A rapid method for determining Mycobacterium tuberculosis based on a bulk acoustic wave impedance biosensor

The bulk acoustic wave impedance biosensor was set up and used to monitor the growth of Mycobacterium tuberculosis (M.TB). This sensor is rapid, simple, sensitive (lower limit is 2×10³ cells ml⁻¹) and cheap (easy to generalize). The typical response curve was different from other bacteria's, such as Escherichia coli, Staphylococcus aureus, Proteus mirabilis. The frequency detection time was used to quantitatively determine M.TB. It was proportional to logarithm of the initial concentration of M.TB in the range of 2×10³-3×10⁷ cells ml⁻¹. The set up sensor was applied to the direct diagnosis of M.TB samples. The interference of other bacteria was eliminated by pretreatment. Our results confirmed that the use of the set up biosensor was reliable, sensitive. It gives out the potential use for determining M.TB.     

Ion chromatographic determination of salicylate in human serum with a bulk acoustic wave sensor as detector

Salicylate is a hydrolysis product of salicylate-containing drugs (such as aspirin) in patients' blood. Monitoring of this ion in blood is helpful for diagnosing of overdosage of these drugs. The present paper describes an ion chromatography (IC) method developed for determination of total salicylate in human serum, in which a hulk acoustic wave (BAW) sensor was used as detector; 0.5 mmoI/L sodium carbonate (Na₂CO₃, pH 8.5) served as mobile phase. Interference in the determination was negligible. The method is simple, rapid, accurate, and precise. Serum salicylate was analyzed using both the proposed IC-BAW method and the classical Trinder spec-trophotometric method, and the results showed that the two method agreed well.     

Electropolymerized m-phenylenediamine as a means to immobilize active protein on thickness-shear-mode quartz crystal

Electropolymerized m-phenylenediamine was used as an active coating for immobilizing urease and lectin on a gold-plated thickness-shear-mode (TSM) crystal. To enhance effectiveness of immobilization. a bilayer polymer film composed of polyaniline and poly-m-phenylenediamine was proposed. Compared with single poly-m-phenylenediamine film, the bilayer polymer film gave better results in terms of immobilizing capacity, stability and reproductivity. On this bilayer-film-coated TSM quartz crystal, the amount of immobilized lectin was estimated about 1.8 mug/cm(2). Detection of purified human erythrocytes is demonstrated as an example of potential application of this lectin-modified TSM biosensor in clinic.     

Bovine serum albumin as a molecular sensor for the discrimination of complex metabolite samples

The potential for using serum albumin (SA) as a broadly applicable molecular sensor was explored in an effort to develop a method for rapid analysis of complex metabolite samples. SA is a protein present at high concentration in blood, which transports a diverse set of compounds including fatty acids, hormones, and drugs. The effectiveness of the bovine ortholog (BSA) as a molecular sensor was tested by analyzing the pool of small molecules bound to the protein after a brief incubation with complex fluids of biological origin. As an initial test, three varietals of red wine were readily distinguished. Further analysis using four varietals of white wine also showed clear separation. In a second analysis using urine, animals in hemorrhagic shock were separated from a group of comparably treated controls. A time course analysis showed that recovery from injury could also be followed using the assay. This finding is significant as there currently is no method or biomarker for predicting the onset of shock. Comparison of samples was based on liquid chromatography mass spectrometry (LCMS) analysis of compounds selectively bound by BSA. Analysis of the samples after protein selection revealed a significant reduction in complexity and clear separation of groups by Principle Component Analysis (PCA). These results show the potential for using cargo-carrying proteins as molecular sensors for screening complex samples without the need for prior knowledge of sample composition or concentration and may streamline elucidation of biomarkers.     

Selective and sensitive homogenous assay of serum albumin with 1-anilinonaphthalene-8-sulphonate as a biosensor

Homogenous selective assay of albumin (ALB) in clinical sera was tested with 1-anilinonaphthalene-8-sulphonate (ANS) as Förster-resonance-energy-transfer (FRET) acceptor of tryptophan residues and biosensor of ALB. Between the excitation at 280 and 350 nm, the ratio of the fluorescence at 470 nm of free ANS in ethanol was about 1.9 while that of the complexes of ALB and ANS was about 3.9, supporting FRET in complexes of ANS and ALB. ANS below 1.0 mM saturated one site of ALB with K_d of about 0.13 μM in 20 mM sodium phosphate buffer at pH 7.0. For selective assay of ALB, 0.30 μM ANS was used to quantify fluorescence of the complexes at 470 nm under the excitation at 280 nm. ALB from 1.8 to 25 nM was quantified, whose lower limit was below 1% than that by bromocresol green assay while one-third than that by immunoturbidimetric assay. Globular proteins at comparable levels gave negligible signals. This new method showed reasonable resistance to other interfering substances in clinical sera. Quantities of ALB in clinical sera by this method were consistent with those by bromocresol green assay and immunoturbidimetric assay. Hence, homogenous assay of ALB with ANS as FRET biosensor was effective.     

Mucin/carbopol matrix to immobilize oxalate oxidase in a urine oxalate amperometric biosensor

An enzymatic amperometric electrode with extended analytical range and improved stability for oxalate determination has been developed. Glutarlaldehyde/mucin/carbopol matrix was used for the crosslinking of the enzyme between polymeric membranes to form a classical laminate construction (sandwich) and compared with the glutaraldehyde/mucin/enzyme and glutaraldehyde/albumin/enzyme.The use of a sulphonated membrane as internal membrane allowed rejection of the most important electrooxidable urine interferents. The recovery assays were highly satisfactory. The wide linear response in the range 2-400 μM after 1/10 urine dilution (corresponding to 20-4000 μM) made it suitable for clinical range. High correlation with the standard spectrophotometric method was obtained (r² = 0.98, y = 0.89x, n = 25).     

Surface acoustic wave biosensor as a tool to study the interaction of antimicrobial peptides with phospholipid and lipopolysaccharide model membranes

Surface acoustic wave biosensors are a powerful tool for the study of biomolecular interactions. The modulation of a surface-confined acoustic wave is utilized here for the analysis of surface binding. Phase and amplitude of the wave correspond roughly to mass loading and viscoelastic properties of the surface, respectively. We established a procedure to reconstitute phospholipid and lipopolysaccharide bilayers on the surface of a modified gold sensor chip to study the mode of action of membrane-active peptides. The procedure included the formation of a self-assembled monolayer of 11-mercaptoundecanol, covalent coupling of carboxymethyl-dextran, and subsequent coating with a poly- l-lysine layer. The lipid coverage of the surface is highly reproducible and homogeneous as demonstrated in atomic force micrographs. Ethanol/triton treatment removed the lipids completely, which provided the basis for continuous sequences of independent experiments. The setup was applied to investigate the binding of human cathelicidin-derived peptide LL32, as an example for antimicrobial peptides, to immobilized phosphatidylserine membranes. The peptide-membrane interaction results in a positive phase shift and an increase in amplitude, indicating a mass increase along with a loss in viscosity. This suggests that the bilayer becomes more rigid upon interaction with LL32.     

Bovine serum albumin as a universal suppressor of non-specific peptide binding in vials prior to nano-chromatography coupled mass-spectrometry analysis

Non-specific binding (NSB) is a well-known problem for any application that deals with ultralow analyte quantities. The modern nano-flow chromatography coupled tandem mass-spectrometry (nanoLC-MS/MS) works with the lowest conceivable analyte concentrations. However, while the NSB problem is widely accepted and investigated for metabolomics and single-peptide medicine-related assays, its impact is not studied for complex peptide mixtures in proteomic applications. In this work peptide NSB to a common plastic autosampler vial was studied for a model mixture of 46 synthetic peptides. A significant NSB level was demonstrated for total peptide concentrations of up to 1 mg mL⁻¹. Different agents were tried for NSB suppression and compatibility with nanoLC-MS/MS analysis: a chaotropic agent, an amino acid mixture, a peptide mixture and a protein solution. The first two were inefficacious. The peptide matrix blocked NSB, however, it also led to analyte ionization suppression in nanoLC-MS/MS. The protein solution (0.1% BSA) efficiently eliminated NSB, while a trap-elute nanoHPLC configuration together with a small-pore reverse-phased sorbent effectively and quantitatively extracted the model peptides and depleted protein material from the sample. Higher protein concentration partially impeded peptide extraction. Thus, the 0.1% BSA solution might be regarded as an effective non-interfering blockader of NSB for sample resuspension and storage in an autosampler prior to LC-MS/MS analysis.     

Graphene oxide as a nanocarrier for loading and delivery of medicinal drugs and as a biosensor for detection of serum albumin

The interaction of graphene oxide (GO), a medicinal drug (10-hydroxy camptothecin (HCPT)), and bovine serum albumin (BSA) was investigated with the aim of developing a method for the analysis of serum albumin proteins. It was demonstrated that HCPT could be readily loaded onto GO via the π–π stacking interaction, and the delivery of HCPT to BSA was improved in the presence of GO; this, in turn, facilitated the binding interaction of HCPT and BSA. Chemometrics methods, multivariate curve resolution-alternating least squares (MCR-ALS) and parallel factor analysis (PARAFAC), were applied to resolve spectral data, and this assisted in the elucidation of the above interaction. GO was found to enhance the fluorescence response of HCPT to BSA, and thus, a low cost fluorescence bio-sensing platform was developed for fluorescence-enhanced detection of BSA based on GO. The satisfactory analytical performance of this biosensor for BSA was attributed to the structure and electronic properties of GO.     

Detection of DNA hybridisation in a diluted serum matrix by surface plasmon resonance and film bulk acoustic resonators

Nanomolar quantities of single-stranded DNA products ~100 nucleotides long can be detected in diluted 1% serum by surface plasmon resonance (SPR) and film bulk acoustic resonators (FBARs). We have used a novel FBAR sensor in parallel with SPR and obtained promising results with both the acoustic and the optical device. Oligonucleotides and a repellent lipoamide, Lipa-DEA, were allowed to assemble on the sensor chip surfaces for only 15 min by dispensing. Lipa-DEA surrounds the analyte-binding probes on the surface and effectively reduces the non-specific binding of bovine serum albumin and non-complementary strands. In a highly diluted serum matrix, the non-specific binding is, however, a hindrance, and the background response must be reduced. Nanomolar concentrations of short complementary oligos could be detected in buffer, whereas the response was too low to be measured in serum. DNA strands that are approximately 100 base pairs long at concentrations as low as 1-nM could be detected both in buffer and in 1% serum by both SPR and the FBAR resonator.     

Bovine serum albumin adsorption on nano-rough platinum surfaces studied by QCM-D

The adsorption of bovine serum albumin (BSA) on platinum surfaces with a root-mean-square roughness ranging from 1.49nm to 4.62nm was investigated using quartz crystal microbalance with dissipation (QCM-D). Two different BSA concentrations, 50microg/ml and 1mg/ml, were used, and the adsorption studies were complemented by monitoring the antibody interaction with the adsorbed BSA layer. The adsorption process was significantly influenced by the surface nano-roughness, and it was observed that the surface mass density of the adsorbed BSA layer is enhanced in a non-trivial way with the surface roughness. From a close examination of the energy dissipation vs. frequency shift plot obtained by the QCM-D technique, it was additionally observed that the BSA adsorption on the roughest surface is subject to several distinct adsorption phases revealing the presence of structural changes facilitated by the nano-rough surface morphology during the adsorption process. These changes were in particular noticeable for the adsorption at the low (50microg/ml) BSA concentration. The results confirm that the nano-rough surface morphology has a significant influence on both the BSA mass uptake and the functionality of the resulting protein layer.     

Monitoring fermentation media by ion-chromatography with a double chamber bulk acoustic wave detector

A reliable ion-chromatographic (IC) method with a novel double chamber bulk acoustic wave (DCBAW) detector was developed for monitoring five important inorganic cations (Na(+), K(+), NH(4)(+), Ca(2+), Mg(2+)) in biological culture media. A Shimpack IC-C1 analytical column with 5 mM hydrochloric acid and 2 mM acetonitrile as mobile phase was used. All investigated inorganic cations could be detected and qualified in the range of 0.1-100 mg/l. Results showed that the consumption velocities of Mg(2+) and K(+) are related to the growth of the cells and decrease fastest during the first 2-5 h. Mg became a growth limiting factor at concentration below 0.1 mg/l. The concentrations of the other cations stayed nearly constant during the whole fermentation process. The simple sample preparation, short analytical time and accurate results made it a useful tool for the on-line monitoring, controlling and optimization of the fermentation process.     

Silicon surface modification with a mixed silanes layer to immobilize proteins for biosensor with imaging ellipsometry

One kind of surface modification method on silicon wafer was presented in this paper. A mixed silanes layer was used to modify silicon surface and rendered the surface medium hydrophobic. The mixed silanes layer contained two kinds of compounds, aminopropyltriethoxysilane (APTES) and methyltriethoxysilane (MTES). A few of APTES molecules in the layer was used to immobilize covalently human immunoglobulin G (IgG) on the silicon surface. The human IgG molecules immobilized covalently on the modified surface could retain their structures well and bind more antibody molecules than that on silicon surface modified with only APTES. This kind of surface modification method effectively improved the sensitivity of the biosensor with imaging ellipsometry.     

Bovine serum albumin unfolding at the air/water interface as studied by dilational surface rheology

Measurements of the surface dilational elasticity close to equilibrium did not indicate significant distinctions in the surface conformation of different forms of bovine serum albumin (BSA) in a broad pH range. At the same time, the protein denaturation in the surface layer under the influence of guanidine hydrochloride led to strong changes in the kinetic dependencies of the dynamic surface elasticity if the denaturant concentration exceeded a critical value. It was shown that the BSA unfolding at the solution surface occurred at lower denaturant concentrations than in the bulk phase. In the former case, the unfolding resulted in the formation of loops and tails at surface pressures above 12 mN/m. The maximal values of the dynamic surface elasticity almost coincided with the corresponding data for the recently investigated solutions of β-lactoglobulin, thereby indicating a similar unfolding mechanism.     

A single DNA aptamer functions as a biosensor for ricin

The use of microorganisms or toxins as weapons of death and fear is not a novel concept; however, the modes by which these agents of bioterrorism are deployed are increasingly clever and insidious. One mechanism by which biothreats are readily disseminated is through a nation's food supply. Ricin, a toxin derived from the castor bean plant, displays a strong thermostability and remains active at acidic and alkaline pHs. Therefore, the CDC has assigned ricin as a category B reagent since it may be easily amendable as a deliberate food biocontaminate. Current tools for ricin detection utilize enzymatic activity, immunointeractions and presence of castor bean DNA. Many of these tools are confounded by complex food matrices, display a limited dynamic range of detection and/or lack specificity. Aptamers, short RNA and single stranded DNA sequences, have increased affinity to their selected receptors, experience little cross-reactivity to other homologous compounds and are currently being sought after as biosensors for bacterial contaminants in food. This paper describes the selection and characterization of a single, dominant aptamer, designated as SSRA1, against the B-chain of ricin. SSRA1 displays one folding conformation that is stable across 4-63 °C (ΔG = -5.05). SSRA1 is able to concentrate at least 30 ng mL(-1) of ricin B chain from several liquid food matrices and outcompetes a currently available ELISA kit and ricin aptamer. Furthermore, we show detection of 25 ng mL(-1) of intact ricin A-B complex using SSRA1 combined with surface enhanced Raman scattering technique. Thus, SSRA1 would serve well as pre-analytical tool for processing of ricin from liquid foods to aid current diagnostics as well as a sensor for direct ricin detection.     

Electropolymerised o-phenylenediamine film as means of immobilising lactate oxidase for a L-lactate biosensor

The immobilisation of L-lactate oxidase at a platinum electrode was achieved by entrapping the enzyme within an o-phenylenediamine film at 0.65 V (vs. Ag/AgCl). Anodic detection of the product of the enzymatic reaction, i.e., hydrogen peroxide, at 0.75 V (vs. Ag/AgCl) was employed for the quantification of L-lactate using amperometric batch and flow injection methods. This technique allows the enzyme to be entrapped in a strongly adherent thin membrane. The sensor exhibits a very fast response time, an active enzyme loading of 5 mU/cm(2) electrode surface, and high sensitivity with a detection limit of 2.46 x 10(-7)M. A sample throughput of 180/hr, precision of 3.53% for 25 injections and linearity up to 1.5mM were obtained in flow injection analysis studies. The one-step procedure for sensor preparation requires 20 min, and the discriminative properties of the polymer film show great promise as a means of excluding interfering compounds commonly found in serum.