Direct Surface Plasmon Resonance Research Articles

Investigation of Charged Small Molecule-Aptamer Interactions with Surface Plasmon Resonance.

Investigating the interactions between small, charged molecules and aptamers using surface plasmon resonance (SPR) is limited by the inherent low response of small molecules and difficulties with nonspecific electrostatic interactions between the aptamer, analyte, and sensor surface. However, aptamers are increasingly being used in sensors for small molecule detection in critical areas like healthcare and environmental safety. The ability to probe these interactions through simple, direct SPR assays would be greatly beneficial and allow for the development of improved sensors without the need for complicated signal enhancement. However, these assays are nearly nonexistent in the current literature and are instead surpassed by sandwich or competitive binding techniques, which require additional sample preparation and reagents. In this work, we develop a method to characterize the interaction between the charged small molecule serotonin (176 Da) and an aptamer with SPR using streptavidin-biotin capture and a high-ionic-strength buffer. Additionally, other methods, such as serotonin immobilization and thiol-coupling of the aptamer, were investigated for comparison. These techniques give insight into working with small molecules and allow for quickly adapting a binding affinity assay into a direct SPR sensor.

Real-Time Ellipsometric Surface Plasmon Resonance Sensor Using Polarization Camera May Provide the Ultimate Detection Limit.

Ellipsometric Surface Plasmon Resonance (SPR) sensors are known for their relatively simple optical configuration compared to interferometric and optical heterodyne phase interrogation techniques. However, most of the previously explored ellipsometric SPR sensors based on intensity measurements are limited by their real-time applications because phase or polarization shifts are conducted serially. Here we present an ellipsometric SPR sensor based on a Kretschmann-Raether (KR) diverging beam configuration and a pixelated microgrid polarization camera. The proposed methodology has the advantage of real-time and higher precision sensing applications. The short-term stability of the measurement using the ellipsometric parameters tanψ and cos(Δ) is found to be superior over direct SPR or intensity measurements, particularly with fluctuating sources such as laser diodes. Refractive index and dynamic change measurements in real-time are presented together with Bovine Serum Albumin (BSA)-anti-BSA antibody binding to demonstrate the potential of the developed sensor for biological sensing applications with a resolution of sub-nM and down to pM with additional optimization. The analysis shows that this approach may provide the ultimate detection limit for SPR sensors.

Electrochemistry-Assisted Surface Plasmon Resonance Biosensor for Detection of CA 15-3.

In this work, we describe an innovative methodology based on combined surface plasmon resonance (SPR) and electrochemical responses (eSPR) in the same immunoassay for screening CA 15-3 cancer biomarker with high sensitivity (and selectivity), in a very simple, label-free, accurate, and fully automated manner. Detection was achieved by performing two simple steps. In the first step, direct SPR was used to monitor CA 15-3 interaction with surface immobilized antibody. Two linear response ranges were obtained and the detection limit achieved is poor (LOD of 21 U mL-1). However, in the second detection step, electrochemical measurements at the SPR gold surface were performed to measure the decrease of redox probe peak current upon antigen-antibody interaction, providing a suitable amplification strategy to lower detection levels of CA 15-3 (LOD of 0.0998 U mL-1), without the need of additional complex and/or expensive amplification steps to enhance the sensitivity. Moreover, selectivity studies were performed against other common cancer biomarkers and the results showed that the eSPR immunosensor is selective for the CA 15-3 protein. Finally, the clinical applicability of the developed eSPR biosensing methodology was successfully applied to detect CA 15-3 in human serum samples at clinically relevant levels due to the high sensitivity of electrochemical readout. The same concept may be further extended to other proteins of interest.

Surface plasmon resonance-based aptasensor for direct monitoring of thrombin in a minimally processed human blood

Optical affinity biosensors are pursued for timely monitoring of thrombin in human blood, which is of urgent need in tailored anticoagulation therapies. However, the unspecific deposition of molecules, cells, and aggregates from the blood at their surface (also termed fouling) severely hinders their development and impedes the deploying of this technology to everyday clinical practice. We addressed this challenge by designing surface plasmon resonance (SPR) sensor chip with an antifouling polymer brush architecture and incorporated thrombin aptamer bioreceptors. Poly[(N-(2-hydroxypropyl)-methacrylamide)-co-(carboxybetaine methacrylamide)] brushes were synthesized on gold sensor chip surface via photoinduced single-electron transfer living radical polymerization and postmodified with three thrombin aptamers (HD1 short, HD1 and HD22). The affinity interaction of the aptamer bioreceptors with thrombin (as well as with other molecules present in the blood) was investigated and changes in their performance when incorporated into the polymer brushes were characterized. The combination of brushes and aptamer bioreceptors allowed for the analysis of medically relevant concentrations of thrombin in the 10 % blood by direct SPR detection format. This is the first time that the optical affinity biosensor is demonstrated for label-free analysis of biomarkers in a minimally processed human blood without a need for pre-separation steps. We believe that this system constitutes a basis for the future affinity biosensor applications that are suitable for the clinical settings and can be readily adapted to detect a range of important biological markers.

Determination of cross-reactivity of poly- and monoclonal antibodies for synthetic cannabinoids by direct SPR and ELISA

One of the main reasons for the rise in popularity of synthetic cannabinoids (SCs) is their ability to remain unrecognized in conventional drug screenings. Due to their structural diversity, caused by the constant introduction of new substances to circumvent legal regulation, antibodies with a wide range of cross-reactivity are necessary for the establishment of a reliable immunological based drug test. Therefore, high-quality binding data are needed to select promising antibody candidates for further development.In this study, we carried out a direct surface plasmon resonance (SPR) method and evaluated its suitability for the characterization of antibody–SC interactions. The cross-reactivity of 22 SCs with three polyclonal antibodies, raised against JWH-018 haptens with different attachment positions of the linker, and two commercial available monoclonal antibodies were determined. These results were compared with the commonly used competitive enzyme-linked immunosorbent assay (ELISA). It could be demonstrated, that direct SPR and competitive ELISA show comparable specificity results for the majority of the measured compounds. However, the reduced manual labor, the real-time analysis and the high information content about the binding events of SPR compared to ELISA, showed that SPR is a valuable tool during the development of antibodies against synthetic cannabinoids, currently the largest group of new psychoactive substances.

Direct surface plasmon resonance immunosensor for in situ detection of benzoylecgonine, the major cocaine metabolite

In this paper the development of the first direct surface plasmon resonance (SPR) immunoassay for the detection of benzoylecgonine (BZE) is described. Immunosensor chips consisting of a high affinity monoclonal anti-BZE-antibody (anti-BZE–Ab) immobilized at high density to a sensor chip were prepared. First, BZE detection in Hepes buffer was achieved by direct, real time monitoring of the binding between BZE in solution and the surface bound antibody. The detection protocol was based on calibration curves obtained from reaction rate data and end point data analysis of sensorgrams registered after injection of a series of known BZE concentrations over the chips. Moreover, immunosensor accuracy, reproducibility, stability and robustness were tested to demonstrate their good performance as reusable devices. The immunosensor was used for BZE detection in oral fluid (OF) showing that, within 180 s, our immunoassay detects BZE concentrations as low as 4 μg/L in filtered OF-buffer (1:4) samples. This value is remarkably lower than current cut off levels established by the Substance Abuse and Mental Health Services Administration. These results manifest the potential use of this direct SPR immunoassay for the in situ sensitive detection of recent cocaine abuse, of utility in roadside drug OF testing. Moreover, it exemplifies the high potential of direct SPR immunoassays for the rapid, sensitive detection of small molecules in contrast with the more established indirect methods.

Subtractive inhibition assay for the detection of E. coli O157:H7 using surface plasmon resonance.

A surface plasmon resonance (SPR) immunosensor was developed for the detection of E. coli O157:H7 by means of a new subtractive inhibition assay. In the subtractive inhibition assay, E. coli O157:H7 cells and goat polyclonal antibodies for E. coli O157:H7 were incubated for a short of time, and then the E. coli O157:H7 cells which bound antibodies were removed by a stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using a BIAcore 3000 biosensor. The results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 cells in a range from 3.0 × 104 to 3.0 × 108 cfu/mL with a detection limit of 3.0 × 104 cfu/mL. Compared with direct SPR by immobilizing antibodies on the chip surface to capture the bacterial cells and ELISA for E. coli O157:H7 (detection limit: both 3.0 × 105 cfu/mL in this paper), the detection limit of subtractive inhibition assay method was reduced by one order of magnitude. The method simplifies bacterial cell detection to protein-protein interaction, which has the potential for providing a practical alternative for the monitoring of E. coli O157:H7 and other pathogens.

Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen–oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.

High-Affinity Small Molecule Inhibitors of T Cell Costimulation: Compounds for Immunotherapy

Costimulatory molecules are important regulators of T cell activation and thus favored targets for therapeutic manipulation of immune responses. One of the key costimulatory receptors is CD80, which binds the T cell ligands, CD28, and CTLA-4. We describe a set of small compounds that bind with high specificity and low nanomolar affinity to CD80. The compounds have relatively slow off-rates and block both CD28 and CTLA-4 binding, implying that they occlude the shared ligand binding site. The compounds inhibit proinflammatory cytokine release in T cell assays with submicromolar potency, and as such, they represent promising leads for the development of novel therapeutics for immune-mediated inflammatory disease. Our results also suggest that other predominantly beta proteins, such as those that dominate the cell surface, may also be accessible as potentially therapeutic targets.