Performance qualification for reproducible Surface Plasmon Resonance analysis.

Surface Plasmon Resonance Biosensors (SPR) are one of the most powerful tools to characterize protein binding, e.g. for drug discovery, like target identification, ligand fishing, assay development, lead selection and manufacturing quality control. However, there is increasing concern about its reproducibility in the light of the reproducibility crisis. Therefore an appropriate analytical instrument qualification (AIQ) is required for quality assurance of SPR instruments. AIQ is a prerequisite for analytical method validation and it is consisting of four parts, Design Qualification (DQ), Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ). PQ regularly executed is supposed to continuously control the performance of the instrument under actual running conditions. In this work a performance qualification method was developed for the SPR instrument Biacore X100. This method is suitable for the routinely control of the instrument performance for antibody-antigen binding measurements. Control charts were designed to get a clearly representable and easy implementable tool to check the critical parameters. These control charts and a straightforward protocol now allow the design and application of an individual performance qualification procedure that can be used in the laboratory routine. They serve as reference for individual standard operation procedures (SOPs).

Improving the Performance of an SPR Biosensor Using Long-Range Surface Plasmon of Ga-Doped Zinc Oxide.

Transparent conducting oxides (TCOs) have appeared in the past few years as potential plasmonic materials for the development of optical devices in the near infrared regime (NIR). However, the performance of biosensors with TCOs has been limited in sensitivity and figure of merit (FOM). To improve the performance of the biosensors with TCOs, a biosensor based on long-range surface plasmon with Ga-doped zinc oxide (GZO) is proposed. It is shown that a larger FOM with a 2~7 times enhancement compared to the traditional surface plasmon polaritons (SPPs) sensor and higher detection accuracy (DA) can be realized in our proposed sensor compared with the surface plasmon resonance (SPR) sensor with GZO. Therefore, this sensor can be used to detect biological activity or chemical reactions in the near infrared region.

Bulk immunoassays for analysis of extracellular vesicles.

There is increasing clinical interest in extracellular vesicles (EV) for diagnostic and treatment purposes. This review provides an overview of bulk immunoassays to analyse EV. Western blot and enzyme-linked immunosorbent assay are still the two predominant bulk immunoassays. Recently, new assays have become available that can detect exposure to EV concentrations that are up to 10,000-fold lower. This is advantageous for applications that detect rare EV. Other important parameters are the detectable concentration range, the required sample volume, whether simultaneous presence of different antigens on a single EV can be detected, size selectivity of each assay and practical considerations. In this review, we will explain the working principles of the traditional and novel assays together with their performance parameters. The most sensitive assays are micro-nuclear magnetic resonance, surface plasmon resonance, and time-resolved fluorescent immunoassay.

Evaluation of calibration-free concentration analysis provided by Biacore™ systems.

Surface Plasmon Resonance biosensors measure the interaction between a molecule in solution and its interaction partner attached to a sensor surface. Under certain conditions, the observed binding rate can be used directly to obtain the concentration of the molecule in solution, without the use of any standard. This type of assay is referred to as Calibration Free Concentration Analysis, CFCA. By examining experimental conditions, including immobilization levels and temperature, for a range of analytes, and by using global analysis of several sample dilutions, conditions that gave the most robust results were identified. These conditions provided the concentration values that were on average ∼15% lower than those obtained using other methods. The accuracy of the concentration determined may be related to how the analyte is distributed in the dextran matrix and to its distance from the gold surface, and may thereby depend on the conversion of the SPR signal to mass. A good precision of CFCA, ∼8% (n = 21), was demonstrated when this method was used to efficiently guide purification procedures of Interferon α-2a. In this paper, the theory behind CFCA and the future developments, as well as the application of CFCA for absolute and relative concentration measurements (including the assessment of the potency of a biotherapeutic medicine) are discussed, and new evaluation tools that broaden the range of applications, are introduced.

Evaluation of antiphospholipid antibody assays using latent class analysis to address the lack of a reference standard.

BACKGROUND: Method evaluation of new assays for the detection of antiphospholipid antibodies (aPL) such as anti-cardiolipin (aCL) or anti-ß2-glycoprotein I (aß2-GPI) is challenging, as no internationally accepted reference material is available yet. Besides a lack of standardization, unacceptable inter-laboratory comparability of established tests is regularly observed. Owing to the absence of a commonly accepted reference standard, the evaluation of two research surface plasmon resonance (SPR) biosensor assays was performed using statistical methods from latent class analysis (LCA). METHODS: aCL and aß2-GPI IgG and IgM were measured in sera from 63 antiphospholipid syndrome patients, fulfilling the Sydney criteria, and in 34 healthy controls with four commercial assays. LCA was performed on the results and sera were assigned to the antibody-positive or antibody-negative group. Sera were subsequently evaluated in the SPR assays for aCL and aß2-GPI. Optimal cutoffs and diagnostic performances of the research systems were established employing the LCA-derived gold standard. RESULTS: With area under the curve results of 0.96 and 0.89 for the detection of aCL and aß2-GPI, the research SPR assays discriminated well between antibody-positive and antibody-negative sera. Their sensitivities and specificities were comparable to the investigated commercial immunoassays. CONCLUSIONS: SPR assays are a suitable tool for the detection of aCL and aß2-GPI with diagnostic performances not different from currently available commercial tests. LCA enabled the calculation of sensitivities and specificities for aPL assays in absence of a reference standard.

Data quality in drug discovery: the role of analytical performance in ligand binding assays.

Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.

Enhancing sensitivity of surface plasmon resonance biosensors by functionalized gold nanoparticles: size matters.

We study how the size of spherical gold nanoparticles (AuNPs) influences their ability to enhance the response of optical biosensors based on surface plasmon resonance (SPR). We present a theoretical model that relates the enhancement generated by the AuNPs to their composition, size, and concentration, thus allowing for accurate predictions regarding the SPR sensor response to various AuNPs. The effect of the AuNP size is also investigated experimentally using an SPR biosensor for the detection of carcinoembryonic antigen (CEA) in which AuNPs covered with neutravidin (N-AuNPs) are used in the last step of a sandwich assay to enhance the sensor response to biotinylated secondary antibody against CEA. The experimental data are in excellent agreement with the results of the theoretical analysis. We demonstrate that the sensor response enhancement generated by the N-AuNPs is determined by (i) the sensor sensitivity to N-AuNP surface density (Sσ) and (ii) the ability of the N-AuNPs to bind to the functionalized surface of the sensor. Our results indicate that, while Sσ increases with the size of the N-AuNP, the ability of the functionalized surface of the sensor to bind the N-AuNPs is affected by steric effects and decreases with the size of N-AuNP.

Ultrasensitive and ultrawide range detection of a cardiac biomarker on a surface plasmon resonance platform.

One of the main challenges in the development of new analytical platforms for ultrasensitive bioaffinity detection is jointly achieving a wide dynamic range in target analyte concentration, especially for approaches that rely on multistep processes as a part of the signal amplification mechanism. In this paper, a new surface-based sandwich assay is introduced for the direct detection of B-type natriuretic peptide (BNP), an important biomarker for cardiac failure, at concentrations ranging from 1 aM to 500 nM. This was achieved using nanoparticle-enhanced surface plasmon resonance (SPR) where a DNA aptamer is immobilized on a chemically modified gold surface in conjunction with the specific adsorption of antiBNP coated gold nanocubes in the presence of the biomarker target. A concentration detection range greater than eleven orders of magnitude was achieved through dynamic control of only the secondary nanoparticle probe concentration. Furthermore, detection at low attomolar concentrations was also achieved in undiluted human serum.

A simple small size and low cost sensor based on surface plasmon resonance for selective detection of Fe(III).

A simple, small size, and low cost sensor based on a Deferoxamine Self Assembled Monolayer (DFO-SAM) and Surface Plasmon Resonance (SPR) transduction, in connection with a Plastic Optical Fiber (POF), has been developed for the selective detection of Fe(III). DFO-SAM sensors based on appropriate electrochemical techniques can be frequently found in the scientific literature. In this work, we present the first example of a DFO-SAM sensor based on SPR in an optical fiber. The SPR sensing platform was realized by removing the cladding of a plastic optical fiber along half the circumference, spin coating a buffer of Microposit S1813 photoresist on the exposed core, and finally sputtering a thin gold film. The hydroxamate siderophore deferoxamine (DFO), having high binding affinity for Fe(III), is then used in its immobilized form, as self-assembled monolayer on the gold layer surface of the POF sensor. The results showed that the DFO-SAM-POF-sensor was able to sense the formation of the Fe(III)/DFO complex in the range of concentrations between 1 µm and 50 µm with a linearity range from 0 to 30 µm of Fe(III). The selectivity of the sensor was also proved by interference tests.

Protein interactomics based on direct molecular fishing on paramagnetic particles: experimental simulation and SPR validation.

We describe an experimental approach for direct molecular fishing of prey protein on the surface of two types of paramagnetic particles (PMP) having different size and composition. Human microsomal cytochrome b(5) (b(5)) and its known partner human cytochrome P450 3A5 (CYP3A5) were used as bait and prey proteins, respectively. For assessing the level of unspecific binding of background proteins, α-fetoprotein (aFP) was used. SPR measurements were applied for quantitative analysis of trapped proteins (CYP3A5 and aFP) after fishing on PMP. It was shown that the described approach of molecular fishing on micro-PMP provides enough prey proteins for LC-MS/MS identification and SPR validation, so this approach can be used for discovery of new protein-protein interactions in the framework of Human Proteome Project.

Optimisation of infrared-assisted extraction of rutin from crude Flos Sophorae Immaturus using response surface methodology and HPLC analysis.

INTRODUCTION: Rutin, one of main constituents in Flos Sophorae Immaturus, has been proven to possess several pharmacological properties such as anti-oxidant, anti-platelet, anti-inflammatory effects and so on. However, optimisation of the extraction of rutin from Flos Sophorae Immaturus has rarely been reported. Thus, it is important to develop an effective method to obtain maximum yields of rutin from Flos Sophorae Immaturus. OBJECTIVE: To develop an infrared-assisted extraction method for maximum rutin yield from crude Flos Sophorae Immaturus using response surface methodology and HPLC analysis. METHODOLOGY: Through single factor experiments, ranges of the main variables (including methanol concentration, liquid:solid ratio, extraction time and infrared power) affecting the extraction yield of rutin were confirmed. A Box-Behnken design consisting of 24 experimental runs and five replicates at zero point was then applied and a regression model was obtained to predict the optimal extraction yield. RESULTS: The ANOVA analysis indicated that the regression equation fits very well with the actual situation. The optimal conditions were as follows: infrared power 204.90 W, liquid:solid ratio 30.00 mL/g, methanol concentration 70.00% and extraction time 4.80 min. Under optimal conditions the predicted maximum yield (125.70 mg rutin/0.5 g raw material) was consistent with the experimental value (126.32 ± 0.67 mg rutin/0.5 g raw material) (n = 3). CONCLUSION: The application of response surface methodology was reliable and feasible in the optimisation of infrared-assisted extraction of rutin from crude Flos Sophorae Immaturus.

Determination of amylose in Iranian rice by multivariate calibration of the surface plasmon resonance spectra of silver nanoparticles.

A simple and non-separative analytical method for selective determination of amylose in Iranian rice has been developed. It was based on the reduction of silver ions by amylose and production of Ag nanoparticles, which exhibit surface plasmon resonance (SPR) spectra in the ultraviolet/visible region. The formation of Ag nanoparticles in the presence of amylose was monitored by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The experimental conditions were optimized to obtain the highest yield for nanoparticle formation. Partial least square (PLS) regression as an efficient multivariate spectral calibration method was employed to make a connection between the SPR spectra of the generated Ag nanoparticles and the amylose content (AC) of the rice starch. The number of PLS latent variables was optimized by leave-one-out cross-validation utilizing prediction residual error sum of square (PRESS). The proposed model exhibited a high ability for prediction of amylose concentration in both standard starch samples and real rice samples prepared from different regions of Iran. The relative errors of prediction were almost lower than ±5% for different real samples and the detection limit was 3.23 weight percent of amylose in rice. In comparison to the reference method (Juliano method), the proposed method is simpler and does not need tedious sample preprocessing steps.

Lead identification and optimization in crude samples using label free resonant acoustic profiling.

Detecting the molecular basis of protein-protein recognition is an essential element in understanding protein function because their ability to form specific complexes with other proteins underlies most cellular processes. The use of labels has limitations, such as changes to the binding kinetics due to the alterations in structure and function that occur with label addition, difficulty in detecting biochemical activities and the need for additional steps in assay development. These issues have driven the development of label-free formats for identifying the full range of biochemical activities. Although optical-based systems dominate the label-free biosensor market, electrochemical, piezoelectric and acoustic devices represent similar but significantly less expensive alternatives. Acoustic biosensors have been employed in the label-free detection of an incredibly broad range of analytes, from interfacial chemistries and lipid membranes, to small molecules and whole cells. Resonant acoustic profiling (RAP) technology offers label-free, real-time analysis of biomolecular interactions and offers an efficient way to optimize the development and production process of recombinant proteins. RAP measures only the physical binding events and is insensitive to refractive index and colour changes. This enables direct measurement in undiluted crude and complex samples, such as cell culture media or periplasmic extracts, without intensive assay calibration. This advantage simplifies experimental design and eliminates expensive time-consuming purification of often limited material, while delivering high content information. In this respect RAP technology reduces costs and increases the throughput and the density of information to optimize and control the processes more effectively.

An efficient LSPR method to quantitatively detect dimethoate: Development, characterization and evaluation.

In recent years, there has been growing concern among consumers about pesticide contamination in fruits. Therefore, rapid, reliable, and consistent detection methods for OPPs, especially dimethoate, are crucially needed. The existing quantitative methods for detecting dimethoate are not suitable for rapid measuring system such as the dimethoate samples from two channels. Hence this paper examines the utilization of a dual-channel system for utilize the absorption variations of the Localized Surface Plasmon Resonance (LSPR) bands of gold nanoparticles (AuNPs) were investigate for detection of dimethoate. Under optimized conditions, the relationship between concentrations of dimethoate and absorbance ratios (A(520)/A(640)) was linearly found in the concentration range of 10-100 nM. Result from the experiment shows that both channels exhibit a linear correlation coefficient as high as 0.97 and a limit of detection (LOD) as low as 5.5 nM. This LSPR detection system was characterized by testing the dimethoate in apple samples and the recovery rates were found to be in the range of 85.90% to 107.37%. The proposed dual-channel LSPR system for detecting dimethoate creating a new approach for detecting organophosphate insecticide in agricultural fields. It could lay the foundation for designing a high-throughput analysis of the insecticides using a wavelength division multiplexing switch (WDMS).

Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

The ongoing outbreak of the novel coronavirus disease (COVID-19) has spread globally and poses a threat to public health in more than 200 countries. Reliable laboratory diagnosis of the disease has been one of the foremost priorities for promoting public health interventions. The routinely used reverse transcription polymerase chain reaction (RT-PCR) is currently the reference method for COVID-19 diagnosis. However, it also reported a number of false-positive or -negative cases, especially in the early stages of the novel virus outbreak. In this work, a dual-functional plasmonic biosensor combining the plasmonic photothermal (PPT) effect and localized surface plasmon resonance (LSPR) sensing transduction provides an alternative and promising solution for the clinical COVID-19 diagnosis. The two-dimensional gold nanoislands (AuNIs) functionalized with complementary DNA receptors can perform a sensitive detection of the selected sequences from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through nucleic acid hybridization. For better sensing performance, the thermoplasmonic heat is generated on the same AuNIs chip when illuminated at their plasmonic resonance frequency. The localized PPT heat is capable to elevate the in situ hybridization temperature and facilitate the accurate discrimination of two similar gene sequences. Our dual-functional LSPR biosensor exhibits a high sensitivity toward the selected SARS-CoV-2 sequences with a lower detection limit down to the concentration of 0.22 pM and allows precise detection of the specific target in a multigene mixture. This study gains insight into the thermoplasmonic enhancement and its applicability in the nucleic acid tests and viral disease diagnosis.

Designing efficient zero calibration point for phase-sensitive surface plasmon resonance biosensing.

This work is related to the development of phase-sensitive methodologies in Surface Plasmon Resonance (SPR) biosensing. We take advantage of a specific angular dependence of phase of light, reflected under SPR geometry, on parameters of the SPR-supporting metal, and propose a polarimetry-based methodology to easily determine the optimal calibration zero point, corresponding to the maximal phase sensitivity. The proposed methodology can significantly facilitate the calibration of the system in field and multi-channel sensing, broaden the dynamic range, as well as contribute to the development of feedback loops.

Calibration and testing with real turbulence of a pyramid sensor employing static modulation.

The pyramid sensor (PS) is an interesting alternative to the Shack-Hartmann wavefront sensor (SH WFS) for astronomical Adaptive Optics (AO) because of its potential advantages in sensitivity and applicability to novel wavefront sensing schemes. The PS uses a pyramidal prism to perform a knife-edge test in two dimensions simultaneously and relies on modulating the position of the prism to increase the linear dynamic range. It has been suggested that this could also be accomplished by a static diffusing element. We test this idea and show that the diffuser produces a modulation effect. We compare the results of our PS to a SH WFS measuring spatial and temporal properties of real turbulence produced in the lab with a hot-air turbulence generator.

"Spot and hop": internal referencing for surface plasmon resonance imaging using a three-dimensional microfluidic flow cell array.

We have developed a novel referencing technique for surface plasmon resonance imaging systems referred to as "spot and hop." The technique enables internal referencing for individual flow cells in a parallel processing microfluidic network. Internal referencing provides the ability to correct for nonspecific binding and instrument drift, significantly improving data quality at each region of interest. The performance of a 48-flow-cell device was demonstrated through a series of studies, including "rise and fall" time, ligand preconcentration, ligand immobilization, analyte binding, and regeneration tests. Interfacing parallel processing fluidics with imaging systems will significantly expand the throughput and applications of array-based optical biosensors while retaining high data quality.

A comparison report of three advanced methods for drug-cyclodextrin interaction measurements.

Three advanced methods, high performance affinity chromatography (HPAC), surface plasmon resonance (SPR) and surface plasmon resonance imaging (SPRi) were compared and evaluated for determining the drug-cyclodextrin (CD) interactions herein. In total, 18 sparingly soluble drugs were selected for this comparative study. The three methods share a unique connection in the working principles and strategies. The same strategies of CD fixation onto solid phase were used in HPAC and SPR for the measurements, whereas, the SPR and SPRi share identical working principles. However, whilst these relationships are evident, no strong correlation was found between kinetic constants obtained from the three methods: Four drugs, namely, prednisolone, pseudolaric acid B, diazepam and gramisetron failed to show any response on SPR, whereas, the kinetics parameters from SPRi and HPAC were successfully measured. From a comparative review of all the kinetic data, random results without any trends were observed (ka, kd and KA) regardless of the relationships between the three methods: It is apparent that the measurement conditions (volume, flow rate, buffers), non-specific adsorption and experimental procedures had a strong impact on the generated data. The relative advantages and limitations of each method are critically presented on the basis of generated data. This comparative study provides a basis to further upgrade these techniques for confident measurement of drug-CDs interactions.

Rapid signal enhancement method for nanoprobe-based biosensing.

The introduction of nanomaterials as detection reagents has enabled improved sensitivity and facilitated detection in a variety of bioanalytical assays. However, high nanoprobe densities are typically needed for colorimetric detection and to circumvent this limitation several enhancement protocols have been reported. Nevertheless, there is currently a lack of universal, enzyme-free and versatile methods that can be readily applied to existing as well as new biosensing strategies. The novel method presented here is shown to enhance the signal of gold nanoparticles enabling visual detection of a spot containing <10 nanoparticles. Detection of Protein G on paper arrays was improved by a 100-fold amplification factor in under five minutes of assay time, using IgG-labelled gold, silver, silica and iron oxide nanoprobes. Furthermore, we show that the presented protocol can be applied to a commercial allergen microarray assay, ImmunoCAP ISAC sIgE 112, attaining a good agreement with fluorescent detection when analysing human clinical samples.