Real-time Surface Plasmon Resonance Research Articles

Single-Nanoparticle Near-Infrared Surface Plasmon Resonance Microscopy for Real-Time Measurements of DNA Hybridization Adsorption

A novel 814 nm near-infrared surface plasmon resonance (SPR) microscope is used for the real-time detection of the sequence-selective hybridization adsorption of single DNA-functionalized gold nanoparticles. The objective-coupled, high numerical aperture SPR microscope is capable of imaging in situ the adsorption of single polystyrene and gold particles with diameters ranging from 450 to 20 nm onto a 90 μm × 70 μm area of a gold thin film with a time resolution of approximately 1-3 s. Initial real-time SPR imaging (SPRI) measurements were performed to detect the accumulation of 40 nm gold nanoparticles for 10 min onto a gold thin film functionalized with a 100% complementary DNA surface at concentrations from 5 pM to 100 fM by counting individual particle binding events. A 100% noncomplementary DNA surface exhibited virtually no nanoparticle adsorption. In contrast, in a second set of SPRI measurements, two component complementary/noncomplementary mixed DNA monolayers that contained a very small percentage of complementary sequences ranging from 0.1 to 0.001%, showed both permanent and transient hybridization adsorption of the gold nanoparticles that could be tracked both temporally and spatially with the SPR microscope. These experiments demonstrate that SPR imaging measurements of single biofunctionalized nanoparticles can be incorporated into bioaffinity biosensing methods at subpicomolar concentrations.

Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.

Real-Time Label-Free Surface Plasmon Resonance Biosensing with Gold Nanohole Arrays Fabricated by Nanoimprint Lithography

In this work we present a surface plasmon resonance sensor based on enhanced optical transmission through sub-wavelength nanohole arrays. This technique is extremely sensitive to changes in the refractive index of the surrounding medium which result in a modulation of the transmitted light. The periodic gold nanohole array sensors were fabricated by high-throughput thermal nanoimprint lithography. Square periodic arrays with sub-wavelength hole diameters were obtained and characterized. Using solutions with known refractive index, the array sensitivities were obtained. Finally, protein absorption was monitored in real-time demonstrating the label-free biosensing capabilities of the fabricated devices.

Rebmab200, a Humanized Monoclonal Antibody Targeting the Sodium Phosphate Transporter NaPi2b Displays Strong Immune Mediated Cytotoxicity against Cancer: A Novel Reagent for Targeted Antibody Therapy of Cancer

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.

An In-situ Real-Time Optical Fiber Sensor Based on Surface Plasmon Resonance for Monitoring the Growth of TiO2 Thin Films

An optical fiber sensor based on surface plasmon resonance (SPR) is proposed for monitoring the thickness of deposited nano-thin films. A side-polished multimode SPR optical fiber sensor with an 850 nm-LD is used as the transducing element for real-time monitoring of the deposited TiO2 thin films. The SPR optical fiber sensor was installed in the TiO2 sputtering system in order to measure the thickness of the deposited sample during TiO2 deposition. The SPR response declined in real-time in relation to the growth of the thickness of the TiO2 thin film. Our results show the same trend of the SPR response in real-time and in spectra taken before and after deposition. The SPR transmitted intensity changes by approximately 18.76% corresponding to 50 nm of deposited TiO2 thin film. We have shown that optical fiber sensors utilizing SPR have the potential for real-time monitoring of the SPR technology of nanometer film thickness. The compact size of the SPR fiber sensor enables it to be positioned inside the deposition chamber, and it could thus measure the film thickness directly in real-time. This technology also has potential application for monitoring the deposition of other materials. Moreover, in-situ real-time SPR optical fiber sensor technology is in inexpensive, disposable technique that has anti-interference properties, and the potential to enable on-line monitoring and monitoring of organic coatings.

Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26

The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV. CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 β-propeller. The atomic details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.

Peptide Nucleic Acid (PNA)–DNA Duplexes: Comparison of Hybridization Affinity between Vertically and Horizontally Tethered PNA Probes

We compare the PNA-DNA duplex hybridization characteristics of vertically tethered and new horizontally tethered PNA probes on solid surfaces. The horizontal 15-mer PNA probe has been synthesized with linker molecules attached at three locations (γ-points) positioned along the PNA backbone that provides covalent attachment of the probe with the backbone aligned parallel to the surface, which is important for DNA hybridization assays that use electric field effect sensors for detection. A radioactive labeled assay and real-time surface plasmon resonance (SPR) biosensor are used to assess the probe surface density, nonspecific binding, and DNA hybridization affinity, respectively, of the new PNA probe configuration. The estimated equilibrium dissociation constants of the horizontally tethered duplex and the vertically tethered duplex are of the same order of magnitude (KD ≈ 5 nM), which indicates a sufficient hybridization affinity for many electronic biosensors that benefit from the horizontal alignment, which minimizes the effects of counterion screening.

Surface Plasmon Resonance Sensogram Based Characterization of Antibodies Raised Against Intact Teliospores and Purified Diagnostic Antigen for Development of Nano-Immunosensor for Fungal Spore Antigen of Karnal Bunt (Tilletia indica) of Wheat

The label free real time surface plasmon resonance based immuno-sensing system was first time demonstrated in the authors’ lab using a mercaptoundecanoic acid modified gold sensor chip. The kinetics of the antigen–antibody interaction was determined for optimization of resonance signal intensity for the detection of antigen(s) of fungal teliospores-causative entities of Karnal bunt disease of wheat incited by Tilletia indica. The approach involves the use of a rabbit polyclonal (anti-teliospore) antibody and mono-specific antibody generated against intact teliospores and purified diagnostic antigen of 28 kDa protein. The sensitivity of each of these two immunosensors constructed with the different concentrations of teliosporic antigen was determined by sensogram analysis which showed detection sensitivity as low as 625 and 156 pg which is equivalent to 2.5 and 1.0 teliospores over the sensor surface by getting an angle of dip indicating greater affinity of raised anti-teliospore antibodies and monospecific antibody respectively. By using kinetic evaluation software, the equilibrium constant (KD) and maximum binding capacity of analyte (Bmax) values were calculated and found to be 3.45 and 331.8 nM respectively using polyclonal and 1.57 and 289.57 nM respectively using monospecific antibody.

C2-Streptavidin Mediates the Delivery of Biotin-Conjugated Tumor Suppressor Protein P53 into Tumor Cells

We have previously generated a recombinant C2-streptavidin fusion protein for the delivery of biotin-labeled molecules of low molecular weight into the cytosol of mammalian cells. A nontoxic moiety of Clostridium botulinum C2 toxin mediates the cellular uptake, whereas the streptavidin unit serves as a binding platform for biotin-labeled cargo molecules. In the present study, we used the C2-streptavidin transporter to introduce biotin-conjugated p53 protein into various mammalian cell lines. The p53 tumor suppressor protein is inactivated in many human cancers by multiple mechanisms and therefore the restoration of its activity in tumor cells is of great therapeutic interest. Recombinant p53 was expressed in insect cells and biotin-labeled. Biotin-p53 retained its specific high-affinity DNA-binding as revealed by gel-shift analysis. Successful conjugation of biotin-p53 to the C2-streptavidin transporter was monitored by an overlay blot technique and confirmed by real-time surface plasmon resonance, providing a KD-value in the low nM range. C2-streptavidin significantly enhanced the uptake of biotin-p53 into African Green Monkey (Vero) epithelial cells as shown by flow cytometry. Using cell fractionation, the cytosolic translocation of biotin-p53 was detected in Vero cells as well as in HeLa cervix carcinoma cells. In line with this finding, confocal microscopy displayed cytoplasmic staining of biotin-p53 in HeLa and HL60 leukemia cells. Internalized biotin-p53 partially colocalized with early endosomes, as confirmed by confocal microscopy. In conclusion, our results demonstrate the successful conjugation of biotin-p53 to C2-streptavidin and its subsequent receptor-mediated endocytosis into different human tumor cell lines.

Parts per Trillion Detection of Ni(II) Ions by Nanoparticle-Enhanced Surface Plasmon Resonance

This paper demonstrates the development of a novel nanoparticle-enhanced surface plasmon resonance (SPR) sensing platform for the selective and sensitive in situ detection of nickel(II) ions at concentrations as low as 50 parts per trillion (211 pM). An enhancement in selectivity was achieved by designing a surface sandwich assay involving two different ligands each selective toward nickel(II) ions, namely, N-[5-(3'-maleimidopropylamido)-1-carboxypentyl]iminodiacetic acid (NTA) and polyhistidine. Maleimido-modified NTA was first immobilized on an alkanedithiol-modified gold thin film, followed by the sequential adsorption of Ni(II) ions. Next, polyhistidine-functionalized quasispherical gold nanoparticles, designed to enhance the SPR sensitivity, were specifically adsorbed onto surface Ni(II)-NTA complexes. This process was monitored by real-time SPR. The ability to detect Ni(II) ions as low as 50 parts per trillion (ppt) is a remarkable improvement compared to other optical and colorimetric techniques utilizing nanoparticles and is comparable to what can be achieved by state-of-the-art inductively coupled plasma mass spectrometry (ICP-MS). The improved selectivity for Ni(II) ions by the sandwich assay approach was confirmed by comparing measurements involving other divalent cations such as Zn(II), Pb(II), and Cu(II), some of which individually possess binding affinities toward either the NTA or histidine moieties.

The over expression of cathelicidin peptide LL37 in head and neck squamous cell carcinoma: The peptide marker for the prognosis of cancer

The over expression of LL37, an antimicrobial peptide belonging~to the cathelicidin family has implication in the progression of human malignancy but the exact role is still not clear. This study aims to elucidate the correlation of LL37 with Head and Neck Squamous Cell Carcinoma (HNSCC) and the consequences of radiotherapy on it. The LL37 levels were quantified in serum samples of control and HNSCC patients at pre-RT, during-RT and post-RT using a real time Surface Plasmon Resonance technology and ELISA. The LL37 of 50 HNSCC patients was significantly (p < 0.0001) threefold higher than the 25 controls and declined with respect to radiation therapy (p < 0.0001) supporting its candidature as a prognostic marker in HNSCC.

TLR2 Is a Primary Receptor for Alzheimer’s Amyloid β Peptide To Trigger Neuroinflammatory Activation

Microglia activated by extracellularly deposited amyloid β peptide (Aβ) act as a two-edged sword in Alzheimer's disease pathogenesis: on the one hand, they damage neurons by releasing neurotoxic proinflammatory mediators (M1 activation); on the other hand, they protect neurons by triggering anti-inflammatory/neurotrophic M2 activation and by clearing Aβ via phagocytosis. TLRs are associated with Aβ-induced microglial inflammatory activation and Aβ internalization, but the mechanisms remain unclear. In this study, we used real-time surface plasmon resonance spectroscopy and conventional biochemical pull-down assays to demonstrate a direct interaction between TLR2 and the aggregated 42-aa form of human Aβ (Aβ42). TLR2 deficiency reduced Aβ42-triggered inflammatory activation but enhanced Aβ phagocytosis in cultured microglia and macrophages. By expressing TLR2 in HEK293 cells that do not endogenously express TLR2, we observed that TLR2 expression enabled HEK293 cells to respond to Aβ42. Through site-directed mutagenesis of tlr2 gene, we identified the amino acids EKKA (741-744) as a critical cytoplasmic domain for transduction of inflammatory signals. By coexpressing TLR1 or TLR6 in TLR2-transgenic HEK293 cells or silencing tlrs genes in RAW264.7 macrophages, we observed that TLR2-mediated Aβ42-triggered inflammatory activation was enhanced by TLR1 and suppressed by TLR6. Using bone marrow chimeric Alzheimer's amyloid precursor transgenic mice, we observed that TLR2 deficiency in microglia shifts M1- to M2-inflammatory activation in vivo, which was associated with improved neuronal function. Our study demonstrated that TLR2 is a primary receptor for Aβ to trigger neuroinflammatory activation and suggested that inhibition of TLR2 in microglia could be beneficial in Alzheimer's disease pathogenesis.

Structural Basis of the Specific Functions of Proteins Secreted from Mammary Gland during Different Progressive Stages such as Proliferative, Early-Lactation, Late-Lactation and Involution Phases

Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India-110029The mammary gland undergoes repeated cycles of pregnancy, lactation and involution. Throughout these cycles, the interactions of mammary gland epithelial cells with their surrounding extracellular matrix (ECM), in particular the specialized ECM known as basement membrane (BM), contributes to the signals required for their proliferation, differentiation and survival. The structural and functional studies of prominent proteins secreted during these various phases have been carried out. The proteins included in these studies are insulin growth factor (IGF), lactoferrin (LF), Lactoperoxidase (LP), peptidoglycan recognition protein (PGRP) and signaling glycoproteins (SPX-40). Three-dimensional structural determinations using X-ray crystallographic methods and binding studies using real time kinetics and surface plasmon resonance of these proteins have shown several features that are important for signaling roles of IGF and SPX-40. The detailed study of various structural characterstics of LF and LP have revealed their new functions and have helped in the understanding of the mechanism of their action. IGF is a signaling component secreted during proliferative phase and its structure showed many novel features pertaining to structure and function relationship. We have also studied the structures of C-terminal halves of LF and their various complexes with tight binders of C-lobe and NSAIDs. It indicates a new role of lactoferrin C-lobe as a protector of side effects of NSAIDs. The structural studies of PGRP have revealed that this protein existed in a tetrameric form with four structurally identical molecules giving rise to a potent and versatile binding site.

Real-time full-spectral imaging and affinity measurements from 50 microfluidic channels using nanohole surface plasmon resonance.

With recent advances in high-throughput proteomics and systems biology, there is a growing demand for new instruments that can precisely quantify a wide range of receptor-ligand binding kinetics in a high-throughput fashion. Here we demonstrate a surface plasmon resonance (SPR) imaging spectroscopy instrument capable of simultaneously extracting binding kinetics and affinities from 50 parallel microfluidic channels. The instrument utilizes large-area (~ cm(2)) metallic nanohole arrays as SPR sensing substrates and combines a broadband light source, a high-resolution imaging spectrometer and a low-noise CCD camera to extract spectral information from every channel in real time with a refractive index resolution of 7.7 × 10(-6) refractive index units. To demonstrate the utility of our instrument for quantifying a wide range of biomolecular interactions, each parallel microfluidic channel is coated with a biomimetic supported lipid membrane containing ganglioside (GM1) receptors. The binding kinetics of cholera toxin b (CTX-b) to GM1 are then measured in a single experiment from 50 channels. By combining the highly parallel microfluidic device with large-area periodic nanohole array chips, our SPR imaging spectrometer system enables high-throughput, label-free, real-time SPR biosensing, and its full-spectral imaging capability combined with nanohole arrays could enable integration of SPR imaging with concurrent surface-enhanced Raman spectroscopy.

Quantification of p38αMAP kinase: A prognostic marker in HNSCC with respect to radiation therapy

BackgroundTranslation of early findings from basic research is aimed to benefit cancer therapeutics. We report the p38α level in serum of head and neck squamous cell carcinoma (HNSCC) patients indicating it as a prognostic marker and established its correlation with radiation therapy (RT). MethodsThe case-controlled study was performed on 120 HNSCC patients from whom 81 patients and 45 controls were statistically analyzed. The p38α estimation was done at pre-RT, during-RT and post-RT using a real time Surface Plasmon Resonance (SPR) technology, ELISA and western blot. ResultsHNSCC patients showed threefold increase in p38α level when compared to control (p value<0.0001). The estimated concentration of p38α in a temporal manner, before-RT, during-RT and post-RT was 0.61ng/μl (95%CI: 0.53–0.69), 0.35ng/μl (95%CI: 0.31–0.38) and 0.30ng/μl (95%CI: 0.26–0.33), respectively. Among the 81 cases, 70 patients (86.42%) showed a declined p38α in response to RT as evaluated by SPR and were responding clinically (clinical tumor regression). ConclusionsThis study showed elevated p38α level at cancer diagnosis and a statistically significant decline during-RT and post-RT periods. Hence, it can emerge as a prognostic marker supporting the candidature of p38α as a suitable serum marker in HNSCC.

Development of a “Modular” Scheme to Describe the Kinetics of Transcript Elongation by RNA Polymerase

Transcript elongation by RNA polymerase involves the sequential appearance of several alternative and off-pathway states of the transcript elongation complex (TEC), and this complicates modeling of the kinetics of the transcription elongation process. Based on solutions of the chemical master equation for such transcription systems as a function of time, we here develop a modular scheme for simulating such kinetic transcription data. This scheme deals explicitly with the problem of TEC desynchronization as transcript synthesis proceeds, and develops kinetic modules to permit the various alternative states of the TECs (paused states, backtracked states, arrested states, and terminated states) to be introduced one-by-one as needed. In this way, we can set up a comprehensive kinetic model of appropriate complexity to fit the known transcriptional properties of any given DNA template and set of experimental conditions, including regulatory cofactors. In the companion article, this modular scheme is successfully used to model kinetic transcription elongation data obtained by bulk-gel electrophoresis quenching procedures and real-time surface plasmon resonance methods from a template of known sequence that contains defined pause, stall, and termination sites.

Development of a Novel Electrochemical Surface Plasmon Resonance Instrument Based on a Mini Three Electrode Flow Cell

The construction and performance of a novel electrochemical (EC) surface plasmon resonance (SPR) instrument based on a mini three electrode flow cell that was designed and manufactured by MEMS technology is described. The working electrode is 50nm gold and counter and reference electrodes, made of Au and Ag/AgCl separately, are 0.6 mm in diameter. The flow channel is made of polydimethylsiloxane (PDMS) with 1mm in width, 200μm in depth, 5μL in volume. The process for activating and modifying the working electrode is given by the real-time SPR detection. The Cyclic voltammetry (CV) and SPR detection are conducted simultaneously in potassium ferricyanide solution system. It is proved that the signal of redox reaction and the change of refractive index have a well correlation by the potential sweep experiment. Four kinds of concentration of potassium ferricyanide are analyzed and the peak oxidizing current, obtained from the curve, is proportional to the respective concentration of the sample. The EC-SPR experiment is also carried out in CuSO4 system and the results is similar to potassium ferricyanide.

Prognostic Significance of Cyclooxygenase-2 and Response to Chemotherapy in Invasive Ductal Breast Carcinoma Patients by Real Time Surface Plasmon Resonance Analysis

Cyclooxygenase-2 (COX-2), an inducible enzyme, has been implicated in the progression and angiogenesis of breast cancer. The aim of the study is to quantify the concentration of COX-2 and its association with clinico-pathological parameters and response to treatment in patients with invasive ductal carcinoma receiving both neo-adjuvant and adjuvant chemotherapy. The level of COX-2 was estimated using a novel biosensor-based surface plasmon resonance technique in serum of 84 patients with breast cancer (48 patients of neo-adjuvant chemotherapy and 36 patients of adjuvant chemotherapy) and 40 age- and gender-matched normal individuals. A significant increase in COX-2 level was observed in patients compared with normal individuals (p>0.0001). The COX-2 level in serum was found to be significantly higher in patients with lymph node involvement (p<0.0061). 68% (33/48) of the patients receiving neo-adjuvant chemotherapy showed significantly (p<0.0025) reduced COX-2 levels. This study shows significant decrease of COX-2 level in patients with breast cancer treated with both neo-adjuvant and adjuvant chemotherapy. Estimation of COX-2 level in serum may serve as a tumor biomarker in patients with breast cancer.

Preliminary approach of real-time monitoring in vitro matrix mineralization based on surface plasmon resonance detection

Matrix mineralization is a terminal process in osteoblast differentiation, and several approaches have been introduced to characterize the process in tissues or cultured cells. However, an analytical technique that quantitates in vitro matrix mineralization of live cells without any labeling or complex treatments is still lacking. In this study, we investigate a simple and enhanced optical method based on surface plasmon resonance (SPR) detection that can monitor the surface-limited refractive index change in real-time. During monitoring MC3T3-E1 cells in vitro culture every 2 days for over 4 weeks, the SPR angle is shifted with a greater resonance change in cells cultured with osteogenic reagents than those without the reagents. In addition, the SPR results obtained have a close relevance with the tendency of conventional mineralization staining and an inductively coupled plasma-based calcium content measure. These results suggest a new approach of a real-time SPR monitoring in vitro matrix mineralization of cultured cells.

Simultaneous purification and surface plasmon resonance characterization of mechanoresponsive, discretely functionalized gold nanoparticles

Discretely functionalized gold nanoparticles (AuNPs) with an amine surface functional group on each AuNP have been synthesized as a crude mixture with a significant amount of nonfunctional AuNPs. By employing the mechanoresponsive nature and reversible self-assembling properties of the mono-functionalized AuNP on a crown ether-coated gold surface, mono-functionalized AuNPs have been successfully isolated from a crude mixture. A real-time surface plasmon resonance (SPR) with organic solvent-compatible microfluidic device/instrumentation has been built together with a three-step denoising procedure for the detection of SPR absorption dip shifts with high sensitivity. With this instrument, pH-driven self-assembly and disassembly processes of the mono-functionalized gold nanoparticles (AuNPs), non-functional/mono-functionalized (crude) AuNPs, and randomly functionalized AuNPs on a crown ether-coated gold surface have been monitored simultaneously. From the real-time SPR traces, in general, the self-assembled AuNPs are stable on the surface with a solvent flow for a long period of time. These AuNPs possess different self-assembling and disassembling properties. In particular, SPR response traces obtained from mono-functionalized AuNPs reveal complete pH-driven self-assembly and disassembly processes on the gold surface. This device has enabled real-time monitoring of mono-functionalized AuNPs characteristics with the exclusion of non-functional AuNPs in a crude mixture. The SPR trace of self-assembled, randomly functionalized AuNPs indicates that only partial AuNPs were disassembled from the surface and eluted away after base treatment. Residue randomly functionalized AuNPs that were intact with the gold surface might possess higher number of functional group on each NP, rendering multivalent binding with enhanced stability towards the base. This observation has also been confirmed by atomic force microscopy (AFM). SPR monitoring of discretely functionalized AuNPs enables a real-time characterization other than the conventional transmission electron microscopy (TEM) and UV/visible absorption spectroscopic methods. The reported mono-functionalized AuNPs may find applications in single molecular detection and analysis of molecular binding events with a quantification capability with higher sensitivity.