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Direct optical detection has proven to be a highly interesting tool in biomolecular interaction analysis to be used in drug discovery, ligand/receptor interactions, environmental analysis, clinical diagnostics, screening of large data volumes in immunology, cancer therapy, or personalized medicine. In this review, the fundamental optical principles and applications are reviewed. Devices are based on concepts such as refractometry, evanescent field, waveguides modes, reflectometry, resonance and/or interference. They are realized in ring resonators; prism couplers; surface plasmon resonance; resonant mirror; Bragg grating; grating couplers; photonic crystals, Mach-Zehnder, Young, Hartman interferometers; backscattering; ellipsometry; or reflectance interferometry. The physical theories of various optical principles have already been reviewed in detail elsewhere and are therefore only cited. This review provides an overall survey on the application of these methods in direct optical biosensing. The “historical” development of the main principles is given to understand the various, and sometimes only slightly modified variations published as “new” methods or the use of a new acronym and commercialization by different companies. Improvement of optics is only one way to increase the quality of biosensors. Additional essential aspects are the surface modification of transducers, immobilization strategies, selection of recognition elements, the influence of non-specific interaction, selectivity, and sensitivity. Furthermore, papers use for reporting minimal amounts of detectable analyte terms such as value of mass, moles, grams, or mol/L which are difficult to compare. Both these essential aspects (i.e., biochemistry and the presentation of LOD values) can be discussed only in brief (but references are provided) in order to prevent the paper from becoming too long. The review will concentrate on a comparison of the optical methods, their application, and the resulting bioanalytical quality.

Localized surface plasmon resonance (LSPR) detection offers highly sensitive label-free detection of biomolecular interactions. Simple and robust surface architectures compatible with real-time detection in a flow-through system are required for broad application in quantitative interaction analysis. Here, we established self-assembly of a functionalized gold nanoparticle (AuNP) monolayer on a glass substrate for stable, yet reversible immobilization of Histidine-tagged proteins. To this end, one-step coating of glass substrates with poly-L-lysine graft poly(ethylene glycol) functionalized with ortho-pyridyl disulfide (PLL-PEG-OPSS) was employed as a reactive, yet biocompatible monolayer to self-assemble AuNP into a LSPR active monolayer. Site-specific, reversible immobilization of His-tagged proteins was accomplished by coating the AuNP monolayer with tris-nitrilotriacetic acid (trisNTA) PEG disulfide. LSPR spectroscopy detection of protein binding on these biocompatible functionalized AuNP monolayers confirms high stability under various harsh analytical conditions. These features were successfully employed to demonstrate unbiased kinetic analysis of cytokine-receptor interactions. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02551-6) contains supplementary material, which is available to authorized users.

In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations. Electronic supplementary material The online version of this article (10.1007/s00216-017-0735-6) contains supplementary material, which is available to authorized users.

This work reports on further development of an optical biosensor for the in vitro detection of mycotoxins (in particular, aflatoxin B1) using a highly sensitive planar waveguide transducer in combination with a highly specific aptamer bioreceptor. This sensor is built on a SiO2–Si3N4–SiO2 optical planar waveguide (OPW) operating as a polarization interferometer (PI), which detects a phase shift between p- and s-components of polarized light propagating through the waveguide caused by the molecular adsorption. The refractive index sensitivity (RIS) of the recently upgraded PI experimental setup has been improved and reached values of around 9600 rad per refractive index unity (RIU), the highest RIS values reported, which enables the detection of low molecular weight analytes such as mycotoxins in very low concentrations. The biosensing tests yielded remarkable results for the detection of aflatoxin B1 in a wide range of concentrations from 1 pg/mL to 1 μg/mL in direct assay with specific DNA-based aptamers. Graphical abstractOptical planar waveguide polarization interferometry biosensor for detection of aflatoxin B1 using specific aptamer. Electronic supplementary material The online version of this article (10.1007/s00216-019-02033-4) contains supplementary material, which is available to authorized users.

In this paper, we investigate whether dissolution in water under autoclaving conditions (140 °C, 20 min) or in dimethyl sulfoxide, DMSO (100 °C, 1 h), is preferable for characterization of amylose. Two types of amylose, potato and maize, were dissolved either in water using an autoclave or in DMSO. On the aqueous solutions obtained, the extent of molecular dissolution of the sample (referred to as the dissolution yield) was determined by enzymatic analysis as well as the molecular properties, such as molar mass and root-mean-square radius, obtained with asymmetrical flow field-flow fractionation coupled to multi-angle light scattering and differential refractive index detection (AF4-MALS-dRI). The results showed that both dissolution methods are efficient at dissolving amylose. However, AF4-MALS-dRI analysis revealed substantial differences. Amylose aqueous solutions obtained by dissolution in DMSO were relatively stable over time, but the dissolution method in autoclave caused some degradation of the molecules, and their solutions display a high tendency to retrograde.

Cereal-based beverages contain a complex mixture of various polymeric macromolecules including polysaccharides, peptides, and polyphenols. The molar mass of polymers and their degradation products affect different technological and especially sensory parameters of beverages. Asymmetrical flow field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS) and refractive index detection (dRI) or UV detection (UV) is a technique for structure and molar mass distribution analysis of macromolecules commonly used for pure compound solutions. The objective of this study was to develop a systematic approach for identifying the polymer classes in an AF4//MALS/dRI/UV fractogram of the complex matrix in beer, a yeast-fermented cereal-based beverage. Assignment of fractogram fractions to polymer substance classes was achieved by targeted precipitations, enzymatic hydrolysis, and alignments with purified polymer standards. Corresponding effects on dRI and UV signals were evaluated according to the detector’s sensitivities. Using these techniques, the AF4 fractogram of beer was classified into different fractions: (1) the low molar mass fraction was assigned to proteinaceous molecules with different degrees of glycosylation, (2) the middle molar mass fraction was attributed to protein–polyphenol complexes with a coelution of non-starch polysaccharides, and (3) the high molar mass fraction was identified as a mixture of the cell wall polysaccharides (i.e., β-glucan and arabinoxylan) with a low content of polysaccharide–protein association. In addition, dextrins derived from incomplete starch hydrolysis were identified in all fractions and over the complete molar mass range. The ability to assess the components of an AF4 fractogram is beneficial for the targeted design and evaluation of polymers in fermented cereal-based beverages and for controlling and monitoring quality parameters.

A combined setup of quartz crystal microbalance and generalized ellipsometry can be used to comprehensively investigate complex functional coatings comprising stimuli-responsive polymer brushes and 3D nanostructures in a dynamic, noninvasive in situ measurement. While the quartz crystal microbalance detects the overall change in areal mass, for instance, during a swelling or adsorption process, the generalized ellipsometry data can be evaluated in terms of a layered model to distinguish between processes occurring within the intercolumnar space or on top of the anisotropic nanocolumns. Silicon films with anisotropic nanocolumnar morphology were prepared by the glancing angle deposition technique and further functionalized by grafting of poly-(acrylic acid) or poly-(N- isopropylacrylamide) chains. Investigations of the thermoresponsive swelling of the poly-(N-isopropylacrylamide) brush on the Si nanocolumns proved the successful preparation of a stimuli-responsive coating. Furthermore, the potential of these novel coatings in the field of biotechnology was explored by investigation of the adsorption of the model protein bovine serum albumin. Adsorption, retention, and desorption triggered by a change in the pH value is observed using poly-(acrylic acid) functionalized nanostructures, although generalized ellipsometry data revealed that this process occurs only on top of the nanostructures. Poly-(N-isopropylacrylamide) is found to render the nanostructures non-fouling properties.

Thermal field-flow fractionation (ThFFF) is used to separate a linear triblock copolymer of polystyrene, poly(tert-butyl acrylate), and poly(methyl methacrylate) by composition. Fractions were collected and subjected to off-line NMR analysis. The resultant mole fraction versus retention time plots for each of the three polymer components confirmed the success of the separation and yielded the composition distribution of the copolymer. The composition distribution was also obtained using a second approach that involved solving a series of equations comprised of polymer thermal diffusion coefficients and quasi-elastic light scattering, differential refractometry, and UV detector responses. Both sets of data showed similar trends of composition variations in each polymer component as a function of retention time. However, discrepancies were observed in the mole fraction values. The ability to compositionally separate and to determine composition distribution of copolymers is important as demonstrated by the presence of diblock impurities in the ThFFF with off-line NMR results.

A novel, highly selective, and sensitive resonance light scattering (RLS) detection approach coupled with high performance liquid chromatography (HPLC) was researched and developed for the synchronous analysis of three kinds of benzimidazole anthelmintics, including mebendazole (MBZ), albendazole (ABZ), and fenbendazole (FBZ) for the first time. In the pH range of 3.5-3.7 Britton-Robinson buffer medium, three kinds of anthelmintics, which were separated by HPLC, reacted with eosin Y (EY) to form 1:1 ion-association complexes, resulting in significantly enhanced RLS signals and the maximum peak located at 335 nm. The enhanced RLS intensity was in proportion to the MBZ, ABZ, and FBZ concentration in the range 0.2-25, 0.2-23, and 0.15-20 μg/mL, respectively. The limit of detection was in the range of 0.064-0.16 μg/mL. In addition, human urine was determined to validate the proposed method by spiked samples and real urine samples. Satisfactory results were obtained by HPLC-RLS method. Graphical Abstract The diagram mechanism of generating resonance between emitted light and scattered light.

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M w) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A 2) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M w of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M w values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M w results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.

The biological application of nanoparticles has resulted in an increased need for the development of robust, accurate, and precise methods for quality control analysis and characterization. Parameters such as particle size, particle shape, and their distributions affect end-use properties such as chemical reactivity, diffusivity, permeability, and transport. Introduced here is a hydrodynamic chromatography (HDC) method utilizing multi-angle static light scattering, quasi-elastic light scattering, differential viscometry, and differential refractometry detection for characterizing nanoscale vesicles. Quadruple-detector HDC was used to determine multiple sizing parameters and their statistical moments and distributions. Molar mass and molar mass averages were determined in a calibrant-independent fashion. Both the sizing parameters and the molar mass were measured across the HDC elution profile. The shape and structure of the nanoparticle were monitored as a function of HDC elution volume through the dimensionless ratio ρ≡R(G,z)/R(H,z). The HDC results were comparable to those obtained by transmission electron microscopy, but more extensive characterization was possible by HDC, which provided distributions of both particle size and particle shape.

Particle size and shape and their distribution directly influence a variety of end-use material properties related to packing, mixing, and transport of powders, solutions, and suspensions. Many of the techniques currently employed for particle size characterization have found limited applicability for broadly polydisperse and/or nonspherical particles. Here, we introduce a quadruple-detector hydrodynamic chromatography (HDC) method utilizing static multiangle light scattering (MALS), quasi-elastic light scattering (QELS), differential viscometry (VISC), and differential refractometry (DRI), and apply the technique to characterizing a series of solid and hollow polystyrene latexes with diameters in the approximate range of 40–400 nm. Using HDC/MALS/QELS/VISC/DRI, we were able to determine a multiplicity of size parameters and their polydispersity and to monitor the size of the particles across the elution profile of each sample. Using self-similarity scaling relationships between the molar mass and the various particle radii, we were also able to ascertain the shape of the latexes and the shape constancy as a function of particle size. The particle shape for each latex was confirmed by the dimensionless ratio ρ ≡ R G,z /R H,z which, in addition, provided information on the structure (compactness) of the latexes as a function of particle size. Solid and hollow polystyrene latex samples were also differentiable using these methods. Extension of this method to nonspherical, fractal objects should be possible.

UV spectrophotometric measurement is a widely accepted and standardized routine analysis for quantitation of highly purified proteins; however, the reliability of the results strictly depends on the accuracy of the employed extinction coefficients. In this work, an experimental estimation of the differential refractive index (dn/dc), based on dry weight measurements, was performed in order to determine accurate extinction coefficients for four biotherapeutic proteins and one synthetic copolymer after separation in a size-exclusion ultra-performance liquid chromatograph coupled to an ultraviolet, multiangle light scattering and refractive index (SE-UPLC-UV-MALS-RI) multidetection system. The results showed small deviations with respect to theoretical values, calculated from the specific amino acid sequences, for all the studied immunoglobulins. Nevertheless, for proteins like etanercept and glatiramer acetate, several considerations, such as glycan content, partial specific volume, polarizability, and higher order structure, should be considered to properly calculate theoretical extinction coefficient values. Herein, these values were assessed with simple approximations. The precision of the experimentally obtained extinction coefficients, and its convergence towards the theoretical values, makes them useful for characterization and comparability exercises. Also, these values provide insight into the absorbance and scattering properties of the evaluated proteins. Overall, this methodology is capable of providing accurate extinction coefficients useful for development studies.

Localized surface plasmon resonances of metallic nanoparticles can be used for biosensing because of their sensitive dependence on the refractive index of the surrounding medium. The binding of molecules to the particles causes a change of the effective refractive index in their close vicinity, which leads to a reversible shift of the resonance. We present simulations and sensing experiments of a plasmon resonance based biosensor that makes use of the narrow antisymmetric resonance in coupled plasmonic vertical dimers. The sensitivity of the antisymmetric resonance is compared with that of a surface lattice resonance for refractive index sensing of bulk and of thin layers of molecules. The functionality of such a sensor surface is demonstrated via a testosterone immunoassay for detection of antibody from a solution by binding to surface-immobilized antigen in a fluidic channel.

In order to simplify the procedure for assembling a surface-plasmon resonance (SPR) sensor, a refractive index matching polymer film was prepared as an alternative to the conventionally used matching oil. The refractive index matching polymer film, the refractive index of which was nearly equal to the prism and sensor chip material (a cover glass) of the SPR sensor, was prepared by casting a tetrahydrofuran solution of poly (vinyl chloride) (PVC) containing equal weights of dioctyl phthalate and tricresyl phosphate. The refractive index matching polymer film was found to have a refractive index of 1.516, which is identical to that of the prism and the cover glass used for the present SPR sensor. The utility of the matching polymer film for the SPR sensor was confirmed by the detection of anti-human albumin, based on an antigen-antibody reaction.

Square hollow waveguides are used to integrate measurement of absorption with chip-based electrophoresis. The 50x50 microm liquid channel and 50x50 microm waveguide are etched as a negative pattern into a silicon master and replicated as a positive in poly-dimethylsiloxane (PDMS). The uniform refractive index of the chip prevents guiding by total internal reflection. Instead, light at 488 nm is guided by reflection at the air-PDMS interface. The waveguide has a 60% efficiency over a distance of 3.2 cm. Separation of fluorescein and the dye BODIPY is demonstrated. A detection limit (S/N=3) of 200 microM fluorescein is obtained using a 50 microm pathlength and a simple photocell detector.

The thickness and refractive index of 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) monolayers Langmuir--Blodgett (LB) deposited on mica were measured in dry air and bulk water using multiple-beam interferometry (MBI). Measurements of thickness using atomic force microscopy (AFM) of identical monolayers, and X-ray reflectivity (XRR) of the monolayers on quartz were taken for comparison. The measurement of the properties of solid-supported monolayers in dry air allows lipid optical properties to be determined free from solvent effects. The thickness and refractive index measured by MBI were 25.5 ± 0.6 A and 1.485 ± 0.007 for DPPE monolayers, and 23.9 ± 0.5 A and 1.478 ± 0.006 for DPPC monolayers in dry air. These thicknesses are consistent with the other techniques used in this work as well as other measurements in the literature. The refractive indices of solid-supported lipid monolayers have not been previously measured. The values are higher than previous measurements on black lipid films done by reflectometry, which is attributed to increased lipid packing density and the absence of hydrocarbon solvents. Applying water to the monolayers had no measurable effect on their properties, indicating that any change in hydration was below detection.

We characterized ultrahigh molecular weight sodium hyaluronate (NaHA) and blended pharmaceutical products containing NaHA using flow field-flow fractionation and multiangle light scattering-differential refractive index (FlFFF-MALS-DRI). NaHA is a water-soluble polysaccharide with a range of molecular weights (MW; 10(5)~10(8) Da) that is found in body fluids and tissues. NaHA is also used commercially in pharmaceutical and cosmetic applications. We used a frit inlet asymmetrical FlFFF channel to separate aqueous polymers according to their hydrodynamic size, and we used on-line measurements of light scattering to obtain the MW distribution (MWD) as well as structural information about NaHA in aqueous solution. In this study, we investigated NaHA and anti-adhesive blend mixtures of NaHA (a commercial NaHA blend mixture containing sodium carboxymethyl cellulose and a new blend with hydroxyethyl starch (HES)) to determine the molecular weight distribution MWD of NaHA and the blend mixtures and to obtain structural information about these compounds in aqueous solution. We also examined the characteristics of NaHA-HES-polylactic-co-glycolic acid film products exposed to gamma radiation for sterilization purposes.

In biomolecular interaction analysis, direct optical detection is attracting increasing interest in academia and industry. Therefore, a previous review has been updated. Optical principles are given in brief, focussing especially on modern and frequently used techniques. Commercialized methods are listed with some specific applications. In addition, some of the many applications found in the literature are listed; others which have been reviewed elsewhere are cited. Overall, the growing interest in direct optical monitoring of biomolecular interaction is demonstrated and future trends are outlined. Because optical methods is a very wide field, the paper concentrates on the currently most common methods, microrefractometry and microreflectivity.

We present a method by which to obtain the absolute, chemical-heterogeneity-corrected molar mass (M) averages and distributions of copolymers and apply the method to a gradient random copolymer of styrene and methyl methacrylate in which the styrene percentage decreases from approximately 30% to 19% as a function of increasing molar mass. The method consists of separation by size-exclusion chromatography (SEC) with detection using multi-angle static light scattering (MALS), differential viscometry (VISC), differential refractometry (DRI), and ultraviolet absorption spectroscopy (UV) and relies on the preferential absorption of styrene over methyl methacrylate at 260 nm. Using this quadruple-detector SEC/MALS/UV/VISC/DRI approach, the percentage of styrene (%St) in each elution slice is determined. This %St is then used to determine the specific refractive index increment, corrected for chemical composition, at each elution slice, which is then used to obtain the molar mass at each slice, corrected for chemical composition. From this corrected molar mass and from the chemical-composition-corrected refractometer response, the absolute, chemical-heterogeneity-corrected molar mass averages and distribution of the copolymer are calculated. The corrected molar mass and intrinsic viscosity at each SEC elution slice are used to construct a chemical-heterogeneity-corrected Mark–Houwink plot. The slice-wise-corrected M data are used, in conjunction with the MALS-determined R G,z of each slice, to construct a conformation plot corrected for chemical heterogeneity. The corrected molar mass distribution (MMD) of the gradient copolymer extends over an approximately 30,000 g/mol wider range than the uncorrected MMD. Additionally, correction of the Mark–Houwink and conformation plots for the effects of chemical heterogeneity shows that the copolymer adopts a more compact conformation in solution than originally concluded.

The standard methods currently used to read out microarrays are fluorescent and chemiluminescent imaging techniques. These methods require labeling of a component with a marker and, usually, only the concentration of the marker molecule is detected. A label-free imaging method that also enables quantitative spectroscopic analysis of the composition and component interaction would be of great advantage. In this article it is shown for the first time that IR mapping ellipsometry enables label-free imaging of a biochip before and after incubation with peptide solution. The measurements prove that IR ellipsometry is a sensitive tool for laterally resolved identification of the different materials and determination of the composition of a biochip. The lateral resolution required was achieved by using radiation from an infrared synchrotron beamline.

Label-free sensing is an important method for many (bio-)chemical applications in fields such as biotechnology, medicine, pharma, ecology and food quality control. The broad range of applications includes liquid refractive index sensing, molecule detection, and the detection of particles or cells. Integrated optics based on the use of waveguide modes offers a great potential and flexibility to tailor the sensor properties to these applications. In this paper, the results of a numerical study are presented, showing that this flexibility is founded on the many degrees of freedom that can be used for the integrated optical chip design, in contrast to other technologies such as those based on surface plasmon resonance, for which the materials' properties limit the range of choices. The applications that are explicitly considered and discussed include (1) bulk refractometry, (2) thin-layer sensing, for example biosensors monitoring molecular adsorption processes occurring within some 10 nm of the chip's surface, (3) thick-layer sensing with processes involving molecules or ions to be monitored within a sensing matrix extending to some 100 nm from the chip's surface, for example hydrogel-based layers and chemo-optically sensitive membranes, and (4) particle sensing with particles or, for example, biological cells to be monitored within probe volumes extending to some 1,000 nm from the chip's surface. The peculiarities for the different types of applications will be discussed, and suitable modeling methods presented. Finally, the application-specific design guidelines supplied will enable the optimization of various types of integrated optical sensors, including interferometers and grating-based sensors.