Reflectometric Interference Spectroscopy Research Articles

Advances in Diagnostic Tools and Therapeutic Approaches for Gliomas: A Comprehensive Review.

Gliomas, a prevalent category of primary malignant brain tumors, pose formidable clinical challenges due to their invasive nature and limited treatment options. The current therapeutic landscape for gliomas is constrained by a "one-size-fits-all" paradigm, significantly restricting treatment efficacy. Despite the implementation of multimodal therapeutic strategies, survival rates remain disheartening. The conventional treatment approach, involving surgical resection, radiation, and chemotherapy, grapples with substantial limitations, particularly in addressing the invasive nature of gliomas. Conventional diagnostic tools, including computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), play pivotal roles in outlining tumor characteristics. However, they face limitations, such as poor biological specificity and challenges in distinguishing active tumor regions. The ongoing development of diagnostic tools and therapeutic approaches represents a multifaceted and promising frontier in the battle against this challenging brain tumor. The aim of this comprehensive review is to address recent advances in diagnostic tools and therapeutic approaches for gliomas. These innovations aim to minimize invasiveness while enabling the precise, multimodal targeting of localized gliomas. Researchers are actively developing new diagnostic tools, such as colorimetric techniques, electrochemical biosensors, optical coherence tomography, reflectometric interference spectroscopy, surface-enhanced Raman spectroscopy, and optical biosensors. These tools aim to regulate tumor progression and develop precise treatment methods for gliomas. Recent technological advancements, coupled with bioelectronic sensors, open avenues for new therapeutic modalities, minimizing invasiveness and enabling multimodal targeting with unprecedented precision. The next generation of multimodal therapeutic strategies holds potential for precision medicine, aiding the early detection and effective management of solid brain tumors. These innovations offer promise in adopting precision medicine methodologies, enabling early disease detection, and improving solid brain tumor management. This review comprehensively recognizes the critical role of pioneering therapeutic interventions, holding significant potential to revolutionize brain tumor therapeutics.

(R)evolution of the Standard Addition Procedure for Immunoassays.

A new method to transfer the standard addition procedure for concentration determination to immunoassays with non-linear calibration curves was developed. The new method was successfully applied to simulated data and benchmarked against a state-of-the-art algorithm, showing a significantly improved performance with improvement factors between 2 and 192. The logit function was used to transform the immunoassay signal response of test samples spiked with known analyte concentrations. The relationship between logit(signal) and log-transformed estimated total analyte concentration is linear if the estimated total analyte concentration is correct. Finally, the new method was validated experimentally using different assays in varying, relevant complex matrices, such as serum, saliva, and milk. Different concentrations of testosterone and amitriptyline between 0.05 and 3.0 µg L-1 were quantified using a binding inhibition assay in combination with reflectometric interference spectroscopy (RIfS) as the transduction principle. The sample concentration was calculated using a numerical method. Samples could be quantified with recoveries between 70 and 118%. The standard addition method accounts for individual matrix interference on the immunoassay by spiking the test sample itself. Although the experiments were carried out using RIfS, the method can be applied to any immunoassay that meets the analytical requirements.

Overview of Optical Biosensors for Early Cancer Detection: Fundamentals, Applications and Future Perspectives

Conventional cancer detection and treatment methodologies are based on surgical, chemical and radiational processes, which are expensive, time consuming and painful. Therefore, great interest has been directed toward developing sensitive, inexpensive and rapid techniques for early cancer detection. Optical biosensors have advantages in terms of high sensitivity and being label free with a compact size. In this review paper, the state of the art of optical biosensors for early cancer detection is presented in detail. The basic idea, sensitivity analysis, advantages and limitations of the optical biosensors are discussed. This includes optical biosensors based on plasmonic waveguides, photonic crystal fibers, slot waveguides and metamaterials. Further, the traditional optical methods, such as the colorimetric technique, optical coherence tomography, surface-enhanced Raman spectroscopy and reflectometric interference spectroscopy, are addressed.

Bare Eye Detection of Bacterial Enzymes of Pseudomonas aeruginosa with Polymer Modified Nanoporous Silicon Rugate Filters

The fabrication, characterization and application of a nanoporous Silicon Rugate Filter (pSiRF) loaded with an enzymatically degradable polymer is reported as a bare eye detection optical sensor for enzymes of pathogenic bacteria, which is devoid of any dyes. The nanopores of pSiRF were filled with poly(lactic acid) (PLA), which, upon enzymatic degradation, resulted in a change in the effective refractive index of the pSiRF film, leading to a readily discernible color change of the sensor. The shifts in the characteristic fringe patterns before and after the enzymatic reaction were analyzed quantitatively by Reflectometric Interference Spectroscopy (RIfS) to estimate the apparent kinetics and its dependence on enzyme concentration. A clear color change from green to blue was observed by the bare eye after PLA degradation by proteinase K. Moreover, the color change was further confirmed in measurements in bacterial suspensions of the pathogen Pseudomonas aeruginosa (PAO1) as well as in situ in the corresponding bacterial supernatants. This study highlights the potential of the approach in point of care bacteria detection.

Real-time detection of per-fluoroalkyl substance (PFAS) self-assembled monolayers in nanoporous interferometers

Identification and quantification of per- and polyfluoroalkyl substances (PFASs) remain challenging due to their chemical diversity, and their inert optical and chemical nature. Here, we present an optical system integrating perfluorosilane-functionalized nanoporous anodic alumina (NAA) interferometers with reflectometric interference spectroscopy (RIfS) for real-time, label-free detection of self-assembled monolayers (SAMs) of perfluorooctanoic acid (PFOA) as a model PFAS. Measured changes in the effective optical thickness (ΔOTeff) of NAA interferometers made it possible to study the fluorous interaction-induced self-assembly of PFOA molecules with perfluorosilane functional molecules of varying length, in real time and in situ. Analysis of key sensing parameters—sensitivity, low limit of detection and linearity—allowed us to determine the most optimal molecular length of perfluorosilanes to maximize immobilization of PFOA onto functional surfaces. Freundlich and Langmuir isotherm models were adapted to experimentally acquired values of ΔOTeff to elucidate the mechanism of PFOA–perfluorosilane interactions. Interpretation of these models suggests that PFOA binds to perfluorosilanes functional groups immobilized onto the inner surface of NAA interferometers through a fluorous interaction-induced Freundlich mechanism. The potential real-life applicability of this system was demonstrated by detecting the formation of PFOA-based SAMs in aqueous matrices of varying complexity (i.e. ultrapure, deionized, tap, and river water). This study provides new insights into how functional surface chemistries can be engineered to maximize sensitivity and selectivity to PFAS, harnessing fluorous interactions—with implications for future deployable systems to detect and remove these emerging toxicants.

Comparison of methods for quantitative biomolecular interaction analysis

In order to perform good kinetic experiments, not only the experimental conditions have to be optimized, but the evaluation procedure as well. The focus of this work is the in-depth comparison of different approaches and algorithms to determine kinetic rate constants for biomolecular interaction analysis (BIA). The different algorithms are applied not only to flawless simulated data, but also to real-world measurements. We compare five mathematical approaches for the evaluation of binding curves following pseudo-first-order kinetics with different noise levels. In addition, reflectometric interference spectroscopy (RIfS) measurements of two antibodies are evaluated to determine their binding kinetics. The advantages and disadvantages of the individual approach will be investigated and discussed in detail. In summary, we will raise awareness on how to evaluate and judge results from BIA by using different approaches rather than having to rely on “black box” closed (commercial) software packages.

Advanced Nanoporous Anodic Alumina-Based Optical Sensors for Biomedical Applications

Close-packed hexagonal array nanopores are widely used both in research and industry. A self-ordered nanoporous structure makes anodic aluminum oxide (AAO) one of the most popular nanomaterials. This paper describes the main formation mechanisms for AAO, the AAO fabrication process, and optical sensor applications. The paper is focused on four types of AAO-based optical biosensor technology: surface-Enhanced Raman Scattering (SERS), surface Plasmon Resonance (SPR), reflectometric Interference Spectroscopy (RIfS), and photoluminescence Spectroscopy (PL). AAO-based optical biosensors feature very good selectivity, specificity, and reusability.

Monitoring of Binding Affinity Between Drugs and Human Serum Albumin Using Reflectometric Interference Spectroscopy with Silica Colloidal Crystal Films

When a drug enters an organism, interactions between the drug and proteins in the organism play a vital role in the storage, transport and metabolism of the drug and also affect its nonspecific toxicity, targeting and pharmacodynamic activity. However, monitoring the interaction process is a great challenge in the research of the absorption, transport and metabolic processes of drugs. In this study, we used reflectometric interference spectroscopy (RIfS) and silica colloidal crystal (SCC) film as a sensing platform to detect the binding affinity between human serum albumin (HSA) and indomethacin. SCC films composed of three silica nanospheres with different diameters were fabricated using the vertical evaporation method. HSA was immobilized covalently on SCC film using a very simple approach, and optical thickness was used as a parameter to evaluate the process of drug absorption and desorption. Finally, the optimal SCC film was selected, and three drugs other than indomethacin (i.e., warfarin, salicylic acid and quinine) were used for the validation of this sensing platform. The results verified that SCC film using RIfS is a simple and real-time sensing platform for detecting the affinity between HSA and drugs, which may be widely used in drug development and clinical testing in the future.

A Novel Protein Corona Characterization based on the Reflectometric Interference Spectroscopy with Silica Colloidal Crystal Films

A Novel Protein Corona Characterization based on the Reflectometric Interference Spectroscopy with Silica Colloidal Crystal Films

Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically ‘open’ and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

Inflammatory biomarker detection in milk using label-free porous SiO2 interferometer

N-acetyl-β-d-glucosaminidase (NAGase) is an established indicative biomarker released upon damage or necrosis of tubular epithelial cells in both humans and animals, indicating severe nephrological disorders and bovine mastitis (BM), respectively. The latter is the most common and costly disease in dairy cattle associated with production losses, elevated somatic cell counts and deteriorated health status. Herein, we report on a reflective based assay for early diagnosis of BM through the analysis of NAGase inherent content found in whole milk samples using a miniaturized optical transducer. Gelatin functionalized porous Si Fabry–Pérot interferometers are employed for monitoring the lysosomal activity in various stages of the inflammation (healthy, subclinical and clinical). The enzymatic reaction products precipitate and accumulate within the porous nanostructure, thus alter the average refractive index monitored using reflectometric interference spectroscopy. The optical assay is calibrated within the clinically relevant concentrations of BM while presenting a dynamic range of 1.04–16.7 μM min−1 and the detection limit of 0.49 μM min−1. The specific optical performance of the biosensor correlates with a gold standard laboratory-based approach, in which escalated somatic cell counts reveal augmented NAGase levels and thus severe pathogenesis. Overall, our study provides new opportunities to develop a convenient bio-diagnostic sensing system for BM detection and classification by addressing the limitations of conventional practices.

In Situ Study of Layer-by-Layer Polyelectrolyte Deposition in Nanopores of Anodic Aluminum Oxide by Reflectometric Interference Spectroscopy

The modification of cylindrical anodic aluminum oxide (AAO) nanopores by alternating layer-by-layer (LBL) deposition of poly(sodium-4-styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) was studied in situ by reflectometric interference spectroscopy (RIfS). In particular, the kinetics of polyelectrolyte deposition inside the pores with a diameter of 37 ± 3 nm and a length of 3.7 ± 0.3 μm were unraveled, and potential differences in the LBL multilayer growth compared to flat silicon substrates as well as the effect of different ionic strengths and different types of ions were investigated. RIfS measures the effective optical thicknesses, which is-for a constant pore length-proportional to the effective refractive index of the AAO sample, from which, in turn, the deposited mass of the polymer or the corresponding layer thickness can be estimated. Compared to the multilayer growth by the LBL deposition on the flat aminosilane-primed silicon wafers, which was assessed by spectroscopic ellipsometry, the thickness increment per deposited bilayer, as well as the dependence of this increment on the ionic strength (0.01-0.15) and the counterion type (Na+ vs Ca2+) inside the aminosilane-primed nanopores, was for the first bilayers to within the experimental error identical. For thicker multilayers, the pore diameter became smaller, which led to reduced thickness increments and eventually virtually completely filled the pores. The observed kinetics is consistent with the mass-transport-limited adsorption of the polyelectrolyte to the charged surface according to a Langmuir isotherm with a negligible desorption rate. In addition to fundamental insights into the buildup of polyelectrolyte multilayers inside the AAO nanopores, our results highlight the sensitivity of RIfS and its use as an analytical tool for probing processes inside the nanopores and for the development of biosensors.

Leaflet-Dependent Distribution of PtdIns[4,5]P2 in Supported Model Membranes.

Supported planar lipid bilayers (SLBs) prepared by spreading of unilamellar vesicles on hydrophilic substrates such as silicon dioxide are frequently used to investigate lipid-protein interactions by means of surface-sensitive methods. In recent years, the receptor lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) became particularly important as a significant number of proteins bind to this lipid at the inner leaflet of the plasma membrane. Here, we investigated how the lipid PtdIns[4,5]P2 distributes between the two leaflets of an SLB on SiO2 surfaces. We prepared SLBs on SiO2 by spreading small unilamellar vesicles and quantified the adsorption of PtdIns[4,5]P2 binding proteins providing information about the accessibility of PtdIns[4,5]P2. We compared protein binding to PtdIns[4,5]P2 in SLBs with that in lipid monolayers on a 1,1,1-trimethyl-N-(trimethylsilyl)silanamine-functionalized SiO2 surface using reflectometric interference spectroscopy and atomic force microscopy. Our results clearly demonstrate that the accessibility of PtdIns[4,5]P2 for protein binding is reduced in SLBs compared to that in supported hybrid membranes, which is discussed in terms of PtdIns[4,5]P2 distribution in the two leaflets of SLBs.

Fast optical method for characterizing plasmonic nanoparticle adhesion on functionalized surfaces

In this paper, a rapid optical method for characterizing plasmonic (gold) nanoparticle (AuNP) adhesion is presented. Two different methods were used for AuNP preparation: the well-known Turkevich method resulted in particles with negative surface charge; for preparing AuNPs with positive surface charge, stainless steel was used as reducing agent. The solid surface for adhesion was provided by a column packed with pristine or surface-modified glass beads. The size of the nanoparticles was studied by transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS); the surface charge of the components was determined by streaming potential measurements. The characterization of adhesion was performed in a flow system by UV-Vis spectroscopy. During the adhesion experiments, the role of the surface charge, the particle size, and the pH were studied, as well as the adhered amount of gold nanoparticles and the surface coverage values. The latter was estimated by theoretical calculations and defined by the quotient of the measured and the maximal adhered amount of nanoparticles, which could be determined by the cross-sectional area of the NPs and the specific surface area of the glass beads. The results are verified by the polarization reflectometric interference spectroscopy (PRIfS) method: silica nanoparticles with diameters of a few hundred (d~450) nanometers were immobilized on the surface of glass substrate by the Langmuir–Blodgett method, the surface was modified similar to the 3D (continuous flow packed column) system, and gold nanoparticles from different pH solutions were adhered during the measurements. These kinds of modified surfaces allow the investigation of biomolecule adsorption in the same reflectometric setup.Graphical abstract

The Theoretical Concept of Polarization Reflectometric Interference Spectroscopy (PRIFS): An Optical Method to Monitor Molecule Adsorption and Nanoparticle Adhesion on the Surface of Thin Films

In this paper, we present an improved reflectometric interference spectroscopy (RIfS) sensor principle which is suitable for thin films. The conventional RIfS technique is an appropriate method to detect interfacial interactions at the solid–gas or solid–liquid interface in the case of thin films with a thickness of a few hundred nanometers, but when a significantly lower layer thickness (~100 nm) is required, the method is barely usable. By applying polarized reflected light and monitoring the ratio of the p- and s-polarized components, a characteristic curve can be obtained with one or a few local extreme value(s) with significantly favorable intensity ratios compared to the conventional method. In this work we studied the effect of film thickness, incident angle and the refractive indices of the thin film, the medium and the substrate. As a main result, it was demonstrated that the sensitivity of the PRIfS method is 4–7 times higher than that of the conventional technique near a critical angle. In simulated adsorption experiments, it was determined that the sensitivity of RIfS is around 550 nm/RIU (refractive index unit), while it is 1825 and 3966 nm/RIU for PRIfS in gas and aqueous phase, respectively.

Label-free detecting oligonucleotide hybridization melting temperature in real-time with a reflectometric interference spectroscopy–based nanosensor system

The accurate determination of melting temperature (Tm) of oligonucleotide hybridization is the primary for various biosensors based on the technique of using surface-tethered nucleic acids as probes. In this paper, a new type of nanosensor based on the reflectometric interference spectroscopy (RIFS) was proposed to determine the Tm in real time. A composite solid nanostructure, i.e., a nanoporous anodic alumina film with a 15 nm top layer of gold sputtered on surface, was employed as the substrate of the RIFS-based nanosensor, and a 30 bp oligonucleotide molecule was bond to the inner wall of pores and hybridized, and then its Tm was detected with raising temperature. The experimental data showed that there were obvious signs of dsDNA molecules denatured and melted into ssDNA molecules at 95 ℃, which was highly consistent with the theoretical analysis. The cost-effective RIFS nanosensor has excellent thermal and physicochemical stabilities, higher determination accuracy, and great potential in the field of biomolecule research.

Silicon nitride sugar chips for detection of Ricinus communis proteins and Escherichia coli O157 Shiga toxins

Lactosides having either an amino-triethylene glycol or an azido-triethylene glycol were designed and synthesized, and the two derivatives were immobilized onto silicon nitride (SiN) surfaces. When a click reaction was applied for the immobilization of the azido-sugar, a Ricinus communis lectin (RCA120) was detected with a higher response by reflectometric interference spectroscopy (RIfS). When an N-hydroxysuccinimide (NHS) method was applied for the sugar immobilization, the response was less than that of the click one. The response of bovine serum albumin (BSA) as the negative control was negligible, but the lactose-SiN chip prepared by the click method suppressed nonspecific binding more effectively than did the chip from the NHS method. Next, we examined an antibody-immobilized SiN chip prepared by the click reaction. The detection response was, however, lower than that of the lactose-SiN chip, meaning that the sugar-chip by the click reaction was superior to the antibody-chip. Finally, to detect Shiga toxins from Escherichia coli O157:H7, globotrisaccharide (Gb3) with an azido-triethylene glycol was synthesized and immobilized onto the SiN chip by the click reaction. The Gb3-SiN chips enabled us to detect the toxins at concentrations less than 100 ng/mL. RCA120, horse gram, gorse lectins and BSA showed no response to the Gb3-SiN chip, showing a high specificity for the toxin.

Interference Effect of Silica Colloidal Crystal Films and Their Applications to Biosensing.

With the aim to develop better and more reliable interference effective substrates, silica colloidal crystal films with different sphere diameters and film thicknesses were successfully made by an improved vertical deposition method and a systematic investigation of their reflectometric interference spectroscopy (RIfS) properties are presented in this work. The influence of silica sphere diameter and film thickness on the RIfS signals was studied. The results showed that the film thickness is the key factor of RIfS signals. An RIfS system was set up by using a silica colloidal crystal film as an interference effective substrate. The influence of film thickness on the response to refractive index changes of the proposed system was also investigated. When the influence of film thickness on RIfS signals and refractive index response we considered together, silica colloidal crystal films with a thickness between 4 and 6 μm were chosen for sensor construction. Monitoring the digestive process of gelatin with trypsin was also demonstrated by combining gelatin-modified silica colloidal crystal films with RIfS. The system showed excellent sensitivity with a wide linear range and could achieve real-time measurement of each process. It has been proved that this is a promising method to construct biosensors using silica colloidal crystal films as interference-sensitive substrates.

Environmental Copper Sensor Based on Polyethylenimine-Functionalized Nanoporous Anodic Alumina Interferometers.

Anthropogenic copper pollution of environmental waters from sources such as acid mine drainage, antifouling paints, and industrial waste discharge is a major threat to our environment and human health. This study presents an optical sensing system that combines self-assembled glutaraldehyde-cross-linked double-layered polyethylenimine (PEI-GA-PEI)-modified nanoporous anodic alumina (NAA) interferometers with reflectometric interference spectroscopy (RIfS) for label-free, selective monitoring of ionic copper in environmental waters. Calibration of the sensing system with analytical solutions of copper shows a linear working range between 1 and 100 mg L-1, and a low limit of detection of 0.007 ± 0.001 mg L-1 (i.e., ∼0.007 ppm). Changes in the effective optical thickness (ΔOTeff) of PEI-GA-PEI-functionalized NAA interferometers are monitored in real-time by RIfS, and correlated with the amount of ionic copper present in aqueous solutions. The system performance is validated through X-ray photoelectron spectroscopy (XPS) and the spatial distribution of copper within the nanoporous films is characterized by time-of-flight-secondary ion mass spectroscopy (TOF-SIMS). The specificity and chemical selectivity of the PEI-GA-PEI-NAA sensor to Cu2+ ions is verified by screening six different metal ion solutions containing potentially interfering ions such as Al3+, Cd2+, Fe3+, Pb2+, Ni2+, and Zn2+. Finally, the performance of the PEI-GA-PEI-NAA sensor for real-life applications is demonstrated using legacy acid mine drainage liquid and tap water for qualitative and quantitative detection of copper ions. This study provides new opportunities to develop portable, cost-competitive, and ultrasensitive sensing systems for real-life environmental applications.

Fluorimetric Detection of G-Quadruplex DNA in Solution and Adsorbed on Surfaces with a Selective Trinuclear Cyanine Dye.

Quadruplex DNA, which is a relevant target for anticancer therapies, may alter its conformation because of interactions with interfaces. In pursuit of a versatile methodology to probe adsorption-induced conformational changes, the interaction between a fluorescent [2.2.2]heptamethinecyanine dye and quadruplex DNA (G4-DNA) was studied in solution and on surfaces. In solution, the cyanine dye exhibits a strong light-up effect upon the association with G4-DNA without interference from double-stranded DNA. In addition, a terminal π-stacking as a binding mode between the cyanine dye and G4-DNA is concluded using NMR spectroscopy. To unravel the effects of adsorption on the conformation of quadruplex-DNA, G4-DNA, and double-stranded and single-stranded DNA were adsorbed to positively charged poly(allylamine) hydrochloride (PAH) surfaces, both in planar and in constrained 55 nm diameter aluminum oxide nanopore formats. All DNA forms showed a very strong affinity to the PAH surfaces as shown by surface plasmon resonance and reflectometric interference spectroscopy. The significant increase of the fluorescence emission intensity of the cyanine light-up probe observed exclusively for surface immobilized G4-DNA affords evidence for the adsorption of G4-DNA on PAH with retained quadruplex conformation.