Compact and low-cost biosensor based on novel approach to spectroscopy of surface plasmons

A high-performance surface plasmon resonance (SPR) sensor based on a novel approach to spectroscopy of surface plasmons is reported. This approach employs a special diffraction grating structure (referred to as surface plasmon resonance coupler and disperser, SPRCD) which simultaneously couples light into a surface plasmon and disperses the diffracted light for spectral readout of SPR signal. The developed SPRCD sensor consists of a miniature cartridge integrating the diffraction grating and microfluidics and a compact optical system which simultaneously acquires data from four independent sensing channels in the cartridge. It is demonstrated that the SPRCD sensor is able to measure bulk refractive index changes as small as 3 × 10⁻⁷ RIU (refractive index units) and to detect short oligonucleotides in concentrations down to 200 pM.     

Characteristics of protein-carbohydrate interactions as a basis for developing novel carbohydrate-based antirejection therapies

The relative shortage of human organs for transplantation is today the major barrier to a broader use of transplantation as a means of treating patients with end-stage organ failure. This barrier could be partly overcome by an increased use of blood group ABO-incompatible live donors, and such trials are currently underway at several transplant centres. If xenotransplantation can be used clinically in the future, the human organ shortage will, in principle, be eradicated. In both these cases, carbohydrate antigens and the corresponding anti-carbohydrate antibodies are the major primary immunological barriers to overcome. Refined carbohydrate-based therapeutics may permit an increased number of ABO-incompatible transplantations to be carried out, and may remove the initial barriers to clinical xenotransplantation. Here, we will discuss the chemical characteristics of protein-carbohydrate interactions and outline carbohydrate-based antirejection therapies as used today in experimental as well as in clinical settings. Novel mucin-based adsorbers of natural anti-carbohydrate antibodies will also be described.     

A novel approach to the generation of high affinity class II-binding peptides

We have found that if core regions crucial for class II binding are incorporated in multiple copies in the same peptide molecule ("reiterative motifs"), marked enhancement of the binding capacity occurs. Isotype specificity (IAd vs IEd binding capacities) is retained in all three antigenic determinants so far analyzed (lambda rep 12-26, OVA 323-339, and hen egg lysozyme 105-120). The mechanism involved in such an effect is not clear, but experiments involving introduction of a peptide spacer between two repeated core regions do not support the notion that the effect is mediated by cross-linking of more than one MHC molecule, favoring the possibility that conformational effects or distinct subsites of interaction on the MHC molecule may be involved. Based on reiterative structures, a peptide molecule composed of only two different amino acids (Ala and His) has been produced that still retains a very high binding affinity. An 125I-radiolabeled form of this peptide has been used to demonstrate that the high binding detected is mediated by the same binding site involved in the interaction of IAd and OVA 323-339. Inhibition of Ag presentation studies further supports the immunologic relevance of the phenomena observed. Finally, we observed naturally occurring clustered binding sites in proximity of immunodominant protein regions, raising the possibility that the phenomenon might have a physiologic counterpart.     

A novel approach for determining cancer genomic breakpoints in the presence of normal DNA

CDKN2A (encodes p16(INK4A) and p14(ARF)) deletion, which results in both Rb and p53 inactivation, is the most common chromosomal anomaly in human cancers. To precisely map the deletion breakpoints is important to understanding the molecular mechanism of genomic rearrangement and may also be useful for clinical applications. However, current methods for determining the breakpoint are either of low resolution or require the isolation of relatively pure cancer cells, which can be difficult for clinical samples that are typically contaminated with various amounts of normal host cells. To overcome this hurdle, we have developed a novel approach, designated Primer Approximation Multiplex PCR (PAMP), for enriching breakpoint sequences followed by genomic tiling array hybridization to locate the breakpoints. In a series of proof-of-concept experiments, we were able to identify cancer-derived CDKN2A genomic breakpoints when more than 99.9% of wild type genome was present in a model system. This design can be scaled up with bioinformatics support and can be applied to validate other candidate cancer-associated loci that are revealed by other more systemic but lower throughput assays.     

A new approach to the design of a sequence with the highest affinity for a molecular surface.

We describe an algorithm to design the primary structures for peptides which must have the strongest binding to a given molecular surface. This problem cannot be solved by a direct combinatorial sorting, because of an enormous number of possible primary and spatial structures. The approach to solve this problem is to describe a state of each residue by two variables: (i) amino acid type and (ii) 3-D coordinate, and to minimize binding energy over all these variables simultaneously. For short chains which have no long-range interactions within themselves, this minimization can be done easily and efficiently by dynamic programming. We also discuss the problem of how to estimate specificity of binding and how to deduce a sequence with maximal specificity for a given surface. We show that this sequence can be deduced by the same algorithm after some modification of energetic parameters.     

Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin-biotin coupling. A biotin-labeled PNA was employed in the latter approach whereas the covalent immobilization included the following steps: A maleimide group was attached to the N-terminal of the PNA using N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). To generate free thiol groups for coupling, a carboxylated dextran matrix of the sensor surface was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) and thiolated by addition of cystamine dihydrochloride followed by reduction with 1, 4-dithioerythrite (DTE). Finally, the modified PNA was coupled to the sulfhydryl groups of the activated dextran matrix. Repetitive hybridizations of a single-stranded synthetic DNA oligomer to the PNAs demonstrated the superior stability of covalent immobilization compared to noncovalent immobilization. Differentiation of point mutations in the analyte molecule was accomplished at 40 degrees C using guanidine thiocyanate concentrations of 1.5-1.7 M. In further experiments, we showed that a perfectly matched PNA allows the detection of a single-stranded DNA at a sensitivity of less than 1% in a background of single-stranded DNA having a single C to T point mutation in the region complementary to the PNA. Consequently, covalently bound PNAs provide a stable and reproducible environment for the development of mutation-specific DNA analysis assays.     

Potential of biosensor technology for the characterization of interactions by quantitative affinity chromatography

This review places the characterization of interactions by biosensor technology in the broader context of their study by quantitative affinity chromatography. The general reluctance to consider biosensor-based characterization as a form of quantitative affinity chromatography on the grounds of a difference in aims of the two techniques reflects a mistaken belief that BIAcore and IAsys studies characterize the kinetics of the chemical reaction responsible for biospecific adsorption of a soluble reactant to an immobilized form of its affinity partner. It now transpires that the association and dissociation rate constants thereby determined refer to thermodynamic characterization of biospecific adsorption in terms of a single-phase model in which affinity sites are distributed uniformly throughout the liquid-phase volume accessible to the partitioning reactant—the model used for characterization of biospecific adsorption by quantitative affinity chromatography. In that light the most important attribute of biosensor technology is its potential for thermodynamic characterization of biospecific adsorption by virtue of its ability to monitor complex formation directly; and hence its potential for the characterization of interactions with affinities that are too strong for study by forms of quantitative affinity chromatography that monitor complex formation on the basis of reactant depletion from the liquid phase. Kinetic as well as thermodynamic analyses of biosensor data are described for attainment of that potential.     

A proteomic approach for evaluating the cell response to a novel histone deacetylase inhibitor in colon cancer cells

Epigenetic inactivation of gene expression is a general phenomenon associated with malignant transformation. Recently, we have found that a novel series of histone deacetylases (HDAC) inhibitors exhibit a broad-spectrum inhibition profile characterized by a marked effect on acetylation of histone and non-histone proteins. RC307, a representative compound of this series, caused a growth-inhibitory effect in colon carcinoma cells HCT116 associated with G2 accumulation and induction of apoptosis. The present study was designed to investigate the effect of RC307 on protein expressions in the HCT116 cells following treatment with cytotoxic drug concentrations. HCT116 cells were cultured in the absence or presence of RC307 and total cell lysates, as well as nuclear proteins, were extracted. The protein samples were then subjected to two-dimensional polyacrylamide gel electrophoresis, and the 2D gel images were compared to discover the protein changes caused by RC307 treatment. A total of 48 and 46 different spots were found to be modulated by RC307 in total lysates and nuclear proteome of HCT116 cell line. The modulated proteins were identified by tandem mass spectrometry. We found that RC307 exposure modulates proteins that are involved in proliferation, cell cycle regulation, apoptosis, gene expression, as well as chromatin and cytoskeleton organization.     

Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor

We have investigated whether surface plasmon resonance (SPR) sensors based on the wavelength interrogation are able to analyze protein interactions on protein arrays. The spectral SPR sensor was self-constructed and its detection limit, expressed as the minimal refractive index variation, was calculated to be 6.6x10(-5) with the signal fluctuation of 1.0x10(-5). The protein array surface was modified by a mixed thiol monolayer to immobilize proteins. Protein arrays were analyzed by the line-scanning mode of the SPR sensor, which scanned every 100 microm along the central line of array spots and the scanned results were presented by color spectra from blue to red. Glutathione S-transferase (GST)-rac1 caused a concentration-dependent increase of SPR wavelength shift on protein arrays. The surface structure of the protein arrays was analyzed by atomic force microscopy. Specific interactions of antigens with antibodies were analyzed on the protein arrays by using three antibodies and eight proteins. These results suggest that the wavelength interrogation-based SPR sensor can be used as the biosensor for the high-throughput analysis of protein interactions on protein arrays.     

A novel approach to estimate the German-wide incidence of testicular cancer

Background: Currently, only 7 out of 16 Federal States of Germany provide testicular cancer incidence rates with an estimated completeness of at least 90% which complicates the regional comparison of incidence rates. The aim of this study was to provide a novel approach to estimate the testicular cancer incidence in Germany by using nationwide hospitalization data. Methods: We used the nationwide hospitalization data (DRG statistics) of the years 2005–2006 including 16,6 million hospitalizations among men. We identified incident testicular cancer cases by the combination of a diagnosis of testicular cancer and an orchiectomy during the same hospitalization and estimated the age-specific and age-standardized (World Standard Population) incidence of testicular cancer across Federal States. We also analyzed available cancer registry data from 2005 to 2006. Results: A total of 8544 hospitalizations indicated incident testicular cancer cases in 2005–2006. The nationwide crude incidence rate of testicular cancer was 10,6 per 100.000 person-years. The ratio of the number of registered cases (cancer registry) to the estimated number of cases based on the hospitalization statistics ranged between 79% and 100%. There was only little variation of the age-standardized DRG-based incidence estimates across Federal States (range: 8,2–10,6 per 100.000 person-years). Discussion: We provided testicular cancer incidence estimates for each of the 16 Federal States of Germany based on hospitalization data for the first time. The low within-population incidence variability in Germany and high between-population incidence variability in Europe may indicate that ecologic factors play a causal role in the European variation of testicular cancer.     

Determination of organophosphorous pesticides by a novel biosensor based on localized surface plasmon resonance

Liquid and gas chromatography are commonly used to measure organophosphorus pesticides. However, these methods are relatively time consuming and require a tedious sample pretreatment. Here, we applied the localized surface plasmon resonance (LSPR) of gold nanoparticles covalently coupled with acetylcholinesterase (AChE) to create a biosensor for detecting an example of serial signals responding to paraoxon in the range of 1-100 ppb by an AChE modified LSPR sensor immersing in a 0.05 mM ACh solution. The underlying mechanism is that paraoxon prevents acetylcholine chloride (ACh) reacting with AChE by destroying the OH bond of serine in AChE. We found that the AChE modified LSPR sensors prepared by incubation with 12.5 mU/mL of AChE in phosphate buffer solution at pH 8.5 room temperature for 14 h have the best linear inhibition response with a 0.234 ppb limit of paraoxon detection. A 14% of inhibition on the sensor corresponds to the change of paraoxon concentration from 1 to 100 ppb. The sensor remained 94% of its original activity after six cycles of inhibition with 500 ppb paraoxon followed with reactivation of AChE by 0.5 mM 2-pyriding-aldoxime methoiodide (2-PAM). In addition, the sensor retains activity and gives reproducible results after storage in dry state at 4 degrees C for 60 days. In conclusion, we demonstrated that the AChE modified LSPR sensors can be used to determine the concentration of paraoxon biosensor with high sensitive and stable characteristics.     

Development of a label-free impedance biosensor for detection of antibody-antigen interactions based on a novel conductive linker

We developed a label-free impedance biosensor based on an innovative conductive linker for detecting antibody-antigen interactions. As the often used conventional long chain thiol is a poor conductor, it is not a suitable material for use in a faradaic biosensor. In this study, we adopted a thiophene-based conductive bio-linker to form a self-assembled monolayer and to immobilize the bio-molecules. We used cyclic voltammetry and impedance spectroscopy to verify the enhanced conductivity properties. Results showed that the electron transfer resistance of this new conductive linker was 3 orders of a magnitude lower than for a case using a conventional long chain thiol linker. With the decreased impedance (i.e. increased faradaic current), we can obtain a higher signal/noise ratio such that the detection limit is improved. Using fluorescence microscopy, we verified that our new conductive linker has a protein immobilization capability similar to a conventional long chain thiol linker. Also, using S100 proteins, we verified the protein interaction detection capability of our system. Our obtained results showed a linear dynamic range from 10 ng/ml to 10 μg/ml and a detection limit of 10 ng/ml. With our new conductive linker, an electrochemical impedance biosensor shows great potential to be used for point-of-care applications.     

Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on oxalate oxidase and SIRE technology

A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.     

Proteasome inhibition: a novel approach to cancer therapy

The central role of the proteasome in controlling the expression of regulators of cell proliferation and survival has led to interest in developing proteasome inhibitors as novel anticancer agents. In vitro and in vivo studies have shown that proteasome inhibitors have activity against a variety of tumor types. One of these agents, PS-341, has been tested in phase I trials in a variety of tumor types; in these trials, PS-341 treatment was well tolerated and preliminary evidence of biological activity was observed in some patients. Phase II trials in several hematological malignancies and solid tumor types are now in progress.     

Value of a multinational approach in determining the causation of cancer

MacMahon and Pugh define epidemiology as the use of knowledge on the frequency and distribution of disease to search for determinants. This paper demonstrates that a multinational approach in cancer epidemiology can be of great value in at least four circumstances: namely, the compilation and standardization of data, the assessment of risk, the pooling of study populations to obtain interpretable results, and the provision of resources for specific epidemiologic investigations. One or two examples are given for each category--the determination of international cancer incidence patterns, the evaluation of the risk posed by chemicals to man, assessment of the effects of low doses of ionizing radiation, determination of the long-term effects of exposure to asbestos substitutes, and studies on the influence of diet on esophageal cancer.     

A simple electrochemical approach to fabricate a glucose biosensor based on graphene-glucose oxidase biocomposite

We report a simple electrochemical approach for the immobilization of glucose oxidase (GOx) on reduced graphene oxide (RGO). The immobilization of GOx was achieved in a single step without any cross linking agents or modifiers. A simple solution phase approach was used to prepare exfoliated graphene oxide (GO), followed by electrochemical reduction to get RGO-GOx biocomposite. The direct electrochemistry of GOx was revealed at the RGO-GOx modified glassy carbon electrode (GCE). The electrocatalytic and electroanalytical applications of the proposed film were studied by cyclic voltammetry (CV) and amperometry. It is notable that the glucose determination has been achieved in mediator-free conditions. RGO-GOx film showed very good stability, reproducibility and high selectivity. The developed biosensor exhibits excellent catalytic activity towards glucose over a wide linear range of 0.1-27mM with a sensitivity of 1.85μAmM(-1)cm(-2). The facile and easy electrochemical approach used for the preparation of RGO-GOx may open up new horizons in the production of cost-effective biosensors and biofuel cells.     

Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach

Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format.