Surface Plasmon Resonance Imaging Chip Research Articles

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential.

Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein.

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (KD) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

Simultaneous and sensitive detection of two pathogenic genes of thrombotic diseases using SPRi sensor with one-step fixation probe by a poly-adenine oligonucleotide approach

Single-base mutations of Factor V Leiden G1691A and Prothrombin gene G20210A are the main genetic risk factors for inherited thrombotic tendency. The establishment for rapid and efficient detection method is of great significance to the prevention of venous thrombosis. In this work, a multiplexed, highly sensitive and regenerable surface plasmon resonance imaging (SPRi) sensor is described to identify and detect the two pathogenic genes by fixing probes in one-step. The probes are fixed by ployA, which is a simpler, faster and lower cost modification method compared with traditional thiol (-SH). PolyA-DNA-AuNPs is used to amplify the signal to improve sensitivity. The detection limit of the sensor is 8 pM, and it has a wide dynamic range between 8 pM and 100 nM and a good linear relationship between 8 pM to 50 pM. The equilibrium dissociation constant (KD) of 3.0 (± 0.3) pM indicates a high binding capacity. Based on the advantages of high-throughput detection, the SPRi chip can simultaneously identify and detect two genes related to thrombotic Diseases. In addition, more than 90% signal intensity can still be obtained on the surface of the chip after being regenerated of 25 times, indicating that this SPRi sensor has good stability and reproducibility. The established SPRi sensor has the advantages of high-throughput, high-sensitivity, label-free and no need for amplification, which is expected to become an effective technical means for real-time online detection of gene point mutations, and can be extended to detect and quantify a wider range of DNA mutation diseases.

Nanoplasmonics-enhanced label-free imaging of endothelial cell monolayer integrity

Surface plasmon resonance imaging (SPRI) is a powerful label-free imaging modality for the analysis of morphological dynamics in cell monolayers. However, classical plasmonic imaging systems have relatively poor spatial resolution along one axis due to the plasmon mode attenuation distance (tens of μm, typically), which significantly limits their ability to resolve subcellular structures. We address this limitation by adding an array of nanostructures onto the metal sensing surface (25 nm thick, 200 nm width, 400 nm period grating) to couple localized plasmons with propagating plasmons, thereby reducing attenuation length and commensurately increasing spatial imaging resolution, without significant loss of sensitivity or image contrast. In this work, experimental results obtained with both conventional unstructured and nanostructured gold film SPRI sensor chips show a clear gain in spatial resolution achieved with surface nanostructuring. The work demonstrates the ability of the nanostructured SPRI chips to resolve fine morphological detail (intercellular gaps) in experiments monitoring changes in endothelial cell monolayer integrity following the activation of the cell surface protease-activated receptor 1 (PAR1) by thrombin. In particular, the nanostructured chips reveal the persistence of small intercellular gaps (<5 μm2) well after apparent recovery of cell monolayer integrity as determined by conventional unstructured surface based SPRI. This new high spatial resolution plasmonic imaging technique uses low-cost and reusable patterned substrates and is likely to find applications in cell biology and pharmacology by allowing label-free quantification of minute cell morphological activities associated with receptor dependent intracellular signaling activity.

Multiplexed assessment of the surface density of DNA probes on DNA microarrays by surface plasmon resonance imaging

In terms of hybridization assays surface plasmon resonance imaging (SPRi) offers high throughput, label-free and real-time monitoring of the binding kinetics. This requires DNA microarrays on bare or modified gold SPRi chips, which are generally premade by an off-line microspotting procedure. Therefore, the surface density of the immobilized probes is not known although it is an essential quality control parameter, especially, when it can vary in a broad range as in case of self-assembled thiol-labeled DNAs on gold surface. Here we show that the small molecular weight ruthenium(III) hexamine complex (RuHex) introduced earlier for electrochemical quantitation of DNA coverage on gold electrodes can be used also in SPRi to assess the surface density of DNA probes in DNA microarrays. A single injection of RuHex solution allows the simultaneous visualization and quantification of the surface density of DNA probes (ranging in this study from 4 × 1011 to 1.7 × 1013 molecules cm−2) on all spots of a microarray made by microspotting thiol labeled short DNA probes both in prehybridized and single-stranded form on a gold SPRi chip. The methodology was applied to determine the effect of the surface density of DNA probes on the hybridization efficiency and kinetics of complementary microRNAs, using hsa-miR-208a-3p as model. Single mismatch duplexes were found to be more effectively destabilized than fully complementary duplexes by steric hindrance at large surface densities of the DNA probes, which offers an effective mean to increase single mismatch selectivity.

SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins.

We report on the direct coupling of surface plasmon resonance imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the investigation of specific, non-covalent interactions, using the example of designed ankyrin repeat proteins (DARPins) and ribosomal protein S6 kinase 2 (RPS6KA2) directly from lysate of SH-SY5Y cells, derived from human bone marrow. Due to an array format, tracing of binding kinetics of numerous DARPins simultaneously and in real time becomes possible. By optimizing both the proteolytic digest directly on the SPRi chip (amount of trypsin, incubation time, and temperature) as well as the MALDI matrix application (concentration of matrix and number of spray cycles), we are able to identify the specific interaction with RPS6KA2 directly from the cell lysate at a surface coverage of only 0.8fmol/mm2. Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.

SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events.

In this paper we review the underlying principles of the surface plasmon resonance (SPR) technique, particularly emphasizing its advantages along with its limitations regarding the ability to discriminate between the specific binding response and the interfering effects from biological samples. While SPR sensors were developed almost three decades, SPR detection is not yet able to reduce the time-consuming steps of the analysis, and is hardly amenable for miniaturized, portable platforms required in point-of-care (POC) testing. Recent advances in near-field optics have emerged, resulting in the development of SPR imaging (SPRi) as a powerful optical, label-free monitoring tool for multiplexed detection and monitoring of biomolecular events. The microarrays design of the SPRi chips incorporating various metallic nanostructures make these optofluidic devices more suitable for diagnosis and near-patient testing than the traditional SPR sensors. The latest developments indicate SPRi detection as being the most promising surface plasmon-based technique fulfilling the demands for implementation in lab-on-a-chip (LOC) technologies.

A Microwell-Printing Fabrication Strategy for the On-Chip Templated Biosynthesis of Protein Microarrays for Surface Plasmon Resonance Imaging.

A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein. In the second step, the protein microwell array is used to contact print one or more protein microarrays onto nitrilotriacetic acid (NTA)-functionalized gold thin film SPRI chips for real-time SPRI surface bioaffinity adsorption measurements. Even though each microwell array element only contains approximately 40 picomoles of protein, the concentration is sufficiently high for the efficient bioaffinity adsorption and capture of the approximately 100 femtomoles of hexahistidine-tagged protein required to create each SPRI microarray element. As a first example, the protein biosynthesis process is verified with fluorescence imaging measurements of a microwell array containing His-tagged green fluorescent protein (GFP), yellow fluorescent protein (YFP) and mCherry (RFP), and then the fidelity of SPRI chips printed from this protein microwell array is ascertained by measuring the real-time adsorption of various antibodies specific to these three structurally related proteins. This greatly simplified two-step synthesis/printing fabrication methodology eliminates most of the handling, purification and processing steps normally required in the synthesis of multiple protein probes, and enables the rapid fabrication of SPRI protein microarrays from DNA templates for the study of protein-protein bioaffinity interactions.

Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor.

We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.

Sensitive detection of multiple mycotoxins by SPRi with gold nanoparticles as signal amplification tags

Detection of multiple toxic mycotoxins is of importance in food quality control. Surface plasmon resonance imaging (SPRi) is an advanced tool for simultaneously multiple detections with accuracy; however, it suffers from limited sensitivity due to the instrumental constraint and small sizes of mycotoxins with only one epitope for an insensitive competitive immunoassay. In this work a gold nanoparticle (AuNP)-enhanced SPRi chip is designed to sensitively detect multiple mycotoxins using a competitive immunoassay format. The sensing surface is constructed by uniformly attaching dense mycotoxin antigens on poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-co-GMA) brush modified SPRi gold chip. After competitive binding in a sample solution containing respective monoclonal antibodies, secondary antibody-conjugated AuNPs are employed to bind with the captured monoclonal antibodies for further amplification of the SPRi signal. Highly specific and sensitive simultaneous detection is achieved for three typical mycotoxins including Aflatoxin B1 (AFB1), Ochratoxin A (OTA) and Zearalenone (ZEN) with low detection limits of 8, 30 and 15pgmL−1 and dynamic ranges covering three orders of magnitude.

Polydopamine-Functionalization of Graphene Oxide to Enable Dual Signal Amplification for Sensitive Surface Plasmon Resonance Imaging Detection of Biomarker

Surface plasmon resonance imaging (SPRi) is one of the powerful tools for immunoassays with advantages of label-free, real-time, and high-throughput; however, it often suffers from limited sensitivity. Herein we report a dual signal amplification strategy utilizing polydopamine (PDA) functionalization of reduced graphene oxide (PDA-rGO) nanosheets for sensitive SPRi immunoassay in serum. The PDA-rGO nanosheet is synthesized by oxidative polymerization of dopamine in a gentle alkaline solution in the presence of graphene oxide (GO) sheets and then is antibody-conjugated via a spontaneous reaction between the protein and the PDA component. In the dual amplification mode, the first signal comes from capture of the antibody-conjugated PDA-rGO to form sandwiched immunocomplexes on the SPRi chip, followed by a PDA-induced spontaneous gold reductive deposition on PDA-rGO to further enhance the SPRi signal. The detection limit as low as 500 pg mL(-1) is achieved on a nonfouling SPRi chip with high specificity and a wide dynamic range for a model biomarker, carcinoembryonic antigen (CEA) in 10% human serum.

Improving surface plasmon resonance imaging of DNA by creating new gold and silver based surface nanostructures

The use of nanoparticles (NPs) can substantially improve the analytical performance of surface plasmon resonance imaging (SPRi) in general, and in DNA sensing in particular. In this work, we report on the modification of the gold surface of commercial biochips with gold nanospheres, silica-coated gold nanoshells, and silver nanoprisms, respectively. The NPs were tethered onto the surface of the chip and functionalized with a DNA probe. The effects of tethering conditions and varying nanostructures on the SPRi signals were evaluated via hybridization assays. The results showed that coupling between planar surface plasmons and electric fields, generated by localized surface plasmons of the NPs, is mandatory for signal enhancement. Silver nanoprisms gave the best results in improving the signal change at a target DNA concentration of <50 nM by +50 % (compared to a conventional SPRi chip). The limit of detection for the target DNA was 0.5 nM which is 5 times less than in conventional SPRi.

Design of a universal biointerface for sensitive, selective, and multiplex detection of biomarkers using surface plasmon resonance imaging

This paper reports on the sensitive, selective, and simultaneous detection of four protein biomarkers involved in metastasis of various cancers, namely Fas, angiopoietin-2 (Ang-2), human epidermal growth factor receptor 2 (HER2), and matrix metallopeptidase-9 (MMP-9) using an antibody-conjugated quantum dot (QD) chip and surface plasmon resonance imaging (SPRi) biosensors. Initially, a self-assembled monolayer film of l-cysteine, using glutaraldehyde as a linker and QDs as signal enhancement moieties, was employed to immobilize anti-Fas for the detection of Fas as a model protein in buffer. The biointerface was characterized using confocal microscopy, atomic force microscopy, and scanning electron microscopy to provide evidence of uniform surface coverage by the QDs. The SPRi signal was enhanced 100-fold to achieve a detection limit of 25 pg mL(-1) after applying biotinylated detection antibody-conjugate streptavidin-modified QDs. Secondly, this signal amplification strategy was applied to sequentially detect Fas, HER2, MMP-9, and Ang-2 at low concentrations on a protein-microprinted/gold-coated SPRi chip. The results showed the absence of cross-reactivity among these proteins and the feasibility of the approach for multiplex detection of biomarkers as required for the accurate diagnosis of various diseases.

Designed Biointerface Using Near-Infrared Quantum Dots for Ultrasensitive Surface Plasmon Resonance Imaging Biosensors

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 μg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.

Protein−Diazonium Adduct Direct Electrografting onto SPRi-Biochip

A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule electroaddressing strategy, previously demonstrated by our team on carbon surfaces, is here valuably involved and adapted to create a straightforward and efficient protein immobilization process onto SPRi-biochips. The proteins, modified with an aryl-diazonium adduct, are addressed to the SPRi chip surface through the electroreduction of the aryl-diazonium. The biomolecule deposition was followed through SPRi live measurements during the electrografting process. A specially designed setup enabled us to directly observe the mass increasing at the sensor surface while the proteins were electrografted. A pin electrospotting method, allowing the achievement of distinct sensing layers on gold SPRi-biochips, was used to generate microarray biochips. The integrity of the immobilized proteins and the specificity of the detection, based on antigen/antibody interactions, were demonstrated for the detection of specific antibodies and ovalbumin. The SPRi detection limit of ovalbumin using the electroaddressing of anti-ovalbumin IgG was compared with two other immobilization procedures, cystamine-glutaraldehyde self-assembled monolayer and pyrrole, and was found to be a decade lower than these ones (100 ng/mL, i.e., 2 nM).