FO-SPR Model for Full-Spectrum Signal Analysis of Back-reflecting FO-SPR Sensors to Monitor MOF Deposition.

In this study, we explore the full-spectrum capabilities of fiber-optic surface plasmon resonance (FO-SPR) for analyzing heterogeneous samples with increased comprehensiveness. Our approach involves refining a literature-derived FO-SPR model to more precisely reflect experimental data obtained using a back-reflecting sensor configuration. Key enhancements in our model include adjustments to the thickness and permittivity of the gold SPR-active layer on the FO-SPR sensor as well as improvements to the angular distribution of light within the system. We apply this optimized model to the investigation of the deposition process of a metal-organic framework (MOF), specifically ZIF-8, using FO-SPR. By closely examining the temporal variations in the FO-SPR signal during MOF layer formation, we simultaneously determine the evolving thickness and refractive index (RI) of the MOF layer, offering a dual-parameter analysis. Our results demonstrate that a full-spectrum analysis of the FO-SPR signal can extract critical information from samples exhibiting radial heterogeneity. This advancement significantly enhances the quantitative assessment of various phenomena that alter the refractive index in the sensor's domain, such as adsorption and binding processes. This work thus represents a significant step forward in the field of FO-SPR sensor technology, promising broad applications in areas requiring the precise detection and analysis of complex samples.

Integrated Signal Amplification on a Fiber Optic SPR Sensor Using Duplexed Aptamers.

Throughout the past decades, fiber optic surface plasmon resonance (FO-SPR)-based biosensors have proven to be powerful tools for both the characterization of biomolecular interactions and target detection. However, as FO-SPR signals are generally related to the mass that binds to the sensor surface, multistep processes and external reagents are often required to obtain significant signals for low molecular weight targets. This increases the time, cost, and complexity of the respective bioassays and hinders continuous measurements. To overcome these requirements, in this work, cis-duplexed aptamers (DAs) were implemented on FO-SPR sensors, which underwent a conformational change upon target binding. This induced a spatial redistribution of gold nanoparticles (AuNPs) upon specific target binding and resulted in an amplified and concentration-dependent signal. Importantly, the AuNPs were covalently conjugated to the sensor, so the principle does not rely on multistep processes or external reagents. To implement this concept, first, the thickness of the gold fiber coating was adapted to match the resonance conditions of the surface plasmons present on the FO-SPR sensors with those on the AuNPs. As a result, the signal obtained due to the spatial redistribution of the AuNPs was amplified by a factor of 3 compared to the most commonly used thickness. Subsequently, the cis-DAs were successfully implemented on the FO-SPR sensors, and it was demonstrated that the DA-based FO-SPR sensors could specifically and quantitatively detect an ssDNA target with a detection limit of 230 nM. Furthermore, the redistribution of the AuNPs was proven to be reversible, which is an important prerequisite for continuous measurements. Altogether, the established DA-based FO-SPR bioassay holds much promise for the detection of low molecular weight targets in the future and opens up possibilities for FO-SPR-based continuous biosensing.

Paving the way towards continuous biosensing by implementing affinity-based nanoswitches on state-dependent readout platforms.

Continuous biosensors provide real-time information about biochemical processes occurring in the environment of interest and are therefore highly desirable in research, diagnostics and industrial settings. Although remarkable progress has been made in the field of biosensing, most biosensors still rely on batch processes and, thus, are not suited to perform continuous measurements. Recently, however, it has been shown that by combining affinity-based nanoswitches with state-dependent readout platforms, the necessity for batch processes can be overcome and affinity-based continuous biosensing can be achieved. In this review, we first provide an overview of affinity-based continuous biosensing and discuss the required components to achieve this goal. More specifically, we summarize the strategies that have been applied to develop and tune both protein and nucleic acid-based switches, as well as readout strategies that can be applied in combination with the former. Afterwards, biosensors in which both elements were already integrated and hence enabled continuous measurements are reviewed. We also discuss the challenges and opportunities associated with each approach and therefore believe this review can help to encourage and guide future research towards continuous biosensing.

A Versatile One-Step Competitive Fiber Optic Surface Plasmon Resonance Bioassay Enabled by DNA Nanotechnology.

Fiber optic surface plasmon resonance (FO-SPR)-based biosensors have emerged as powerful tools for biomarker detection due to their ability for real-time analysis of biomolecular interactions, cost-effectiveness, and user-friendliness. However, as (FO-)SPR signals are determined by the mass of the target molecules, the detection of low-molecular-weight targets remains challenging and currently requires tedious labeling and preparation steps. Therefore, in this work, we established a new concept for low-molecular-weight target detection by implementing duplexed aptamers on an FO-SPR sensor. In this manner, we enabled one-step competitive detection and could achieve significant signals, independent of the weight of the target molecules, without requiring labeling or preprocessing steps. This was demonstrated for the detection of a small molecule (ATP), protein (thrombin), and ssDNA target, thereby reaching detection limits of 72 µM, 36 nM, and 30 nM respectively and proving the generalizability of the proposed bioassay. Furthermore, target detection was successfully achieved in 10-fold diluted plasma, which demonstrated the applicability of the assay in biologically relevant matrices. Altogether, the developed one-step competitive FO-SPR bioassay opens up possibilities for the detection of low-molecular-weight targets in a fast and straightforward manner.

Unraveling the effect of the aptamer complementary element on the performance of duplexed aptamers: a thermodynamic study.

Duplexed aptamers (DAs) are widespread aptasensor formats that simultaneously recognize and signal the concentration of target molecules. They are composed of an aptamer and aptamer complementary element (ACE) which consists of a short oligonucleotide that partially inhibits the aptamer sequence. Although the design principles to engineer DAs are straightforward, the tailored development of DAs for a particular target is currently based on trial and error due to limited knowledge of how the ACE sequence affects the final performance of DA biosensors. Therefore, we have established a thermodynamic model describing the influence of the ACE on the performance of DAs applied in equilibrium assays and demonstrated that this relationship can be described by the binding strength between the aptamer and ACE. To validate our theoretical findings, the model was applied to the 29-mer anti-thrombin aptamer as a case study, and an experimental relation between the aptamer-ACE binding strength and performance of DAs was established. The obtained results indicated that our proposed model could accurately describe the effect of the ACE sequence on the performance of the established DAs for thrombin detection, applied for equilibrium assays. Furthermore, to characterize the binding strength between the aptamer and ACEs evaluated in this work, a set of fitting equations was derived which enables thermodynamic characterization of DNA-based interactions through thermal denaturation experiments, thereby overcoming the limitations of current predictive software and chemical denaturation experiments. Altogether, this work encourages the development, characterization, and use of DAs in the field of biosensing.

Advancements in SPR biosensing technology: An overview of recent trends in smart layers design, multiplexing concepts, continuous monitoring and in vivo sensing.

Inspired by the rapid progress and existing limitations in surface plasmon resonance (SPR) biosensing technology, we have summarized the recent trends in the fields of both chip-SPR and fiber optic (FO)-SPR biosensors during the past five years, primarily regarding smart layers design, multiplexing, continuous monitoring and in vivo sensing. Versatile surface chemistries, biomaterials and nanomaterials have been utilized thus far to generate smart layers on SPR platforms and as such achieve oriented immobilization of bioreceptors, improved fouling resistance and sensitivity enhancement, collectively aiming to improve the biosensing performance. Furthermore, often driven by the desires for time- and cost-effective quantification of multiple targets in a single measurement, efforts have been made to implement multiplex bioassays on SPR platforms. While this aspect largely remains difficult to attain, numerous alternative strategies arose for obtaining parallel analysis of multiple analytes in one single device. Additionally, one of the upcoming challenges in this field will be to succeed in using SPR platforms for continuous measurements and in vivo sensing, and as such match up other biosensing platforms where these goals have been already conquered. Overall, this review will give insight into multiple possibilities that have become available over the years for boosting the performance of SPR biosensors. However, because combining them all into one optimal sensor is practically not feasible, the final application needs to be considered while designing an SPR biosensor, as this will determine the requirements of the bioassay and will thus help in selecting the essential elements from the recent progress made in SPR sensing.

DNA-only, microwell-based bioassay for multiplex nucleic acid detection with single base-pair resolution using MNAzymes.

In disease diagnostics, single- and multiplex nucleic acid (NA) detection, with the potential to discriminate mutated strands, is of paramount importance. Current techniques that rely on target amplification or protein-enzyme based signal amplification are highly relevant, yet still plagued by diverse drawbacks including erroneous target amplification, and the limited stability of protein enzymes. As a solution, we present a multicomponent nucleic acid enzymes (MNAzymes)-based system for singleplex and multiplex detection of NA targets in microwells down to femtomolar (fM) concentrations, without the need for any target amplification or protein enzymes, while operating at room temperature and with single base-pair resolution. After successful validation of the MNAzymes in solution, their performance was further verified on beads in bulk and in femtoliter-sized microwells. The latter is not only a highly simplified system compared to previous microwell-based bioassays but, with the detection limit of 180 fM, it is to-date the most sensitive NAzyme-mediated, bead-based approach, that does not rely on target amplification or any additional signal amplification strategies. Furthermore, we demonstrated, for the first time, multiplexed target detection in microwells, both from buffer and nasopharyngeal swab samples, and presented superior single base-pair resolution of this assay. Because of the design flexibility of MNAzymes and direct demonstration in swab samples, this system holds great promise for multiplexed detection in other clinically relevant matrices without the need for any additional NA or protein components. Moreover, these findings open up the potential for the development of next-generation, protein-free diagnostic tools, including digital assays with single-molecule resolution.

Re-engineering 10-23 core DNA- and MNAzymes for applications at standard room temperature.

DNA- and MNAzymes are nucleic acid-based enzymes (NAzymes), which infiltrated the otherwise protein-rich field of enzymology three decades ago. The 10-23 core NAzymes are one of the most widely used and well-characterized NAzymes, but often require elevated working temperatures or additional complex modifications for implementation at standard room temperatures. Here, we present a generally applicable method, based on thermodynamic principles governing hybridization, to re-engineer the existing 10-23 core NAzymes for use at 23 °C. To establish this, we first assessed the activity of conventional NAzymes in the presence of cleavable and non-cleavable substrate at 23 °C as well as over a temperature gradient. These tests pointed towards a non-catalytic mechanism of signal generation at 23 °C, suggesting that conventional NAzymes are not suited for use at this temperature. Following this, several novel NAzyme-substrate complexes were re-engineered from the conventional ones and screened for their performance at 23 °C. The complex with substrate and substrate-binding arms of the NAzymes shortened by four nucleotides on each terminus demonstrated efficient catalytic activity at 23 °C. This has been further validated over a dilution of enzymes or enzyme components, revealing their superior performance at 23 °C compared to the conventional 10-23 core NAzymes at their standard operating temperature of 55 °C. Finally, the proposed approach was applied to successfully re-engineer three other new MNAzymes for activity at 23 °C. As such, these re-engineered NAzymes present a remarkable addition to the field by further widening the diverse repertoire of NAzyme applications.