Tumor marker detection is essential for the therapy efficiency of early stage tumors and the evaluation of disease progression. Osteopontin (OPN) is supposed to be closely related to several kinds of tumors. In the present study, we describe a label-free electrochemical detection of OPN based on a specific reaction between OPN and its relevant antibody. An artificial three-dimensional (3D) scaffold structure consisting of 11-mercaptoundecanoic acid/6-mercapto-1-hexanol, dextran amino and synthetic peptides was designed as a substrate for the immobilization of the antibody. This substrate was characterized using cyclic voltammetry, atomic force microscopy and Fourier transform infrared reflection spectroscopy. Antibody immobilization and OPN detection were conducted using electrochemical impedance spectroscopy (EIS). The low limit of detection was 0.17 nM. The concentration of cancer risk (5.77 nM) can be selectively detected with a high EIS signal. The fabricated 3D OPN sensor is proposed for application in clinical analysis.
Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. It has been identified as an ideal biomarker for staging of many kinds of cancers, so more specific and intense signal is desirable for VEGF biosensors so that the sensors may have more valuable clinical application. Herein, we report a highly sensitive and selective surface plasmon resonance (SPR) sensor for VEGF detection by using two DNA aptamers which target different VEGF domains used as the capture and detection probe, respectively. Moreover, by making use of carboxyl-coated polystyrene microspheres, 3'-NH2 immobilized aptamer and 3'-NH2 modified primer DNA are loaded through amidation onto the sensing layer for further rolling circle amplification (RCA) process to amplify the SPR signal. With the well-designed sensing platform, VEGF can be determined in a linear range from 100 pg mL(-1) to 1 ?g mL(-1) with a detection limit of 100 pg mL(-1). Due to its high specificity and desirable sensitivity, this RCA assisted SPR method may be a useful tool for the assay of VEGF in the future. What is more, by replacing the sensing element, i.e., the aptamer of VEGF used in this work, more biosensors for sensitive detection of other biomarkers proteins can be fabricated based on the strategy proposed in this study.
Three calixarene (Cal-4) derivatives which separately contain ethylester (1), carboxylic acid (2), and crownether (3) at the lower rim with a common reactive thiol at the upper rim were synthesized and constructed to self-assembled monolayers (SAMs) on Au films. After spectroscopic characterization of the monolayers, surface coverage and orientation of antibody immobilized on the Cal-4 derivative SAMs were studied by surface plasmon resonance (SPR) technique. Experimental results revealed that the antibody could be immobilized on the Cal-4 derivatives spontaneously. The orientation of absorbed antibody on the Cal-4 derivative SAMs is related to the SAM's dipole moment. The possible orientations of the antibody immobilized on the Cal-4 derivative 1 SAM are lying-on or side-on, while on the Cal-4 derivative 2 and Cal-4 derivative 3 head-on and end-on respectively. These experimental results demonstrate the surface dipole moment of Cal-4 derivative appears to be an important factor to antibody orientation. Cal-4 derivatives are useful in developing site direct protein chips.
Diagnosis of apoptosis is essential to the early detection of therapy efficiency and the evaluation of disease progression. Caspase-3 is supposed to be closely related to cellular apoptosis. We describe here a label-free surface plasmon resonance (SPR) detection of apoptosis based on caspase-3 activity assay through enzyme digestion. An artificial peptide sequence was designed as a substrate of caspase-3 and immobilized on a gold disk through covalent binding. The 4Lys part at the end of the pentadecyl-peptide was designed to form a unique peptide array through electrostatic repulsion. The immobilization of the peptide on the gold surface was carefully characterized by SPR and atomic force microscopy. The catalytic conditions of caspase-3 were optimized with electrochemical impedance spectroscopy. The detection limit of caspase-3 was found at a concentration of 1 pg mL(-1). The activity of caspase-3 in apoptotic cells could also be measured sensitively by the one-step and intuitional SPR response decrease. The fabricated simple and convenient caspase-3 sensor is proposed for application in clinical analysis.
Arginine plays an important role in cell division and the functioning of the immune system. We describe a novel method by which arginine can be identified using an artificial monolayer based on surface plasmon resonance (SPR). The affinity of arginine binding its recognition molecular was compared to that of lysine. In fabrication of an arginine sensing interface, a calixcrown ether monolayer was anchored onto a gold surface and then characterized by Fourier Transform infrared reflection absorption spectroscopy, atomic force microscopy, and cyclic voltammetry. The interaction between arginine and its host compound was investigated by SPR. The calixcrown ether was found to assemble as a monolayer on the gold surface. Recognition of calixcrown monolayer was assessed by the selective binding of arginine. Modification of the SPR chip with the calixcrown monolayer provides a reliable and simple experimental platform for investigation of arginine under aqueous conditions.
Oligonucleotide chips targeting the bacterial internal transcribed spacer region (ITS) of the 16S-23S rRNA gene, which contains genus- and species-specific regions, were developed and evaluated. Forty-three sequences were designed consisting of 1 universal, 3 Gram stain-specific, 9 genus-specific, and 30 species-specific probes. The specificity of the probes was confirmed using bacterial type strains including 54 of 52 species belonging to 18 genera. The performance of the probes was evaluated using 825 consecutive samples that were positive by blood culture in broth medium. Among the 825 clinical specimens, 708 (85.8%) were identified correctly by the oligonucleotide chip. Most (536 isolates, or 75.7%) were identified as staphylococci, Escherichia coli, or Klebsiella pneumoniae. Thirty-seven isolates (4.5%) did not bind to the corresponding specific probes. Most of these also were staphylococci, E. coli, or K. pneumoniae and accounted for 6.3% of total number of the species. Sixty-two specimens (7.5%) did not bind the genus- or species-specific probes because of lack of corresponding specific probes. Among them, Acinetobacter baumannii was the single most frequent isolate (26/62). The oligonucleotide chip was highly specific and sensitive in detecting the causative agents of bacteremia directly from positive blood cultures.
A phenylalanine sensing system was constructed with photochromic spiroxazine derivative via surface plasmon resonance (SPR). Recognition-functional spiroxazine monolayer was formed on Au surface by self-assembly. After spectroscopic characterizations of monolayer, various concentrations of d- and l-phenylalanine were employed as analytes on UV addressable ring-opened spiroxazine monolayer. The different SPR angle shift derived from interaction between d- and l-phenylalanine and spiroxazine monolayer can be explained by the different dipole moment of ionic complexes. Computer simulations using Molecular Orbital PACkage AM1 approximation reinforced the reliability of the experimental results. To confirm the long-time stability of spiroxazine monolayer, we measured SPR response of spiroxazine monolayer with repetitive UV-on and off. These experimental results suggest that it can be applicable to simple analysis of interaction between zwitterionic recognition-functional molecule and analyte.
Spiroxazines are a class of photochromic compounds whose molecular structures are alterable upon exposure to UV/visible light. The typical reaction of spiroxazines is the conversion between the non-polar ring-closure form and polar ring-open form. In this work, copolymer of 1,6-heptadiyne derivatives containing a spiroxazine was used as photochromic material. Precise photochromic properties of the copolymers thin-film were evaluated through measuring dielectric constant, optical constant and thickness by surface plasmon resonance (SPR) with multi-solvent approach. The change in structure under UV-light irradiation is accompanied by increase of dielectric constant. However, dielectric constant and UV-induced thickness change of the photochromic polymer thin-film are independent of film thickness.
Semiconductor nanomaterials have attracted considerable attention in the design of high efficiency PL up-conversion in heterojunctions or nanostructures at extremely low continuous wave (cw)-excitation intensity. In this study, bioconjugated hybrids were constructed using CdTe and Au nanoparticles (NPs), where two-fold PL enhancement was observed in the solution state. These results are in accordance with theoretical predictions of the local-field effects associated with the combined influence of strong localization of the collective plasmon modes in metallic-semiconducting hybrids and multi-photon absorption into its localized plasmon modes. The feasibility of the nanohybrids as sensors was demonstrated by breaking the bioconjugation through thermal stress, which induced a rapid decrease in luminescence intensity. It is believed that the phenomena is applicable to high-compacted optoelectronic devices and sensing systems that take advantage of both quantum confinement effects and nonlinear optical properties.
The Fabry-Perot fringe pattern is determined by wavelength shifts in interferometric reflectance spectroscopy, which is a function of the refractive index (n) and thickness (l) of the porous silicon (PSi). In this paper, we demonstrated the shifts of wavelength as systematic controlling of adsorbed concentration of a protein on the PSi substrate. In order to correlate the wavelength shift of the Fabry-Perot fringe pattern with the protein adsorption, adsorbed concentrations of avidin (66 kDa) on the PSi substrate were measured by three independent methods: first, by means of bromophenol blue, which is applied to surface-confined avidin; second, with fluorescence intensity measurement of FITC-tagged avidin; and finally by utilizing Time of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS). It was found that the wavelength shift is directly proportional to the surface-adsorbed avidins over the wide range of surface concentration on the PSi substrate, with a rate of 0.1 nm red-shift at avidin concentration change of 1.0 fmol/cm2.
Hydroxyapatite (HAp) was coated on scratched areas of a human tooth and HAp disks by the immersion method in a HAp colloidal solution (< or =20 microm of average diameter dispersed in DI water). The surface morphologies of the scratched area after immersion for 1-3 months were investigated showing that the damaged surfaces were remarkably recovered. Then, the mechanical property and chemical stability of the HAp coating layers on both specimens were determined via the Vickers hardness test and concentration measurement of extracted Ca2+ ions, respectively, after strong acidic treatment. The cellular behavior of mouse calvaria-derived pre-osteoblastic cells (MC3T3-E1) was also examined on the HAp layers regenerated on micro-scratched HAp disks for the purpose of their potential applications on maxillofacial bone conservation and reconstruction for prosthetic dentistry, and artificial disk preparation of a vertebral column. The notable loss of Ca2+ ions under a highly acidic condition was not observed in the layers coated by HAp adsorption, indicating that the coating surface was well adhered with the original surfaces of the respective specimen. Moreover, the HAp adsorption did not adversely affect the adhesion, growth and proliferation of MC3T3-E1 cells on the coated HAp layers for up to 21 days. These results suggest that the HAp coating on the scratched areas of the tooth would be effectively applicable for the development of long-term prevention of micro-cleavage and tooth health supporters to reduce discoloration and further maxillofacial and orthopedic applications.
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