DNA aptamer-linked sandwich structure enhanced SPRi sensor for rapid, sensitive, and quantitative detection of SARS-CoV-2 spike protein.

The harm and impact of the COVID-19 pandemic have highlighted the importance of fast, sensitive, and cost-effective virus detection methods. In this study, we developed a DNA aptamer sensor using nanoparticle-enhanced surface plasmon resonance imaging (SPRi) technology to achieve efficient labeling-free detection of SARS-CoV-2 S protein. We used the same DNA aptamer to modify the surface of the SPRi sensor chip and gold nanoparticles (AuNPs), respectively, for capturing target analytes and amplifying signals, achieving ideal results while greatly reducing costs and simplifying the preparation process. The SPRi sensing method exhibits a good linear relationship (R2 = 0.9926) in the concentration range of 1-20 nM before adding AuNPs to amplify the signal, with a limit of detection (LOD) of 0.32 nM. After amplifying the signal, there is a good linear relationship (R2 = 0.9829) between the concentration range of 25-1000 pM, with a LOD of 5.99 pM. The simulation results also verified the effectiveness of AuNPs in improving SPRi signal response. The SPRi sensor has the advantage of short detection time and can complete the detection within 10 min. In addition, the specificity and repeatability of this method can achieve excellent results. This is the first study to simultaneously capture a viral marker protein and amplify the signal using polyadenylic acid (polyA)-modified DNA aptamers on the SPR platform. This scheme can be used as a fast and inexpensive detection method for diagnosis at the point of care (POC) to combat current and future epidemics caused by the virus.

Synthesis of obovatol and related neolignan analogues as α-glucosidase and α-amylase inhibitors.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy), enzymes responsible for the hydrolysis of carbohydrates. In recent decades, many natural compounds and their bioinspired analogues have been studied as α-Glu and α-Amy inhibitors. However, no studies have been devoted to the evaluation of α-Glu and α-Amy inhibition by the neolignan obovatol (1). In this work, we report the synthesis of 1 and a library of new analogues. The synthesis of these compounds was achieved by implementing methodologies based on: phenol allylation, Claisen/Cope rearrangements, methylation, Ullmann coupling, demethylation, phenol oxidation and Michael-type addition. Obovatol (1) and ten analogues were evaluated for their in vitro inhibitory activity towards α-Glu and α-Amy. Our investigation highlighted that the naturally occurring 1 and four neolignan analogues (11, 22, 26 and 27) were more effective inhibitors than the hypoglycemic drug acarbose (α-Amy: 34.6 µM; α-Glu: 248.3 µM) with IC5O value of 6.2-23.6 µM toward α-Amy and 39.8-124.6 µM toward α-Glu. Docking investigations validated the inhibition outcomes, highlighting optimal compatibility between synthesized neolignans and both the enzymes. Concurrently circular dichroism spectroscopy detected the conformational changes in α-Glu induced by its interaction with the studied neolignans. Detailed studies through fluorescence measurements and kinetics of α-Glu and α-Amy inhibition also indicated that 1, 11, 22, 26 and 27 have the greatest affinity for α-Glu and 1, 11 and 27 for α-Amy. Surface plasmon resonance imaging (SPRI) measurements confirmed that among the compounds studied, the neolignan 27 has the greater affinity for both enzymes, thus corroborating the results obtained by kinetics and fluorescence quenching. Finally, in vitro cytotoxicity of the investigated compounds was tested on human colon cancer cell line (HCT-116). All these results demonstrate that these obovatol-based neolignan analogues constitute promising candidates in the pursuit of developing novel hypoglycemic drugs.

A New Approach to the Quantification of Fibroblast Growth Factor 23-An Array Surface Plasmon Resonance Imaging Biosensor.

A new biosensor based on the "surface plasmon resonance imaging (SPRi)" detection technique for the quantification of "fibroblast growth factor 23 (FGF23)" has been developed. FGF23 is mainly produced in bone tissues as a phosphaturic hormone that forms a trimeric complex with "fibroblast growth factor receptor 1 (FGFR1)" and αKlotho upon secretion. FGF23 stimulates phosphate excretion and inhibits the formation of active vitamin D in the kidneys. FGF23 has been shown to play a role in bone carcinogenesis and metastasis. The newly developed method, based on the array SPRi biosensor, was validated-the precision, accuracy, and selectivity were acceptable, and yielded less than ±10% recovery. The rectilinear response of the biosensor ranges from 1 to 75 pg/mL. The limit of detection was 0.033 pg/mL, and the limit of quantification was 0.107 pg/mL. The biosensor was used to determine FGF23 concentrations in the blood plasma of healthy subjects and patients with "clear cell" renal cell carcinoma (ccRCC). The obtained results were compared with those measured through an "enzyme-linked immunosorbent assay (ELISA)". The determined Pearson correlation coefficients were 0.994 and 0.989, demonstrating that the newly developed biosensor can be used as a competitive method for the ELISA.

Multiplexing Biosensor for the Detection of Extracellular Vesicles as Biomarkers of Tissue Damage and Recovery after Ischemic Stroke.

The inflammatory, reparative and regenerative mechanisms activated in ischemic stroke patients immediately after the event cooperate in the response to injury, in the restoration of functions and in brain remodeling even weeks after the event and can be sustained by the rehabilitation treatment. Nonetheless, patients' response to treatments is difficult to predict because of the lack of specific measurable markers of recovery, which could be complementary to clinical scales in the evaluation of patients. Considering that Extracellular Vesicles (EVs) are carriers of multiple molecules involved in the response to stroke injury, in the present study, we have identified a panel of EV-associated molecules that (i) confirm the crucial involvement of EVs in the processes that follow ischemic stroke, (ii) could possibly profile ischemic stroke patients at the beginning of the rehabilitation program, (iii) could be used in predicting patients' response to treatment. By means of a multiplexing Surface Plasmon Resonance imaging biosensor, subacute ischemic stroke patients were proven to have increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) and translocator protein (TSPO) on the surface of small EVs in blood. Besides, microglia EVs and endothelial EVs were shown to be significantly involved in the intercellular communications that occur more than 10 days after ischemic stroke, thus being potential tools for the profiling of patients in the subacute phase after ischemic stroke and in the prediction of their recovery.

Discrimination of deletion to point cytokine mutants based on an array of cross-reactive receptors mimicking protein recognition by heparan sulfate.

Differential sensing of proteins based on cross-reactive arrays and pattern recognition is a promising technique for the detection and identification of proteins. In this study, a rational biomimetic strategy has been used to prepare sensing materials capable of discriminating structurally similar proteins, such as deletion and point mutants of a cytokine, by mimicking the biological properties of heparan sulfate (HS). Using the self-assembly of two disaccharides, lactose and sulfated lactose at various ratios on the surface of a chip, an array of combinatorial cross-reactive receptors has been prepared. Coupling with surface plasmon resonance imaging (SPRi), the obtained cross-reactive array is very efficient for protein sensing. It is able to detect HS binding proteins (HSbps) such as IFNγ at nanomolar concentrations. Moreover, such a system is capable of discriminating between IFNγ and its mutants with good selectivity.

Interaction between Nanoparticles, Membranes and Proteins: A Surface Plasmon Resonance Study.

Regardless of the promising use of nanoparticles (NPs) in biomedical applications, several toxic effects have increased the concerns about the safety of these nanomaterials. Although the pathways for NPs toxicity are diverse and dependent upon many parameters such as the nature of the nanoparticle and the biochemical environment, numerous studies have provided evidence that direct contact between NPs and biomolecules or cell membranes leads to cell inactivation or damage and may be a primary mechanism for cytotoxicity. In such a context, this work focused on developing a fast and accurate method to characterize the interaction between NPs, proteins and lipidic membranes by surface plasmon resonance imaging (SPRi) technique. The interaction of gold NPs with mimetic membranes was evaluated by monitoring the variation of reflectivity after several consecutive gold NPs injections on the lipidic membranes prepared on the SPRi biochip. The interaction on the membranes with varied lipidic composition was compared regarding the total surface concentration density of gold NPs adsorbed on them. Then, the interaction of gold and silver NPs with blood proteins was analyzed regarding their kinetic profile of the association/dissociation and dissociation constants (koff). The surface concentration density on the membrane composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and cholesterol (POPC/cholesterol) was 2.5 times higher than the value found after the injections of gold NPs on POPC only or with dimethyldioctadecylammonium (POPC/DDAB). Regarding the proteins, gold NPs showed preferential binding to fibrinogen resulting in a value of the variation of reflectivity that was 8 times higher than the value found for the other proteins. Differently, silver NPs showed similar interaction on all the tested proteins but with a variation of reflectivity on immunoglobulin G (IgG) 2 times higher than the value found for the other tested proteins.

Identification of amino acid residues involved in the interaction between peste-des-petits-ruminants virus haemagglutinin protein and cellular receptors.

Peste-des-petits-ruminants virus (PPRV) haemagglutinin (H) protein mediates binding to cellular receptors and then initiates virus entry. To identify the key residues of PPRV H (Hv) protein of the Nigeria 75/1 strain involved in binding to receptors, interaction of the Hv and mutated Hv (mHv) proteins with receptors (SLAM and Nectin 4) and their mutants (mSLAM1, mSLAM2, mSLAM3 and mNectin 4) was investigated using surface plasmon resonance imaging (SPRi) and coimmunoprecipitation (co-IP) assays. The results showed that the Hv protein failed to interact with mSLAM3, but interacted at a strong or medium intensity with SLAM, mSLAM2, Nectin 4 and mNectin 4, and at a low level with mSLAM1. The mHv protein was unable to interact with SLAM and its mutants, but bound to Nectin 4 and mNectin 4 with medium and weak intensity, respectively. Further analysis showed that the Hv protein could precipitate mSLAM1, mSLAM2 and mNectin 4, but not mSLAM3. The mHv protein failed to coprecipitate with SLAM and its mutants. The binding activities of mNectin 4 and Nectin 4 to mHv were less than 30.36 and 51.94 % of the wild-type levels, respectively. Based on the results obtained, amino acids at positions R389, L464, I498, R503, R533, Y541, Y543, F552 and Y553 of H protein and I61, H62, L64, K76, K78, E123, H130, I210, A211, S226 and R227 in SLAM were identified to be essential for the specificity of H-SLAM interaction, while the critical residues of H-Nectin 4 interaction require further study. These findings would improve our understanding of the invasive mechanisms of PPRV.

Surface plasmon resonance biosensor using hydrogel-AuNP supramolecular spheres for determination of prostate cancer-derived exosomes.

Based on the hydrogel-AuNP supramolecular sphere (H-Au), a label-free and real-time surface plasmon resonance imaging biosensor has been developed for highly sensitive and specific determination of prostate cancer cell-derived exosomes. After integrating the signal amplification effect of the mass cumulative hydrogel and the LSPR effect of AuNPs with high specific aptamer, the SPRi biosensor for exosome detection exhibited a wide linear range from 1.00 × 105 to 1.00 × 107 particles/mL with a limit of detection of 1.00 × 105 particles/mL. Most importantly, with a strong correlation between the SPRi signal and the t-PSA value measured by the clinical chemiluminescence immunoassay, this biosensor displayed excellent practicability for human serum analysis, which exhibits great potential applications in disease diagnosis and bioanalysis. Prostate cancer has been one of the most threatening diseases in human life and health nowadays. In particular, as cancer metastasizes, it is more likely to cause fracture, paraplegia, and even fatal consequences. However, the predominant t-PSA test needs further improvement for the deficiencies of limited specificity and sensitivity, which is prone to false positive. As one of the noninvasive markers of liquid biopsies, exosome has the potential to be a substitute for t-PSA, which can provide specific and predictive information in disease diagnosis and prognosis. Herein, based on the hydrogel-AuNP supramolecular sphere (H-Au), a label-free and real-time surface plasmon resonance biosensor has been developed for highly sensitive and specific detection of prostate cancer cell-derived exosomes. After integrating the signal amplification effect of mass cumulative hydrogel and LSPR effect of AuNPs with high specific aptamer, this developed SPRi biosensor for exosome detection exhibited a wide linear range from 1.00 × 105 to 1.00 × 107 particles/mL with a limit of detection down to 1.00 × 105 particles/mL. Most importantly, with a strong correlation between the SPRi signal and the t-PSA value measured by the clinical chemiluminescence immunoassay, this biosensor displayed excellent practicability in human serum, which exhibited great potential applications in disease diagnosis and bioanalysis.

Immunoassay utilizing imaging surface plasmon resonance for the detection of cyclopiazonic acid (CPA) in maize and cheese.

α-Cyclopiazonic acid (CPA) is a tremorgenic mycotoxin produced by Aspergillus and Penicillium fungal species, commonly found on agricultural commodities or fermented food products. A sensitive and rapid imaging surface plasmon resonance (iSPR) assay was developed to detect CPA in maize and cheese by combining an indirect competitive immunoassay and signal amplification based upon a secondary antibody (Ab2) conjugated with gold nanoparticles. Matrix-matched calibration curves were used to determine CPA content in maize and cheese samples. Recoveries, at two spiking levels in maize and cheese, were 89 to 126%, with standard deviations of repeatability (RSDr) of less than 16%. The limits of detection were 17 and 6 µg/kg in maize and cheese, respectively. To separate the CPA-contaminated samples from uncontaminated samples, a cutoff validation level of 40 µg/kg was introduced. The assay was applied to samples of naturally contaminated maize and was compared with competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA). This is the first report to detect CPA using an immuno-biosensor iSPR format.

A New Analytical Method for Determination of Cathepsin L Based on the Surface Plasmon Resonance Imaging Biosensor.

The purpose of this study was to develop a new method for a determination of the cathepsin L-biosensor based on the Surface Plasmon Resonance Imaging technique. The cathepsin L is an endopeptidase, which degrades proteins and plays an important role in various processes occurring in the human body. The detection technique, Surface Plasmon Resonance Imaging, is an optical, label-free technique, which can be used for quantitative determination of the different proteins. In order to bind the enzyme, the cathepsin L inhibitor-RKLLW-NH2 was used. The validation process showed that parameters: precision, accuracy, and selectivity of the method were acceptable. The analytically useful range of the standard curve was 0.50 ng/mL-15.00 ng/mL. The detection and quantification limit of method was 1.67 pg/mL and 5.07 pg/mL, respectively. The usefulness of the developed method was confirmed by the determination of the cathepsin L concentration in the blood plasma of some healthy persons and in the blood plasma of patients. The obtained results were compared with the results obtained by the ELISA. It was found that the correlation between these two methods was very strong, what suggest that the developed method can be used as the competitive method to the ELISA.

Label-free screening of foodborne Salmonella using surface plasmon resonance imaging.

It is estimated that 95% of the foodborne infections are caused by 15 major pathogens. Therefore, rapid and effective multiplex screening techniques for these pathogens with improved efficiencies could benefit public health at lower costs. Surface plasmon resonance imaging (SPRi) provides a label-free, multiplex analytical platform for pathogen screening. In this study, we have developed a singleplex immunoassay for Salmonella to evaluate the potential of SPRi in pathogen detection. Anti-Salmonella and control ligands were arrayed onto the SPRi sensor chip in a microarray format. The influences of ligand immobilization pH and concentration were optimized, and a pause flow protocol was adopted to improve assay rapidity and sensitivity. The method shows good specificity against 6 non-Salmonella species and was able to detect 5 of 6 Salmonella serotypes, including 3 serotypes most frequently associated with outbreaks. Limits of detection were found to be 2.1 × 106 CFU/mL in phosphate-buffered saline and 7.6 × 106 CFU/mL in the presence of chicken rinse matrix with 8.9 × 107 CFU/mL of indigenous microflora. The condition of antibody array regeneration was optimized for sequential sample injections. Finally, the SPRi immunoassay was used to detect Salmonella directly from artificially spiked chicken carcass rinse samples. As low as 6.8 CFU/mL of Salmonella could be detected after overnight enrichment in buffered peptone water, demonstrating the potential in streamlined pathogen screening with minimal sample preparation and without detection labels. Graphical abstract ᅟ.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 106 and 17.6 × 106 CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed.

Utility of cystatin C as a potential bladder tumour biomarker confirmed by surface plasmon resonance technique.

BACKGROUND & OBJECTIVES: The determination of cystatin C (cysC) may be helpful in diagnosis and monitoring of cancer because the pathogenesis of cancer is linked with an increased activity of cysteine peptidases (cathepsins) and a decrease of cysC concentration. This study was aimed to examine the utility of cysC as a marker of bladder cancer (BCa) to be used in the diagnosis. METHODS: This study was conducted with 90 patients with BCa and 27 healthy people. Patients with other cancers, inflammation process and impaired renal function were excluded from the study. The concentrations of cysC in the plasma and urine were measured by surface plasmon resonance imaging technique. RESULTS: The concentration of cysC in the serum taken from the patients with BCa [0.35±0.02 µg/ml (range: 0.20-0.78 µg/ml)] was significantly (P<0.001) lower than the serum cysC concentration of the healthy people [0.68±0.05 µg/ml (range: 0.52-0.89 µg/ml)]. The urinary cysC concentration of the BCa patients [0.19±0.01 µg/ml (range: 0.09-0.34 µg/ml)] was not significantly different from the urinary cysC concentration of the healthy people [0.24±0.02 µg/ml (range: 0.16-0.33 µg/ml)]. Receiver operating characteristic (ROC) curve showed that BCa patients with cysC concentration <0.54 µg/ml [sensitivity: 87%; specificity: 92%; area under the curve (AUC) of ROC: 0.927; P=0.02] could be optimally separated from healthy people. The ROC curve further showed that superficial low-grade patients with cysC concentration lower than 0.36 µg/ml (sensitivity: 0.63%; specificity: 0.58%; AUC of ROC: 0.635; P=0.08) could not be optimally separated from high-risk tumour patients. INTERPRETATION & CONCLUSIONS: BCa patients have lower serum cysC concentration than the control group. Serum cysC may be considered as a potential marker of BCa but not its aggressiveness.

Real-time Simutaneous Separation and Detection of Chemicals using Integrated Micro Column and Surface Plasmon Resonance Imaging Micro-GC.

An integrated and miniaturized Micro-Gas Chromatography with real-time imaging capability for simultaneous chemical separation and detection was developed. Surface Plasmon Resonance imaging (SPRi) was used as a sensitive and real-time imaging based detector for various gaseous chemical mixtures and good gas chromatographs were obtained. The system integrated a home-made miniaturized molecular sieve packed spiral micro-channel column with the SPRi imaging chip and real-time chemical separation and detection were demonstrated using alkanes. The chemical separation processes were simulated using COMSOL and matched well with experimental results. The system enabled the study of chemical separation processes in real-time by miniaturizing and integrating the Micro-GC separation and detection units. This approach can be expanded to multidimensional GC development.

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 0.8 fmol/mm2. Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.

Global identification of hnRNP A1 binding sites for SSO-based splicing modulation.

BACKGROUND: Many pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5' splice site can be blocked by SSOs to activate the exon. CONCLUSIONS: The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.

A Versatile Electronic Tongue Based on Surface Plasmon Resonance Imaging and Cross-Reactive Sensor Arrays-A Mini-Review.

Nowadays, there is a strong demand for the development of new analytical devices with novel performances to improve the quality of our daily lives. In this context, multisensor systems such as electronic tongues (eTs) have emerged as promising alternatives. Recently, we have developed a new versatile eT system by coupling surface plasmon resonance imaging (SPRi) with cross-reactive sensor arrays. In order to largely simplify the preparation of sensing materials with a great diversity, an innovative combinatorial approach was proposed by combining and mixing a small number of easily accessible molecules displaying different physicochemical properties. The obtained eT was able to generate 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL), which is also called 3D image, with valuable kinetic information, for the discrimination and classification of samples. Here, diverse applications of such a versatile eT have been summarized. It is not only effective for pure protein analysis, capable of differentiating protein isoforms such as chemokines CXCL12α and CXCL12γ, but can also be generalized for the analysis of complex mixtures, such as milk samples, with promising potential for monitoring the deterioration of milk.

Plasmonic Imaging of the Interfacial Potential Distribution on Bipolar Electrodes.

Bipolar electrochemistry is based on the gradient distribution of free-electron density along an electrically isolated electrode, which causes a positive electrode potential at one end and a negative potential at the other, allowing for wide applications in analytical chemistry and materials science. To take full advantage of its wireless and high-throughput features, various types of optical probes, such as pH indicators and fluorescence and electrochemiluminescence reagents, have often been used to indirectly monitor the interfacial electron transfer through chromogenic or fluorogenic reactions. Herein, we report the first probe-free imaging approach that can directly visualize the distribution of the interfacial potential in bipolar electrodes, providing essential information for the validation and development of the theory and applications of bipolar electrochemistry. This approach is based on the sensitive dependence of surface plasmon resonance imaging on the local electron density in the electrode, which enables the direct mapping of potential with a spatial resolution close to the optical diffraction limit, a temporal resolution of 50 ms, and a sensitivity of 10 mV. In addition, in contrast to previous optical readouts that relied on faradaic reactions, the present work achieved the impedance-based measurements under non-faradaic conditions. It is anticipated that this technique will greatly expand the application of bipolar electrochemistry as a platform for chemical and biosensing.

Application of surface plasmon resonance imaging to monitoring G protein-coupled receptor signaling and its modulation in a heterologous expression system.

BACKGROUND: G protein-coupled receptors (GPCRs) are ubiquitous surface proteins mediating various biological responses and thus, important targets for therapeutic drugs. GPCRs individually produce their own signaling as well as modulate the signaling of other GPCRs. Real-time observation of GPCR signaling and modulation in living cells is key to molecular study of biological responses and pharmaceutical development. However, fluorescence imaging, the technique widely used for this purpose, requires a fluorescent dye which may inhibit biological responses or a fluorescent-tagged target protein created through time-consuming genetic manipulation. In this study, we applied two-dimensional surface plasmon resonance (SPR) imaging to monitoring the translocation of protein kinase C (PKC), a major GPCR-coupled signaling molecule in the widely used HEK293 cell lines and examined whether the signaling of, and, modulation between heterologously expressed GPCRs can be measured without fluorescent labeling. RESULTS: We cultured HEK293 cells on the gold-plated slide glass and evoked SPR at the interface between the cell's plasma membrane and the gold surface with incident light. The translocation of activated native PKC to the plasma membrane is expected to alter the incident angle-SPR extent relation, and this could be detected as a change in the intensity of light reflection from the specimen illuminated at a fixed incident angle. Direct activation of PKC with 12-O-tetradecanoylphorbol-13-acetate increased the reflection intensity. This increase indeed reported PKC translocation because it was reduced by a pre-treatment with bisindolylmaleimide-1, a PKC inhibitor. We further applied this technique to a stable HEK293 cell line heterologously expressing the GPCRs type-1 metabotropic glutamate receptor (mGluR1) and adenosine A1 receptor (A1R). (RS)-3,5-dihydroxyphenylglycine, a mGluR1 agonist, increased the reflection intensity, and the PKC inhibitor reduced this increase. A pre-treatment with (R)-N(6)-phenylisopropyladenosine, an A1R-selective agonist suppressed mGluR1-mediated reflection increase. These results suggest that our technique can detect PKC translocation initiated by ligand binding to mGluR1 and its modulation by A1R. CONCLUSIONS: SPR imaging turned out to be utilizable for monitoring GPCR-mediated PKC translocation and its modulation by a different GPCR in a heterologous expression system. This technique provides a powerful yet easy-to-use tool for molecular study of biological responses and pharmaceutical development.

Determination of collagen type IV by Surface Plasmon Resonance Imaging using a specific biosensor.

Serum collagen type IV (COLIV) is a promising tumor marker. High COLIV concentrations have been found in the serum of patients with colorectal, gastric, lung, liver and breast cancers. The aim of this work was to develop a biosensor for use with the Surface Plasmon Resonance Imaging (SPRI) technique for COLIV determination. The biosensor consists of glass covered with gold and immobilized monoclonal mouse anti-human collagen type IV antibody via cysteamine linker. The biosensor works selectively within a dynamic response range between 10 and 300 ng mL-1, with LOD 2.4 ng mL-1 and LOQ 8 ng mL-1. The precision of determination is 4.7% at a 150 ng mL-1 COLIV spike and 8.0% at a 20 ng mL-1 spike, with recoveries of 101% and 106% respectively. A 100-fold excess of collagen I, albumin, laminin and fibronectin is tolerated. The average COLIV blood plasma concentration of healthy donors determined by the developed method was 69 ± 10 ng mL-1, while the median of six results available in the literature was approximately 80 ng mL-1. The average COLIV blood plasma concentration of breast cancer patients was 360 ± 68 ng mL-1, showing the high potential of COLIV as a marker of this type of cancer.