High-throughput Surface Plasmon Resonance Research Articles

High-throughput analysis system of interaction kinetics for data-driven antibody design

Surface plasmon resonance (SPR) is widely used for antigen–antibody interaction kinetics analysis. However, it has not been used in the screening phase because of the low throughput of measurement and analysis. Herein, we proposed a high-throughput SPR analysis system named “BreviA” using the Brevibacillus expression system. Brevibacillus was transformed using a plasmid library containing various antibody sequences, and single colonies were cultured in 96-well plates. Sequence analysis was performed using bacterial cells, and recombinant antibodies secreted in the supernatant were immobilized on a sensor chip to analyze their interactions with antigens using high-throughput SPR. Using this system, the process from the transformation to 384 interaction analyses can be performed within a week. This system utility was tested using an interspecies specificity design of an anti-human programmed cell death protein 1 (PD-1) antibody. A plasmid library containing alanine and tyrosine mutants of all complementarity-determining region residues was generated. A high-throughput SPR analysis was performed against human and mouse PD-1, showing that the mutation in the specific region enhanced the affinity for mouse PD-1. Furthermore, deep mutational scanning of the region revealed two mutants with > 100-fold increased affinity for mouse PD-1, demonstrating the potential efficacy of antibody design using data-driven approach.

Parameter estimation and identifiability analysis for a bivalent analyte model of monoclonal antibody-antigen binding

Surface plasmon resonance (SPR) is an extensively used technique to characterize antigen-antibody interactions. Affinity measurements by SPR typically involve testing the binding of antigen in solution to monoclonal antibodies (mAbs) immobilized on a chip and fitting the kinetics data using 1:1 Langmuir binding model to derive rate constants. However, when it is necessary to immobilize antigens instead of the mAbs, a bivalent analyte (1:2) binding model is required for kinetics analysis. This model is lacking in data analysis packages associated with high throughput SPR instruments and the packages containing this model do not explore multiple local minima and parameter identifiability issues that are common in non-linear optimization. Therefore, we developed a method to use a system of ordinary differential equations for analyzing 1:2 binding kinetics data. Salient features of this method include a grid search on parameter initialization and a profile likelihood approach to determine parameter identifiability. Using this method we found a non-identifiable parameter in data set collected under the standard experimental design. A simulation-guided improved experimental design led to reliable estimation of all rate constants. The method and approach developed here for analyzing 1:2 binding kinetics data will be valuable for expeditious therapeutic antibody discovery research.

Regenerable and high-throughput surface plasmon resonance assay for rapid screening of anti-SARS-CoV-2 antibody in serum samples

Current serological antibody tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require enzyme or fluorescent labels, and the titer well plates cannot be reused. By immobilizing histidine (His)-tagged SARS-CoV-2 spike (S1) protein onto tris‒nitrilotriacetic acid (tris-NTA) sensor and using the early association phase for mass-transfer-controlled concentration determination, we developed a rapid and regenerable surface plasmon resonance (SPR) method for quantifying anti-SARS-CoV-2 antibody. On a five-channel SPR instrument and with optimized S1 protein immobilization density, each of the four analytical channels is sequentially used for multiple measurements, and all four channels can be simultaneously regenerated once they have reached a threshold value. Coupled with a programmable autosampler, each sensor can be regenerated at least 20 times, enabling uninterrupted assays of more than 800 serum samples. The accuracy and speed of our method compare well with those of the enzyme-linked immunosorbent assay (ELISA), and the detection limit (0.057 μg mL−1) can easily meet the requirement for screening low antibody levels such as those in convalescent patients. In addition, our method exhibits excellent channel-to-channel (RSD = 1.9%) and sensor-to-sensor (RSD = 2.1%) reproducibility. Obviation of an enzyme label drastically reduced the assay cost, rending our method (<60 cents) much more cost effective than those of commercial ELISA kits ($4.4–11.4). Therefore, our method offers a cost-effective and high-throughput alternative to the existing methods for serological measurements of anti-SARS-CoV-2 antibody levels, holding great promise for rapid screening of clinical samples without elaborate sample pretreatments and special reagents.

Chimeric Antigen Receptor (CAR) T Cells Specific for CD229: A Potentially Curative Approach for Multiple Myeloma

Background: Chimeric Antigen Receptor (CAR) T cells targeting B Cell Maturation Antigen (BCMA) have proven effective in Multiple Myeloma (MM). However, BCMA only shows substantial expression in a subgroup of patients and it is only expressed on terminally differentiated plasma cells. We have previously shown that the surface antigen CD229 is homogenously expressed on the tumor cells of all MM patients and that it is also expressed on MM precursor cells, which are thought to be responsible for relapses after chemotherapy. In addition, we have shown that CD229 is essential for the survival of MM cells potentially preventing the patient's tumor cells from losing its expression under the selective pressure of a CAR T cell immunotherapy.Methods and Results: Considering CD229 a target with curative potential, we aimed at generating CD229-specific CAR T cells as a novel adoptive immunotherapy for MM. We generated the first fully human single-chain variable fragment (scFv) domains for use in our CD229 CAR constructs using antibody phage display. We selected 23 unique CD229 binders and using a high-throughput surface plasmon resonance (SPR) assay, we determined that our antibodies had low nanomolar affinities to CD229 and that they did not bind to other SLAM receptors. When expressed as CARs using a CD8α hinge and transmembrane domain with a CD3ζ signaling and a 4-1BB co-stimulatory domain, 15 / 23 constructs showed high surface expression and CD229 binding. From these clones we then selected clone 2D3, which we confirmed to strongly and specifically bind to 293T cells transfected with a CD229 construct. Using a commercially available antibody we had previously observed that in addition to MM cells, CD229 is also expressed on T cells, B cells, and NK cells. However, our clone 2D3 only showed minor staining of some resting T cells and B cells, and no staining of NK cells and activated T cells. However, it showed strong binding to MM and Burkitt's lymphoma cell lines and even stronger staining of primary myeloma cells. We found that the differential binding may be related to a difference in the expression of CD229 isoforms in these cell types.Using lentiviral gene transfer we engineered primary human T cells expressing a CD229 CAR based on 2D3. We then compared our CD229 CAR to a well-characterized CD19 CAR regarding CAR T cell manufacturing and function. Importantly, we found that CD229 CAR T cells expanded almost identically to CD19 CAR T cells (Fig. 1A). In addition, we determined PD-1 expression during CAR T cell manufacturing and did not observe any signs of sustained spontaneous T cell activation in the absence of antigen, called tonic signaling (Fig. 1B). Analyzing the T cell phenotype during CAR T cell production we found that CD229 CAR T cell phenotypes mirrored those of CD19 CAR T cells, including preferential expansion of CD8+ CAR T cells.We next determined the cytotoxic activity of our CAR T cells using various target cells. CD229 CAR T cells showed strong and specific cytotoxic activity against K562 cells transduced with a CD229 expression construct. We also observed strong cytotoxic activity against CD229+ MM cell lines, such as U-266 (Fig. 1C). We did not observe any cytotoxic activity of our CD229 CAR T cells against normal activated T cells or NK cells, with only limited cytotoxicity observed against B cells and some resting T cells. Importantly, we also did not observe any cytotoxicity against purified CD34+ hematopoietic stem cells. Finally, we determined the in vivo efficacy of CD229 CAR T cells using immunocompromised NOD.Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice after engraftment with U-266 myeloma cells. We found that CD229 CAR T cells had completely eradicated MM cells expressing luciferase after only 18 days, while mice treated with control CD19 CAR T cells or PBS still showed strong bioluminescence signal (Fig. 1D).Conclusions: We have developed the first fully human antibodies against CD229, as well as CD229 CAR T cells and have demonstrated their strong cytotoxic activity and selectivity for MM cells in vitro and in vivo. We plan to initiate clinical trials evaluating the safety and efficacy of our CAR T cells in patients with relapsed/refractory MM and other types of lymphomas. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19.

Characterizing Epitope Binding Regions of Entire Antibody Panels by Combining Experimental and Computational Analysis of Antibody: Antigen Binding Competition.

Vaccines and immunotherapies depend on the ability of antibodies to sensitively and specifically recognize particular antigens and specific epitopes on those antigens. As such, detailed characterization of antibody–antigen binding provides important information to guide development. Due to the time and expense required, high-resolution structural characterization techniques are typically used sparingly and late in a development process. Here, we show that antibody–antigen binding can be characterized early in a process for whole panels of antibodies by combining experimental and computational analyses of competition between monoclonal antibodies for binding to an antigen. Experimental “epitope binning” of monoclonal antibodies uses high-throughput surface plasmon resonance to reveal which antibodies compete, while a new complementary computational analysis that we call “dock binning” evaluates antibody–antigen docking models to identify why and where they might compete, in terms of possible binding sites on the antigen. Experimental and computational characterization of the identified antigenic hotspots then enables the refinement of the competitors and their associated epitope binding regions on the antigen. While not performed at atomic resolution, this approach allows for the group-level identification of functionally related monoclonal antibodies (i.e., communities) and identification of their general binding regions on the antigen. By leveraging extensive epitope characterization data that can be readily generated both experimentally and computationally, researchers can gain broad insights into the basis for antibody–antigen recognition in wide-ranging vaccine and immunotherapy discovery and development programs.

Direct Comparison of Label-Free Biosensor Binding Kinetics Obtained on the Biacore 8K and the Carterra LSA.

Funding pressure on the pharmaceutical industry to deliver new medicines to the market under aggressive timelines has led to a demand for analytical tools with higher detection sensitivity, increased throughput, and automation to speed up research and discovery efforts and converge upon clinically fit leads faster. In the quest for therapeutic antibodies, the early adoption of interaction analysis platforms utilizing surface plasmon resonance (SPR) detection provides insightful molecular-level information about the binding properties of antibody libraries that are key to understanding an antibody's mechanism of action and can guide the library-to-leads triage. Here, we sought to compare the binding kinetics obtained on two state-of-the-art high-throughput SPR platforms in an independent study conducted by unrelated groups located on different continents. We show that when experiments were performed by skilled users adhering to SPR best practices and allowed freedom in their assay design, the two platforms yielded near-identical results, establishing them both as reliable tools in accelerating the characterization of antibody libraries in providing critical information needed to advance leads to the clinic.

Eliminating Bottlenecks in mAb Drug Discovery

Genetic Engineering & Biotechnology NewsVol. 40, No. 7 Drug Discovery TutorialEliminating Bottlenecks in mAb Drug DiscoveryTwist Biopharma's approach to antibody discovery incorporates phage display to generate massive antibody libraries, and high-throughput surface plasmon resonance technology to accelerate screeningAaron Sato, PhDAaron Sato, PhDAaron Sato, PhD, is CSO at Twist Biopharma, a division of Twist Bioscience, based in South San Francisco, CA. Twist Bioscience utilizes its silicon-based DNA synthesis platform to generate DNA and DNA-based products for the development of diagnostics, therapeutics, bio-based chemicals, food, DNA-based data storage, and leverages this platform through Twist Biopharma specifically for antibody drug discovery. Website: www.twistbiopharma.com.Search for more papers by this authorPublished Online:10 Jul 2020https://doi.org/10.1089/gen.40.07.13AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 40Issue 7Jul 2020 Information© 2020 by GEN PublishingTo cite this article:Aaron Sato, PhD.Eliminating Bottlenecks in mAb Drug Discovery.Genetic Engineering & Biotechnology News.Jul 2020.48-49.http://doi.org/10.1089/gen.40.07.13Published in Volume: 40 Issue 7: July 10, 2020PDF download

Diagnosis of Mild Cognitive Impairment and Alzheimer's Disease by the Plasma and Serum Amyloid-beta 42 Assay through Highly Sensitive Peptoid Nanosheet Sensor.

Alzheimer's disease (AD) is the most common neurodegenerative disorder with a continuous pathophysiological process starting from the preclinical and mild cognitive impairment (MCI) phases to the dementia phase. Early diagnosis is prerequisite for the early intervention of AD but meanwhile challenging. Amyloid-beta 1-42 (Aβ42) plays a crucial part in AD pathology. Positron-emission tomography (PET) imaging of Aβ42 in the brain and the measurement of Aβ42 in the cerebrospinal fluid (CSF) have been adopted for the auxiliary diagnosis of AD, but their widespread clinical application has been limited due to the radiation and the high-cost of PET and the invasive lumbar puncture for collecting CSF. Noninvasive and cost-effective blood-based assay is desirable for the early diagnosis of AD. Here, a label-free assay for the quantification of blood Aβ42 was developed using the high-throughput surface plasmon resonance imaging method with the aid of an antibody-mimetic peptoid nanosheet equipping Aβ42-recognizing loops. We demonstrated that this nanosheet-based sensor system could distinguish the plasma and sera from normal individuals and patients suffering AD and amnestic MCI with high sensitivity and specificity, preceding the diagnostic performance of the Aβ42-recognizing molecule and the antibody specific to Aβ42. This work provides a label-free, cost-effective, highly sensitive, and high-throughput blood-based assay for early detection of AD.

Detection of Parkinson's Disease through the Peptoid Recognizing α-Synuclein in Serum.

Parkinson's disease (PD) is a severe neurodegenerative disease and there is great need for developing a biochemical detection method to precisely diagnose it. Alpha-synuclein (α-syn) participates in the main pathology of PD and serves as an important biomarker of PD. Here, we identified peptoid ASBP-7 that had high affinity and specificity to α-syn by screening a peptoid library using the high-throughput surface plasmon resonance imaging method. We confirmed that ASBP-7 could significantly distinguish PD sera from the normal ones through identifying α-syn in the serum. We also demonstrated the high sensitivity of this system in detecting PD serum. This work provides a method for the blood-based, label-free, high-throughput analysis of PD serum, and holds great potential for the early diagnosis and dynamic monitoring of PD.

Automatic Spot Identification Method for High Throughput Surface Plasmon Resonance Imaging Analysis.

An automatic spot identification method is developed for high throughput surface plasmon resonance imaging (SPRi) analysis. As a combination of video accessing, image enhancement, image processing and parallel processing techniques, the method can identify the spots in SPRi images of the microarray from SPRi video data. In demonstrations of the method, SPRi video data of different protein microarrays were processed by the method. Results show that our method can locate spots in the microarray accurately regardless of the microarray pattern, spot-background contrast, light nonuniformity and spotting defects, but also can provide address information of the spots.

Systematic Profiling of Histone Readers in Arabidopsis thaliana

Histone post-translational modifications (PTMs) and their recognition by histone readers exert crucial functions in eukaryotes. Despite extensive studies, conservation and diversity of histone PTM regulation between animals and plants remain less explored because of a lack of systematic knowledge of histone readers in plants. Based on a high-throughput surface plasmon resonance imaging (SPRi) platform, we report the lab-on-chip profiling of interactions between 204 putative reader domains and 11 types of histone peptides in Arabidopsis thaliana. Eleven reader hits were then chosen for histone combinatorial readout pattern profiling. Systematic analysis of histone PTM recognition in Arabidopsis thaliana reveals that plant and human histone readers share conservation in domain types and recognition mechanisms. The differences in particular histone mark recognition by transcription regulator EML1 and DNA damage repair factor MSH6 indicate plant-specific histone PTMs function in Arabidopsis thaliana acquired during evolution.

Abstract 3774: A high-throughput process for the development and characterization of chimeric antigen receptors (CARs)

Abstract INTRODUCTION: Chimeric antigen receptors (CARs) expressed by T cells bind to surface antigens via a single-chain variable fragment (scFv) and are able to elicit potent anti-tumor efficacy. To our knowledge no rapid CAR development platforms exist to facilitate the high-throughput generation and characterization of novel CARs to allow head-to-head comparisons and to determine the influence of scFv properties on CAR T cell activation, phenotype, and persistence. EXPERIMENTAL PROCEDURES: We have established a rapid process for the development of functional novel CARs using fully human antibody phage display, vector systems for bacterial and mammalian expression, as well as high-throughput functional and biochemical characterization assays. We validated individual parts of the workflow using different tumor antigens and completed the entire workflow for one model antigen expressed on the surface of multiple myeloma cells. An initial population of scFvs against the model antigen was selected from a large naïve fully-human antibody phage display library by panning. The reactivity of the polyclonal phage population was then confirmed by time-resolved fluorescence before reformatting into various monoclonal binder formats. We established a high-throughput flow cytometry assay simultaneously determining CAR surface expression and antigen binding. In addition, we adapted protocols for small-scale lentiviral transduction and expansion of primary CAR T cells followed by a sensitive, high-throughput luciferase-based cytotoxicity assay. Finally, in collaboration with Wasatch Microfluidics we performed high-throughput surface plasmon resonance measurements as well as epitope binning of candidate binding domains. RESULTS: We obtained 1,323 scFvs after two rounds of antibody phage selection. The subsequent bacterial screen of 163 clones identified 23 unique monoclonal binders. In a comparison of all 23 binders in different formats we determined that none of the traditional screening formats, including soluble scFv or scFv-Fc fusion constructs, correlated with CAR binding. CAR constructs with high expression and antigen binding were found to have affinities in the low nanomolar range (18-22nM) and evidenced strong killing of human cancer cell lines spontaneously expressing the antigen (53-79% killing at an effector:target ratio of 10:1), but not healthy cells expressing lower levels of the antigen. Importantly, in a murine xenograft model of multiple myeloma we observed specific killing of tumor cells and no overt toxicities. CONCLUSION: We show that our process allows the generation, screening, as well as functional and biochemical characterization of novel CARs within 2 months. Our approach enables researchers to easily carry out head-to-head comparisons allowing comprehensive lead identification and the determination of binding domain properties which may shape CAR T cell function and phenotype. Citation Format: Sabarinath Venniyil Radhakrishnan, Adam Miles, Djordje Atanackovic, Tim Luetkens. A high-throughput process for the development and characterization of chimeric antigen receptors (CARs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2017-3774

Extending the throughput of Biacore 4000 biosensor to accelerate kinetic analysis of antibody-antigen interaction

The surface plasmon resonance (SPR) biosensors are being routinely used in different stages of drug discovery and development. However, the lack of high throughput SPR biosensors continues to be a primary bottleneck for the rapid kinetic screening of large panels of monoclonal antibodies (mAbs). To further increase the throughput of the Biacore 4000 biosensor, we have developed three kinetic screening assays to characterize mAb-antigen interactions – (i) 16-mAb capture kinetic, (ii) single cycle kinetic (SCK), and (iii) parallel kinetic (PK). The performance of all three kinetic assays was evaluated by characterizing the binding of kinetically diverse human mAbs to four antigens with molecular weights of 14kD, 29kD, 38kD, and 48kD and binding affinities ranging from 130pM to 200 nM. The binding rate constants measured using all three kinetic assays were reproducible across multiple experiments and correlated with the values generated using the conventional 8-mAb capture kinetic assay on the Biacore 4000 (R2 > 0.94). Moreover, the 16-mAb capture assay decreased experiment time and analyte consumption by 35% and 50%, respectively. This work illustrates the significance of the 16-mAb capture kinetic, SCK, and PK assays to increase the throughput of Biacore 4000 and to support rapid kinetic screening of mAbs.

Highly selective capture of minicircle DNA biopharmaceuticals by a novel zinc-histidine peptide conjugate

Abstract The use of minicircle DNA (mcDNA) biomolecules as a pharmaceutical product holds remarkable potential due to their improved therapeutic efficacy in comparison with standard non-viral gene expression vectors. However, mcDNA translation into clinical application is still highly restricted due to the lack of robust technologies for minicircles detection and purification. In this study, the potential of a zinc-binding histidine-based peptide to function as a novel ligand for mcDNA recovery was investigated by using high-throughput surface plasmon resonance (SPR) analysis. The histidine-based peptide successfully bound zinc cationic ions and had affinity towards mcDNA biomolecules as confirmed by the dynamic binding responses obtained in SPR experiments. Notably, the obtained results indicate that not only zinc-peptide ligands are able to bind mcDNA with very high affinity (KD = 4.21 × 10−10 M), but also that this interaction is mostly dependent on buffer type. In general, the findings indicated that Zn2+ bound peptide has high affinity to mcDNA in low ionic strength buffers, whereas with high salt buffers no binding is detected. Overall, the novel zinc-binding peptide has shown to have suitable properties for mcDNA binding and recovery under experimental conditions that assure genetic material stability. More importantly, the straightforward approach of employing simple biomimetic ligands for mcDNA capture will contribute for development of new technologies to purify DNA biopharmaceuticals.

Improved polarization contrast method for surface plasmon resonance imaging sensors by inert background gold film extinction

To improve the performance of high-throughput surface plasmon resonance (SPR) imaging sensors, the imperfections of the conventional gold film polarization contrast method and the bare prism polarization contrast method are discussed, and a background extinction method is proposed. Thick gold film is coated as the background area, surrounding gold sensing spots, to form a “gold microwell array”. By blocking the light reflected from the background area with appropriate parameters, an opposite-oriented SPR curve is obtained, which can be exploited to achieve a high signal-to-noise ratio in trace amount detection. The contrast between the sensing spots and the background is effectively improved, and the background is inert to the change of solution. Influences of parameter errors on the SPR curve are also investigated, and results show that extinction adjustment in this method is easy to be realized. The practicability of the background extinction method is proved by an experiment using a home-built SPR imaging sensor.

Assessment of a multiplexing high throughput immunochemical SPR biosensor in measuring multiple proteins on a single biosensor chip

Multiplexed immunochemical detection platforms offer the potential to decrease labour demands, increase sample throughput and decrease overall time to result. A prototype four channel multiplexed high throughput surface plasmon resonance biosensor was previously developed, for the detection of food related contaminants. A study focused on determining the instruments performance characteristics was undertaken. This was followed by the development of a multiplexed assay for four high molecular weight proteins. The protein levels were simultaneously evaluated in serum samples of 10-week-old veal calves (n=24) using multiple sample preparation methods. Each of the biosensor's four channels were shown to be independent of one another and produced multiplexed within run repeatability (n=6) ranging from 2.0 to 6.7%CV, for the four tested proteins, whilst between run reproducibility (n=4) ranged from 1.5 to 8.9%CV. Four calibration curves were successfully constructed before serum sample preparation was optimised for each protein. Multiplexed concentration analysis was successfully performed on four channels revealing that each proteins concentration was consistent across the twenty-four tested animals. Signal reproducibility (n>19) on a further long term study revealed coefficient of variation ranging from 1.1% to 7.3% and showed that the multiplexed assay was stable for at least 480 cycles. These findings indicate that the performance characteristics fall within the range of previously published data for singleplex optical biosensors and that the multiplexing biosensor is fit-for-purpose for simultaneous concentration analysis in many different types of applications such as the multiplexed detection of markers of growth-promoter abuse and multiplexed detection of residues of concern in food safety.

Contrast-enhancing polarization control method for surface plasmon imaging sensor

Based on the surface plasmon resonance (SPR) polarization control theory, a prism polarization control method is proposed. The method works by suppressing the light reflected from the bare prism used for high-throughput SPR array sensors. The contrast of SPR imaging sensors can be enhanced while maintaining high sensitivity. The effects of the proposed method on sensor performance, which is attributable to parameters such as incident angle, wavelength, polarizer angle, and gold film thickness, are analyzed to determine the best set of parameters. The effects of parameter errors on the SPR curve are also simulated and compared with those of the gold film polarization control method. Finally, an experiment is set up to validate the theoretical analysis and provide a solid foundation for the design of a high-sensitivity SPR imaging sensor.

A high-throughput surface plasmon resonance biosensor based on differential interferometric imaging

A new high-throughput surface plasmon resonance (SPR) biosensor based on differential interferometric imaging is reported. The two SPR interferograms of the sensing surface are imaged on two CCD cameras. The phase difference between the two interferograms is 180°. The refractive index related factor (RIRF) of the sensing surface is calculated from the two simultaneously acquired interferograms. The simulation results indicate that the RIRF exhibits a linear relationship with the refractive index of the sensing surface and is unaffected by the noise, drift and intensity distribution of the light source. The affinity and kinetic information can be extracted in real time from continuously acquired RIRF distributions. The results of refractometry experiments show that the dynamic detection range of SPR differential interferometric imaging system can be over 0.015 refractive index unit (RIU). High refractive index resolution is down to 0.45 RU (1 RU = 1 × 10−6 RIU). Imaging and protein microarray experiments demonstrate the ability of high-throughput detection. The aptamer experiments demonstrate that the SPR sensor based on differential interferometric imaging has a great capability to be implemented for high-throughput aptamer kinetic evaluation. These results suggest that this biosensor has the potential to be utilized in proteomics and drug discovery after further improvement.

Bragg-Scattered Surface Plasmon Microscopy: Theoretical Study

We present a new approach to surface plasmon microscopy with high refractive index sensitivity and spatial resolution that is not limited by the propagation length of surface plasmons. It is based on a nanostructured metallic sensor surface supporting Bragg-scattered surface plasmons. We show that these non-propagating surface plasmon modes are excellently suited for spatially resolved observations of refractive index variations on the sensor surface owing to their highly confined field profile perpendicular to as well as parallel to the metal interface. The presented theoretical study reveals that this approach enables reaching similar refractive index sensitivity as regular surface plasmon resonance (SPR) microscopy and offers the advantage of improved spatial resolution when observing dielectric features with lateral size <10 μm for the wavelength around 800 nm and gold as the SPR-active metal. This paper demonstrates the potential of Bragg-scattered surface plasmon microscopy for high-throughput SPR biosensing with high-density microarrays.