Combining plasmonic and electrochemical biosensing methods.

The idea of combining electrochemical (EC) and plasmonic biosensor methods was introduced almost thirty years ago and the potential of electrochemical-plasmonic (EC-P) biosensors has been highlighted ever since. Despite that, the use of EC-P biosensors in analytics has been rather limited so far and the search for unique applications of the EC-P method continues. In this paper, we review the advances in the field of EC-P biosensors and discuss the features and benefits they can provide. In addition, we identify the main challenges for the development of EC-P biosensors and the limitations that prevent EC-P biosensors from more widespread use. Finally, we review applications of EC-P biosensors for the investigation and quantification of biomolecules, and for the study of biomolecular and cellular processes.

Linking aberrant glycosylation of plasma glycoproteins with progression of myelodysplastic syndromes: a study based on plasmonic biosensor and lectin array.

Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease-myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.

Using surface plasmon resonance, capillary electrophoresis and diffusion-ordered NMR spectroscopy to study drug release kinetics.

Nanomedicines, including polymer nanocarriers with controlled drug release, are considered next-generation therapeutics with advanced therapeutic properties and reduced side effects. To develop safe and efficient nanomedicines, it is crucial to precisely determine the drug release kinetics. Herein, we present application of analytical methods, i.e., surface plasmon resonance biosensor technology (SPR), capillary electrophoresis, and 1H diffusion-ordered nuclear magnetic resonance spectroscopy, which were innovatively applied for drug release determination. The methods were optimised to quantify the pH-triggered release of three structurally different drugs from a polymer carrier. The suitability of these methods for drug release characterisation was evaluated and compared using several parameters including applicability for diverse samples, the biological relevance of the experimental setup, method complexity, and the analysis outcome. The SPR method was the most universal method for the evaluation of diverse drug molecule release allowing continuous observation in the flow-through setting and requiring a small amount of sample.

Performance of label-free optical biosensors: What is figure of merit (not) telling us?

Here we study the analytical performance of label-free optical biosensors with respect to analyte-induced refractive index changes that can be measured by a biosensor (refractive index resolution). We present an analytical model that interrelates the refractive index resolution and the parameters of the optical platform of a biosensor. We demonstrate that the figure of merit (FOM), which has been widely used to design optical platforms of label-free optical biosensors, is not an appropriate metric to guide the design or predict the performance of label-free optical biosensors. Therefore, we propose an extended definition of FOM that addresses its limitations. We confirm the validity of the proposed approach by both numerical simulations and experiments. Finally, we show that the analytical model of the refractive index resolution not only makes it possible to predict the performance of a biosensor but also provides strategies for achieving optimal performance.

Advances and applications of nanophotonic biosensors.

Nanophotonic devices, which control light in subwavelength volumes and enhance light-matter interactions, have opened up exciting prospects for biosensing. Numerous nanophotonic biosensors have emerged to address the limitations of the current bioanalytical methods in terms of sensitivity, throughput, ease-of-use and miniaturization. In this Review, we provide an overview of the recent developments of label-free nanophotonic biosensors using evanescent-field-based sensing with plasmon resonances in metals and Mie resonances in dielectrics. We highlight the prospects of achieving an improved sensor performance and added functionalities by leveraging nanostructures and on-chip and optoelectronic integration, as well as microfluidics, biochemistry and data science toolkits. We also discuss open challenges in nanophotonic biosensing, such as reducing the overall cost and handling of complex biological samples, and provide an outlook for future opportunities to improve these technologies and thereby increase their impact in terms of improving health and safety.

Anchored linear oligonucleotides: the effective tool for the real-time measurement of uracil DNA glycosylase activity.

Base excision repair is one of the important DNA repair mechanisms in cells. The fundamental role in this complex process is played by DNA glycosylases. Here, we present a novel approach for the real-time measurement of uracil DNA glycosylase activity, which employs selected oligonucleotides immobilized on the surface of magnetic nanoparticles and Förster resonance energy transfer. We also show that the approach can be performed by surface plasmon resonance sensor technology. We demonstrate that the immobilization of oligonucleotides provides much more reliable data than the free oligonucleotides including molecular beacons. Moreover, our results show that the method provides the possibility to address the relationship between the efficiency of uracil DNA glycosylase activity and the arrangement of the used oligonucleotide probes. For instance, the introduction of the nick into oligonucleotide containing the target base (uracil) resulted in the substantial decrease of uracil DNA glycosylase activity of both the bacterial glycosylase and glycosylases naturally present in nuclear lysates.

Detecting attomolar concentrations of microRNA related to myelodysplastic syndromes in blood plasma using a novel sandwich assay with nanoparticle release.

Microribonucleic acids (miRNAs) are short noncoding ribonucleic acids that have been linked with a multitude of human diseases including lung, breast, and hematological cancers. In this work, we present a novel, extremely sensitive assay for the label-free optical biosensor-based detection of miRNAs, which is based on the oligonucleotide-triggered release of nanoparticles from a sensor surface. We combine this assay (herein referred to as the nanoparticle-release (NPR) assay) with a surface plasmon resonance biosensor and show that the assay is able to enhance the specific sensor response associated with the binding of target miRNA while suppressing the interfering effects caused by the non-specific binding. We apply the assay to the detection of miRNAs related to myelodysplastic syndromes (miR-125b, miR-16) in blood plasma and demonstrate that the assay enables detection of miR-125b with a limit of detection (LOD) of 349 aM (corresponding to the lowest detectable amounts of 419 zmol). The achieved LOD is better by a factor of ∼100 when compared to the conventional nanoparticle-enhanced sandwich assay. Moreover, we demonstrate that the NPR assay may be combined with time-division multiplexing for the multiplexed miRNA detection.

Study of Biomolecular Interactions of Mitochondrial Proteins Related to Alzheimer's Disease: Toward Multi-Interaction Biomolecular Processes.

Progressive mitochondrial dysfunction due to the accumulation of amyloid beta (Aß) peptide within the mitochondrial matrix represents one of the key characteristics of Alzheimer's disease (AD) and appears already in its early stages. Inside the mitochondria, Aß interacts with a number of biomolecules, including cyclophilin D (cypD) and 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10), and affects their physiological functions. However, despite intensive ongoing research, the exact mechanisms through which Aß impairs mitochondrial functions remain to be explained. In this work, we studied the interactions of Aß with cypD and 17ß-HSD10 in vitro using the surface plasmon resonance (SPR) method and determined the kinetic parameters (association and dissociation rates) of these interactions. This is the first work which determines all these parameters under the same conditions, thus, enabling direct comparison of relative affinities of Aß to its mitochondrial binding partners. Moreover, we used the determined characteristics of the individual interactions to simulate the concurrent interactions of Aß with cypD and 17ß-HSD10 in different model situations associated with the progression of AD. This study not only advances the understanding of Aß-induced processes in mitochondria during AD, but it also provides a new perspective on research into complex multi-interaction biomolecular processes in general.

Actuated plasmonic nanohole arrays for sensing and optical spectroscopy applications.

Herein, we report a new approach to rapidly actuate the plasmonic characteristics of thin gold films perforated with nanohole arrays that are coupled with arrays of gold nanoparticles. The near-field interaction between the localized and propagating surface plasmon modes supported by the structure was actively modulated by changing the distance between the nanoholes and nanoparticles and varying the refractive index symmetry of the structure. This approach was applied by using a thin responsive hydrogel cushion, which swelled and collapsed by a temperature stimulus. The detailed experimental study of the changes and interplay of localized and propagating surface plasmons was complemented by numerical simulations. We demonstrate that the interrogation and excitation of the optical resonance to these modes allow the label-free SPR observation of the binding of biomolecules, and is applicable for in situ SERS studies of low molecular weight molecules attached in the gap between the nanoholes and nanoparticles.

Interactions of 17ß-Hydroxysteroid Dehydrogenase Type 10 and Cyclophilin D in Alzheimer's Disease.

The nucleus-encoded 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid ß peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17ß-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17ß-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (ka 2.0 × 105 M1s-1, kd 5.8 × 104 s-1, and KD 3.5 × 10-10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17ß-HSD10-cypD complexes were decreased and those of total amyloid ß increased. Moreover, the levels of 17ß-HSD10-cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17ß-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid ß. Levels of 17ß-HSD10-cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

Ionic Environment Affects Biomolecular Interactions of Amyloid-ß: SPR Biosensor Study.

In early stages of Alzheimer's disease (AD), amyloid beta (Aß) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17ß-hydroxysteroid dehydrogenase 10 (17ß-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aß affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aß (Aß1-40, Aß1-42) with cypD and 17ß-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K+ and Mg2+ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aß1-40 and Aß1-42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aß1-40 to cypD and 17ß-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K+ and increased concentrations of Mg2+ promote the interaction of both mitochondrial proteins with Aß1-42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.

Analyte transport to micro- and nano-plasmonic structures.

The study of optical affinity biosensors based on plasmonic nanostructures has received significant attention in recent years. The sensing surfaces of these biosensors have complex architectures, often composed of localized regions of high sensitivity (electromagnetic hot spots) dispersed along a dielectric substrate having little to no sensitivity. Under conditions such that the sensitive regions are selectively functionalized and the remaining regions passivated, the rate of analyte capture (and thus the sensing performance) will have a strong dependence on the nanoplasmonic architecture. Outside of a few recent studies, there has been little discussion on how changes to a nanoplasmonic architecture will affect the rate of analyte transport. We recently proposed an analytical model to predict transport to such complex architectures; however, those results were based on numerical simulation and to date, have only been partially verified. In this study we measure the characteristics of analyte transport across a wide range of plasmonic structures, varying both in the composition of their base plasmonic element (microwires, nanodisks, and nanorods) and the packing density of such elements. We functionalized each structure with nucleic acid-based bioreceptors, where for each structure we used analyte/receptor sequences as to maintain a Damköhler number close to unity. This method allows to extract both kinetic (in the form of association and dissociation constants) and analyte transport parameters (in the form of a mass transfer coefficient) from sensorgrams taken from each substrate. We show that, despite having large differences in optical characteristics, measured rates of analyte transport for all plasmonic structures match very well to predictions using our previously proposed model. These results highlight that, along with optical characteristics, analyte transport plays a large role in the overall sensing performance of a nanoplasmonic biosensor.

In vitro study of interaction of 17ß-hydroxysteroid dehydrogenase type 10 and cyclophilin D and its potential implications for Alzheimer's disease.

In early stages of Alzheimer's disease (AD), amyloid-ß (Aß) accumulates in neuronal mitochondria where it interacts with a number of biomolecules including 17beta-hydroxysteroide dehydrogenase 10 (17ß-HSD10) and cyclophilin D (cypD). It has been hypothesized that 17ß-HSD10 interacts with cypD preventing it from opening mitochondrial permeability transition pores and that its regulation during AD may be affected by the accumulation of Aß. In this work, we demonstrate for the first time that 17ß-HSD10 and cypD form a stable complex in vitro. Furthermore, we show that factors, such as pH, ionic environment and the presence of Aß, affect the ability of 17ß-HSD10 to bind cypD. We demonstrate that K+ and Mg2+ ions present at low levels may facilitate this binding. We also show that different fragments of Aß (Aß1-40 and Aß1-42) affect the interaction between 17ß-HSD10 and cypD differently and that Aß1-42 (in contrast to Aß1-40) is capable of simultaneously binding both 17ß-HSD10 and cypD in a tri-complex.

Hsp70 Trap Assay for Detection of Misfolded Subproteome Related to Myelodysplastic Syndromes.

The onset and progression of numerous serious diseases (e.g., various types of malignancies, neurodegenerative diseases, and cardiac diseases) are, on a molecular level, associated with protein modifications and misfolding. Current methods for the detection of misfolded proteins are not able to detect the whole misfolded subproteome and, moreover, are rather laborious and time consuming. Herein, we report on a novel simple method for the detection of misfolded proteins employing a surface plasmon resonance (SPR) biosensor and heat shock protein 70 (Hsp70) that recognizes and traps misfolded proteins in a nucleotide-dependent manner. We use this method for the detection of misfolded proteins in blood plasma of patients with various subtypes of myelodysplastic syndromes (MDS) and healthy donors. Our results reveal significantly elevated levels of misfolded proteins in the two stages of MDS that are most affected by oxidative stress: low-risk (RARS) and intermediate-risk (RCMD) patients. This approach can be extended to a variety of diseases and provides unique insights into the thus far unexplored area of blood proteome.

A New Approach for the Diagnosis of Myelodysplastic Syndrome Subtypes Based on Protein Interaction Analysis.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies with a high risk of transformation to acute myeloid leukemia (AML). MDS are associated with posttranslational modifications of proteins and variations in the protein expression levels. In this work, we present a novel interactomic diagnostic method based on both protein array and surface plasmon resonance biosensor technology, which enables monitoring of protein-protein interactions in a label-free manner. In contrast to conventional methods based on the detection of individual biomarkers, our presented method relies on measuring interactions between arrays of selected proteins and patient plasma. We apply this method to plasma samples obtained from MDS and AML patients, as well as healthy donors, and demonstrate that even a small protein array comprising six selected proteins allows the method to discriminate among different MDS subtypes and healthy donors.

High-performance biosensor exploiting a light guidance in sparse arrays of metal nanoparticles.

We introduce a new approach to plasmonic biosensing with superior biosensing properties based on spectroscopy of an electromagnetic mode guided by a monolayer of sparsely distributed colloidal plasmonic nanoparticles. The theoretical prediction of optical and sensing performance is confirmed by an experimental study in which adsorption of biomolecules on the sensor surface is studied. An unprecedentedly high figure of merit related to surface refractive index changes (FOMS) is demonstrated for distances of the biomolecules from the sensor surface up to 30 nm, which makes this approach a promising candidate for localized biosensing.

Advances in Surface Plasmon Resonance Imaging and Microscopy and Their Biological Applications.

Surface plasmon resonance microscopy and imaging are optical methods that enable observation and quantification of interactions of nano- and microscale objects near a metal surface in a temporally and spatially resolved manner. This review describes the principles of surface plasmon resonance microscopy and imaging and discusses recent advances in these methods, in particular, in optical platforms and functional coatings. In addition, the biological applications of these methods are reviewed. These include the detection of a broad variety of analytes (nucleic acids, proteins, bacteria), the investigation of biological systems (bacteria and cells), and biomolecular interactions (drug-receptor, protein-protein, protein-DNA, protein-cell).

Peptide Functionalization of Gold Nanoparticles for the Detection of Carcinoembryonic Antigen in Blood Plasma via SPR-Based Biosensor.

Nanoparticles functionalized with specific biological recognition molecules play a major role for sensor response enhancement in surface plasmon resonance (SPR) based biosensors. The functionalization procedure of such nanoparticles is crucial, since it influences their interactions with the environment and determines their applicability to biomolecular detection in complex matrices. In this work we show how the ζ-potential (Zpot) of bio-functionalized gold spherical NPs (Bio-NPs) is related to the SPR sensor response enhancement of an immune-sandwich-assay for the detection of the carcinoembryonic antigen (CEA), a cancer marker for colorectal carcinomas. In particular, we prepare bio-functional nanoparticles by varying the amount of peptide (either streptavidin or antibody against CEA) bound on their surface. Specific and non-specific sensor responses, reproducibility, and colloidal stability of those bio-functional nanoparticles are measured via SPR and compared to ζ-potential values. Those parameters are first measured in buffer solution, then measured again when the surface of the biosensor is exposed to blood plasma, and finally when the nanoparticles are immersed in blood plasma and flowed overnight on the biosensor. We found that ζ-potential values can guide the design of bio-functional NPs with improved binding efficiency and reduced non-specific sensor response, suitable reproducibility and colloidal stability, even in complex matrixes like blood plasma.

Biomolecular charges influence the response of surface plasmon resonance biosensors through electronic and ionic mechanisms.

Surface plasmon resonance (SPR) biosensors have become an important label-free optical biomolecular sensing technology and a "gold standard" for retrieving information on the kinetics of biomolecular interactions. Even though biomolecules typically contain an abundance of easily ionizable chemical groups, there is a gap in understanding of whether (and how) the electrostatic charge of a biomolecular system influences the SPR biosensor response. In this work we show that negative static charge present in a biomolecular layer on the surface of an SPR sensor results in significant SPR spectral shifts, and we identify two major mechanisms responsible for such shifts: 1) the formation of an electrical double layer (ionic mechanism), and 2) changes in the electron density at the surface of a metal (electronic mechanism). We show that under low ionic strength conditions, the electronic mechanism is dominant and the SPR wavelength shift is linearly proportional to the surface concentration of biomolecular charges. At high ionic strength conditions, both electric and ionic mechanisms contribute to the SPR wavelength shift. Using the electronic mechanism, we estimated the pKa of surface-bound carboxylic groups and the relative concentration of the carboxyl-terminated alkanethiols in a binary self-assembled monolayer of alkanethiols. The reported sensitivity of SPR to surface charge is especially important in the context of biomolecular sensing. Moreover, it provides an avenue for the application of SPR sensors for fast, label-free determination of the net charge of a biomolecular coating, which is of interest in material science, surface chemistry, electrochemistry, and other fields.