI regulation by a small non-peptide biologically active compound, isatin.

Quantitation of Infliximab and Detection of Antidrug Antibodies in Serum by Use of Surface Plasmon Resonance.

Monitoring infliximab (IFX) concentrations and the presence of antidrug antibodies (ADA) is important for patient management. We developed a method to measure IFX and ADA in serum in a single injection using surface plasmon resonance (SPR). Using the Bio-Rad ProteOn XPR36, tumor necrosis factor-α and IFX were covalently immobilized onto separate lanes of a chip surface. Diluted serum was injected over both lanes, followed by an injection of goat antihuman antibody. The binding response was used to quantify IFX or detect ADA. The analytical performance of the assay was determined. Using 50 patient samples, SPR results were compared with results from a reporter gene assay (RGA). For the quantification of IFX, the functional sensitivity was 0.5 μg/mL. The total precision was <10% for all concentrations tested. IFX concentrations measured by SPR correlated well with RGA (R = 0.862), but a bias was observed (slope = 0.61). SPR detected 14 ADA-positive samples. Compared with RGA for ADA detection, there were 6 true-positive, 8 false-positive, 5 false-negative, and 31 true-negative findings. SPR can be used to measure biological drug concentrations and detect ADA in serum. This technique may provide complementary information to current methods used to detect ADA.

Temperature-compensated magnetic field sensor based on surface plasmon resonance and directional resonance coupling in a D-shaped photonic crystal fiber

A D-shaped and magnetic fluids (MF) infiltrated photonic crystal fiber (PCF) based Surface Plasmon Resonance (SPR) sensor is proposed for magnetic field sensing. By inserting a defective cavity filled with toluene solution in the cladding layer, the temperature-compensated mechanism can be achieved based on the dependence of the MF refractive index (RI) on temperature and magnetic field. By the combined use of SPR and directional resonance coupling in the designed PCF, a dual-parameter matrix method is utilized to solve the temperature-induced problem. The relationship between resonant wavelength and external magnetic-field can be derived with a high sensitivity 0.87nm/mT. As the Au-Ag composite film is coating on the plane surface of the photonic crystal fiber, the proposed sensor has the advantage of simplification and easy fabrication.

Use of Surface Plasmon Resonance and Biolayer Interferometry for the Study of Protein–Protein Interactions on the Example of an Enzyme of a Glycosyl Hydrolase Subtype (EC 3.2.1) and Specific Antibodies to It

Two previously obtained, full-size, fully human antibodies that reversibly bind the active form of an enzyme belonging to the subtype EC 3.2.1, which is used for substitutive enzymatic therapy in lysosomal storage diseases, have been characterized by surface plasmon resonance and biolayer interferometry methods. It was shown under normal physiological conditions that the designed antibodies specifically bound with the antigen (KD ~ 10–8 M) and rapidly dissociated at neutral pH in 60% ethylene glycol while leaving the enzymatic activity unchanged. Dan ue to their properties, the developed antibodies can be used in industry as affinity ligand in the isolation of therapeutic substances of the enzyme.

Nanomaterial-based biosensors for detection of prostate specific antigen.

Screening serum for the presence of prostate specific antigen (PSA) belongs to the most common approach for the detection of prostate cancer. This review (with 156 refs.) addresses recent developments in PSA detection based on the use of various kinds of nanomaterials. It starts with an introduction into the field, the significance of testing for PSA, and on current limitations. A first main section treats electrochemical biosensors for PSA, with subsections on methods based on the use of gold electrodes, graphene or graphene-oxide, carbon nanotubes, hybrid nanoparticles, and other types of nanoparticles. It also covers electrochemical methods based on the enzyme-like activity of PSA, on DNA-, aptamer- and biofuel cell-based methods, and on the detection of PSA via its glycan part. The next main section covers optical biosensors, with subsections on methods making use of surface plasmon resonance (SPR), localized SPR and plasmonic ELISA-like schemes. This is followed by subsections on methods based on the use of fiber optics, fluorescence, chemiluminescence, Raman scattering and SERS, electrochemiluminescence and cantilever-based methods. The most sensitive biosensors are the electrochemical ones, with lowest limits of detection (down to attomolar concentrations), followed by mass cantilever sensing and electrochemilumenescent strategies. Optical biosensors show lower performance, but are still more sensitive compared to standard ELISA. The most commonly applied nanomaterials are metal and carbon-based ones and their hybrid composites used for different amplification strategies. The most attractive sensing schemes are summarized in a Table. The review ends with a section on conclusions and perspectives.

Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein.

Vitamin D3 and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D3 metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D3 and three metabolites: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 25-hydroxyvitamin D3 (25OHD3) and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD3 and 24,25(OH)2D3 had higher affinity (0.3μM) than 1,25(OH)2D3 (5μM), with the higher affinity of 25OHD3 and 24,25(OH)2D3 due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D3 (NO2-446), structurally closer to 1,25(OH)2D3, showing the highest value with a K D of 50μM. (24R)-1,25-dihydroxyvitamin-24-buthyl-28-norvitamin D2 (Bu-471) and (24R)-1,25-dihydroxyvitamin-24-phenyl-28-norvitamin D2 (Ph-491), structurally similar, had affinities of 180 and 170μM, respectively. (22R,23E)-1-hydroxy-22-ethenyl-25-methoxy-23-dehydrovitamin D3 (MeO-455) and 1-hydroxy-20(R)-[5(S)-(2,2-dimethyltetrahydropyran-5-yl)]-22,23-dinor vitamin D3 (Oxan-429) had affinities of 310 and 100μM, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction. Graphical Abstract Label-free, real time binding study between vitamin D binding protein immmobilized on the surface of a SPR sensor chip and the vitamin D, metabolites and analogues passed over it as analytes.