Integrated capillary fluorescence DNA biosensor

Covalent attachment of dsDNA molecules inside a glass capillary without the need for hybridization is described. It is shown that the glass capillary has a surface density of 2.5 x 10(13) molecules/cm(2) with specific binding capacity of 62.5%. The resulting substrate was used to develop a biosensor for determining fluorescent organic analytes and metal binding with DNA. The biosensor combines highly specific immobilization chemistry with a capillary-geometry flow cell arrangement. The results show that fluorescent dyes are retained in the dsDNA-modified surface and that exposure to concentrations of nickel and lead ions resulted in a recoverable, highly reproducible diminishment of the fluorescence intensity.     

Self-assembled monolayer-assisted silicon nanowire biosensor for detection of protein-DNA interactions in nuclear extracts from breast cancer cell

The large number of estrogen receptor (ER) binding sites of various sequence patterns requires a sensitive detection to differentiate between subtle differences in ER-DNA binding affinities. A self-assembled monolayer (SAM)-assisted silicon nanowire (SiNW) biosensor for specific and highly sensitive detection of protein-DNA interactions, remarkably in nuclear extracts prepared from breast cancer cells, is presented. As a typical model, estrogen receptor element (ERE, dsDNA) and estrogen receptor alpha (ERα, protein) binding was adopted in the work. The SiNW surface was coated with a vinyl-terminated SAM, and the termination of the surface was changed to carboxylic acid via oxidation. DNA modified with amine group was subsequently immobilized on the SiNW surface. Protein-DNA binding was finally investigated by the functionalized SiNW biosensor. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were employed to characterize the stepwise functionalization of the SAM and DNA on bare silicon surface, and to visualize protein-DNA binding on the SiNW surface, respectively. We observed that ERα had high sequence specificity to the SiNW biosensor which was functionalized with three different EREs including wild-type, mutant and scrambled DNA sequences. We also demonstrate that the specific DNA-functionalized SiNW biosensor was capable of detecting ERα as low as 10 fM. Impressively, the developed SiNW biosensor was able to detect ERα-DNA interactions in nuclear extracts from breast cancer cells. The SAM-assisted SiNW biosensor, as a label-free and highly sensitive tool, shows a potential in studying protein-DNA interactions.     

Investigations of the antioxidant properties of plant extracts using a DNA-electrochemical biosensor

In this work, the results of a method based on an electrochemical biosensor to detect DNA damage in vitro for the evaluation of the antioxidant properties of plant extracts are reported. The biosensor consisted of a dsDNA immobilized on a screen-printed electrode surface (SPE). DNA damage was promoted by the generation of the *OH radicals via Fenton-type reaction. The interaction of the radical species with immobilised DNA in the absence and presence of antioxidants was evaluated by means of changes in the guanine oxidation peak obtained by square wave voltammetry. The results demonstrated that the DNA-based biosensor is suitable as a rapid screening test for the evaluation of antioxidant properties of samples.     

Using exonuclease III to enhance electrochemical detection of natural DNA damage in layered films

The natural double-stranded DNA (dsDNA) was immobilized on electrode surface by layer-by-layer assembly, forming PSS/PDDA/dsDNA films (PSS, poly(styrene-sulfonate); PDDA, poly(diallyldimethylammonium)), and used to detect DNA damage electrochemically. The DNA lesion induced by the alkylating agent methyl methanesulfonate (MMS) could be detected by cyclic voltammetry with ruthenium(II) tris(2,2'-bipyridyl) (Ru(bpy)(3)(2+)) in solution. After treated by E. coli exonuclease III enzyme, the electrocatalytic oxidation peak of the films was further amplified and greatly enhanced because the enzyme could convert those apurinic sites caused by MMS in the damaged dsDNA into single-stranded DNA regions and make more guanines in the DNA become exposed. This approach provided a novel idea for constructing DNA biosensor in sensitive screening of genetoxic chemicals in vitro.     

Invasive cleavage reactions on DNA-modified diamond surfaces

Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions. An important application of these surfaces is for surface invasive cleavage reactions, in which target DNA strands added to the solution may result in specific cleavage of surface-bound probe oligonucleotides, permitting analysis of single nucleotide polymorphisms (SNPs). Our previous work demonstrated the feasibility of performing such cleavage reactions on planar gold surfaces using PCR-amplified human genomic DNA as target. The sensitivity of detection in this earlier work was substantially limited by a lack of stability of the gold surface employed. In the present work, detection sensitivity is improved by a factor of approximately 100 (100 amole of DNA target compared with 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface.     

Nanocrystalline diamond modified gold electrode for glucose biosensing

Boron-doped diamond has drawn much attention in electrochemical sensors. However there are few reports on non-doped diamond because of its weak conductivity. Here, we reported a glucose biosensor based on electrochemical pretreatment of non-doped nanocrystalline diamond (N-NCD) modified gold electrode for the selective detection of glucose. N-NCD was coated on gold electrode and glucose oxidase (GOx) was immobilized onto the surfaces of N-NCD by forming amide linkages between enzyme amine residues and carboxylic acid groups on N-NCD. The anodic pretreatment of N-NCD modified electrode not only promoted the electron transfer rate in the N-NCD thin film, but also resulted in a dramatic improvement in the reduction of the dissolved oxygen. This performance could be used to detect glucose at negative potential through monitoring the current change of oxygen reduction. The biosensor effectively performs a selective electrochemical analysis of glucose in the presence of common interferents, such as ascorbic acid (AA), acetaminophen (AP) and uric acid (UA). A wide linear calibration range from 10 microM to 15 mM and a low detection limit of 5 microM were achieved for the detection of glucose.     

Boron δ-doped (111) diamond solution gate field effect transistors

A solution gate field effect transistor (SGFET) using an oxidised boron δ-doped channel on (111) diamond is presented for the first time. Employing an optimised plasma chemical vapour deposition (PECVD) recipe to deposit δ-layers, SGFETs show improved current-voltage (I-V) characteristics in comparison to previous similar devices fabricated on (100) and polycrystalline diamond, where the device is shown to operate in the enhancement mode of operation, achieving channel pinch-off and drain-source current saturation within the electrochemical window of diamond. A maximum gain and transconductance of 3 and 200μS/mm are extracted, showing comparable figures of merit to hydrogen-based SGFET. The oxidised device shows a site-binding model pH sensitivity of 36 mV/pH, displaying fast temporal responses. Considering the biocompatibility of diamond towards cells, the device's highly mutable transistor characteristics, pH sensitivity and stability against anodic oxidation common to hydrogen terminated diamond SGFET, oxidised boron δ-doped diamond SGFETs show promise for the recording of action potentials from electrogenic cells.     

A lipid-based method for the preparation of a piezoelectric DNA biosensor

A piezoelectric DNA biosensor was prepared by immobilizing DNA probes on a quartz crystal microbalance (QCM) using a lipid-based method. A QCM electrode was coated with a hybrid bilayer membrane composed of an octadecanethiol monolayer and a lipid monolayer containing biotinylated lipids to establish biotin groups on the electrode surface. A DNA biosensor was prepared by sequentially immobilizing avidin and the biotinylated probe. The DNA biosensor was stable throughout repeated surface regeneration and showed higher sensitivity than that prepared by the conventional chemical method using diimide. We also optimized the surface regeneration conditions and flow rate for flow injection analysis.     

Electroimmobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purification

Proinsulin C-peptide was electroimmobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.     

Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine

An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds.     

Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment.

Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH(3)I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF(3)/methanol. The TLC method was found to be less reliable than the new CH(3)I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH(3)I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction.We conclude that the CH(3)I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC.     

Reversible surface thiol immobilization of carboxyl group containing haptens to a BIAcore biosensor chip enabling repeated usage of a single sensor surface

We describe a reversible immobilization method for carboxyl group containing haptens that makes the repeated usage of a BIAcore biosensor chip possible. Haptens which are immobilized according to the surface thiol method can be removed completely from the sensor surface again by a reducing step. In the first part of our study, analogues of the herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were immobilized in succession to a biosensor surface of a BIAcore surface plasmon resonance instrument according to the thiol coupling method. Direct kinetic analysis of these ligands to a polyclonal anti-2,4-dichlorophenoxyacetic acid antibody were performed using these biosensor surfaces. In the second part of the study, different amounts of 2,4-dichlorophenoxyacetic acid were sequentially immobilized onto the same biosensor surface in order to generate a calibration plot for 2,4-dichlorophenoxyacetic acid. Using this plot, the quantitative detection of the herbicide down to a concentration of 0.1 microg/mL, the maximum admissible concentration of pesticides in drinking water, is possible.     

Electrochemical nucleic acid hybridization biosensor based on poly(L-Aspartic acid)-modified electrode for the detection of short oligonucleotide sequences related to hepatitis C virus 1a

Abstract

This work describes, for the first time, the fabrication of poly(L-aspartic acid) (PAA) film modified pencil graphite electrode (PGE) for the detection of hepatitis C Virus 1a (HCV1a). The presence of PAA on the electrode surface can provide free carboxyl groups for covalent binding of biomolecules. The PGE surface was first coated with PAA via electropolymerization of the L-aspartic acid, and avidin was subsequently attached to the PAA modified electrode by covalent attachment. Biotinylated HCV1a probes were immobilized on avidin/PAA/PGE via avidin-biotin interaction. The morphology of PAA/PGE was examined using a scanning electron microscope. The hybridization events were monitored with square wave voltammetry using Meldola’s blue (MDB). Compared to non-complementary oligonucleotide sequences, when hybridization was carried out between the probe and its synthetic targets or the synthetic polymerase chain reaction analog of HCV1a, the highest MDB signal was observed. The linear range of the biosensor was 12.5 to 100?nM and limit of detection was calculated as 8.7?nM. The biosensor exhibited favorable stability over relatively long-term storage. All these results suggest that PAA-modified electrode can be used to nucleic acid biosensor application and electropolymerization of L-aspartic acid can be considered as a good candidate for the immobilization of biomolecules.     

Stimulatory effect of serum albumin on the proliferation of serum-free SV40-transformed Balb/c 3T3 cells

Commercial serum albumins have been found to be able to stimulate the proliferation of Balb/c 3T3 cells transformed by SV40, but not that of the normal counterpart. The effect is most pronounced with crystalline samples of albumin depleted of both globulin and fatty acid components, and depends on conditions used for the attachment and on seeding density. Physical and chemical treatments aimed to remove tightly bound impurities do not abolish the activity of fatty acid free serum albumin, thus supporting the idea that albumin per se is mitogenic towards these cells.     

Electrochemical DNA biosensor for screening of chlorinated benzene pollutants

In this work, an electrochemical DNA biosensor based on double-stranded DNA modified Au electrode (dsDNA/Au) was proposed for the rapid screening and detection of chlorinated benzenes pollutants, in which redox-active methylene blue (MB) was used to amplify the interaction between dsDNA and the target analyte. Using hexachlorobenzene (HCB) as a model analyte of chlorinated benzenes, the biosensor demonstrated a linear response with the logarithm of HCB concentrations from 100 pmol L(-1) to 100 nmol L(-1). The obtained detection limit was 30 pmol L(-1), which was remarkably superior to other biosensors. The interaction mechanism of the biosensor with HCB was proposed based on systematical characterization by cyclic voltammetry (CV), differential pulse voltammetry (DPV), UV-vis spectrometry and electrochemical quartz crystal microbalance (EQCM). Further studies revealed that the biosensor could screen chlorinated benzenes in the presence of 100 fold amount of other co-existing chemicals (ethyl acetate and sodium oxalate, etc.), and the response signal of the biosensors for different chlorinated benzenes was correlative to their respective toxicity. The proposed biosensor proved to be a promising "alarm" tool for rapid screening of chlorinated benzenes in real water samples.     

Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening

Many serotype 3 reoviruses bind to two different host cell molecules, sialic acid and an unidentified protein, using discrete receptor-binding domains in viral attachment protein, final sigma1. To determine mechanisms by which these receptor-binding events cooperate to mediate cell attachment, we generated isogenic reovirus strains that differ in the capacity to bind sialic acid. Strain SA+, but not SA-, bound specifically to sialic acid on a biosensor chip with nanomolar avidity. SA+ displayed 5-fold higher avidity for HeLa cells when compared with SA-, although both strains recognized the same proteinaceous receptor. Increased avidity of SA+ binding was mediated by increased k(on). Neuraminidase treatment to remove cell-surface sialic acid decreased the k(on) of SA+ to that of SA-. Increased k(on) of SA+ enhanced an infectious attachment process, since SA+ was 50-100-fold more efficient than SA- at infecting HeLa cells in a kinetic fluorescent focus assay. Sialic acid binding was operant early during SA+ attachment, since the capacity of soluble sialyllactose to inhibit infection decreased rapidly during the first 20 min of adsorption. These results indicate that reovirus binding to sialic acid enhances virus infection through adhesion of virus to the cell surface where access to a proteinaceous receptor is thermodynamically favored.     

A long-term operating algal biosensor for the rapid detection of volatile toxic compounds

A method for the stable long-termimmobilization of microalgal cultures wasdeveloped. Immobilized Klebsormidiumcultures were used in a biosensor systemfor air monitoring. The measurement ofbiosensor response was performed usingseveral parameters obtained from the PAMchlorophyll fluorescence technique. To testbiosensor response on toxic compoundsmethanol and formaldehyde, classified asvolatile organic compounds (VOC), were usedin concentrations relevant to human health.Our results showed that quantitativedetection of methanol vapour by thebiosensor is possible within minutes atconcentrations from 75 to 350 ppm.Additionally, due to reversibility of thebiosensor response signal and long-termstability, the biosensor was operationalfor 30 days with repeated exposure periodsto methanol vapour. We conclude that thealgal biosensor, in principle, is suitableto detect volatile toxic compounds such asmethanol and formaldehyde.     

Surface plasmon resonance biosensor for dopamine using D3 dopamine receptor as a biorecognition molecule

In modern biomedical technology, development of high performance sensing methods for dopamine (DA) is a critical issue because of its vital role in human metabolism. We report here, a new kind of bioaffinity sensor for DA based on surface plasmon resonance (SPR) using a D(3) dopamine receptor (DA-RC) as a recognition element. A conjugate of DA was synthesized using bovine serum albumin (BSA) protein and was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The biosensor surface was constructed by the immobilization of the DA-BSA conjugate onto an SPR gold surface by physical adsorption. Atomic force microscopy (AFM) investigations revealed that the DA-BSA conjugate was homogeneously distributed over the sensor surface. Specific interaction of the DA-RC with the immobilized DA-BSA conjugate was studied by SPR. Based on the principle of indirect competitive inhibition, the biosensor could detect DA in a linear dynamic range from 85 pg/ml (ppt) to 700 ng/ml (ppb). The biosensor was highly specific for DA and showed no significant interference from potent interferences such as ascorbic acid (AA), uric acid (UA) and other DA analogues viz., 3,4 dihydroxyphenyl acetic acid (DOPAC) and 3-(3,4 dihydroxyphenyl)-alanine (DOPA). The sensor surface displayed a high level of stability during repeated regeneration and affinity reaction cycles. Since this biosensor is simple, effective and is based on utilization of natural receptor, our study presents an encouraging scope for development of portable detection systems for in-vitro and in-vivo measurement of DA in clinical and medical diagnostics.     

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.