Lectin-modified piezoelectric biosensors for bacteria recognition and quantification

The use of lectins for microorganism biosensors fabrication is proposed. Lectins are immobilised onto a gold-plated quartz crystal for direct piezoelectric label-free transduction of the bacteria–lectin binding event using an electrochemical quartz crystal microbalance (EQCM). Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin immobilisation method and the biosensor performance. Adsorption on nonpolarised and polarised (−0.200 V) gold-coated quartz crystals and immobilisation through avidin–biotin binding were checked for Con A surface attachment. Lectin–bacteria binding was evaluated in all cases. With a crystal modified with Con A via avidin–biotin immobilisation we obtained a linear calibration plot between 5.0 × 10⁶ and 2.0 × 10⁷ cfu mL⁻¹ by measuring frequency changes with E. coli concentration 1 h after bacteria addition. A remarkable increase in sensitivity was achieved when the analytical solution contained free biotinylated Con A, as a consequence of multiple lectin adhesion to Escherichia coli cell wall, which produced an accumulation of Con A–E. coli conjugates in the form of multilayers at the electrode surface. A detection limit of approximately 1.0 × 10⁴ cfu mL⁻¹ was achieved. Moreover nonspecific adsorptions were minimised. Using Con A and lectin from Arachis hypogaea, different response profiles were achieved for Escherichia coli, Staphylococcus aureus and Mycobacterium phlei, thus demonstrating the feasibility of bacteria discrimination. An approach involving filtering of free and lectin-bound bacteria and introduction of a filter in the measuring cell allowed a significant frequency change to be obtained for an E. coli concentration of 1.0 × 10³ cfu mL⁻¹ in order to further increase the sensitivity and discriminate between viable and nonviable cells; an approach using electrochemical measurements of bacterial catalase activity was also checked.     

Ion-current signal optimization for lipid membrane-based biosensors

The conductivities of bilayer lipid membranes are greatly affected by their lipid composition. Thus it is possible to prepare membranes having substantially different background or residual ion currents as observed when a direct voltage is applied across the membrane. Physical perturbation of the membrane by phloretin, valinomycin or receptor molecules provides current increases, which can be maximized by proper choice of residual current and membrane lipid chemistry. A compromise between provision of an efficient ion current pathway and minimization of residual current is necessary to optimize signal-to-noise ratio.     

Molecular rotors--fluorescent biosensors for viscosity and flow

Viscosity is a measure of the resistance of a fluid against gradients in flow (shear rate). Both flow and viscosity play an important role in all biological systems from the microscopic (e.g., cellular) to the systemic level. Many methods to measure viscosity and flow have drawbacks, such as the tedious and time-consuming measurement process, expensive instrumentation, or the restriction to bulk sample sizes. Fluorescent environment-sensitive dyes are known to show high sensitivity and high spatial and temporal resolution. Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation and therefore exhibit two competing deexcitation pathways: fluorescence emission and non-radiative deexcitation from the TICT state. Since TICT formation is viscosity-dependent, the emission intensity of molecular rotors depends on the solvent's viscosity. Furthermore, shear-stress dependency of the emission intensity was recently described. Although the photophysical processes are widely explored, the practical application of molecular rotors as sensors for viscosity and the fluid flow introduce additional challenges. Intensity-based measurements are influenced by fluid optical properties and dye concentration, and solvent-dye interaction requires calibration of the measurement system to a specific solvent. Ratiometric dyes and measurement systems help solve these challenges. In addition, the combination of molecular rotors with specific recognition groups allows them to target specific sites, for example the cell membrane or cytoplasm. Molecular rotors are therefore emerging as new biosensors for both bulk and local microviscosity, and for flow and fluid shear stress on a microscopic scale and with real-time response.     

Electrochemical biosensors for monitoring the recognition of glycoprotein-lectin interactions

Despite the wide applicability and specificity of lectins to carbohydrate moieties, there are few lectin specific biosensors. This is attributed to the difficulty in defining the relevant experimental parameters to measure for sensing. We hereby describe the development of direct and indirect electrochemical sensors to determine the exact trace amounts of probarley lectin (ProBL) and its conversion product wheat germ agglutinin (WGA). In addition to WGA, the antigens (ProBL) employed in this study were over expressed in bacteria, isolated from protein bodies, and purified using immobilized N-acetylglusamine in order to obtain correctly folded active lectins. The amperometric immunosensor uses cell lines producing monoclonal antibody (mAB) to the pro-region of ProBL over expressed from Escherichia coli. The efficacy and sensing characteristics of the lectin were optimized using monoclonal antibody to WGA and the resulting sensor was found to detect only ProBL in the linear range 10⁻³-10² μg mL⁻¹ and a detection limit of 10⁻³ μg mL⁻¹.     

Single-walled carbon nanotube biosensors using aptamers as molecular recognition elements

We report the real-time detection of protein using SWNT-FET-based biosensors comprising DNA aptamers as molecular recognition elements. Anti-thrombin aptamers that are highly specific to serine protein thrombin were immobilized on the sidewall of a SWNT-FET using CDI-Tween linking molecules. The binding of thrombin aptamers to SWNT-FETs causes a rightward shift of the threshold gate voltages, presumably due to the negatively charged backbone of the DNA aptamers. While the addition of thrombin solution causes an abrupt decrease in the conductance of the thrombin aptamer immobilized SWNT-FET, no noticeable change was observed with elastase.     

Graphene as signal amplifier for preparation of ultrasensitive electrochemical biosensors

Early diagnosis of diseases with minimal cost and time-consumption has become achievable due to recent advances in the development of biosensors. These devices use biorecognition elements for the selective interaction with an analyte and the signal read-out is obtained via different types of transducers. The operational characteristics of biosensors have been reported as improving substantially when a diverse range of nanomaterials is employed. This review presents the construction of electrochemical biosensors based on graphene, atomically thin 2D carbon crystals, a nanomaterial currently the subject of intensive studies. Here, the most attractive directions for graphene applications in biosensor preparation are discussed, including novel detection and amplification schemes exploiting graphene’s unique electrochemical, physical and chemical properties. There is probably a very bright future for graphene-based biosensors, but much further work is required to fulfill the high expectations.     

Molecular recognition effects in polyaniline

Polyaniline (PANI) is known to dissolve in strong acids, such as sulphonic acids. PANI, in its electrically conductive form, is generally regarded to be poorly soluble in low-acidic solvents and to be infusible, closely resembling fully aromatic rigid rod polymers. We show that “less” acidic solvents and plasticizers can be found based on phenyl-phenyl interactions in combination with hydrogen bonding. The requirement is that the interactions are strong enough and, importantly, sterically match the complementary moieties of the sulphonic acid doped PANI. Dihydroxybenzenes and bisphenols are examples of such low-acidic compounds. This type of molecular recognition allows solution and melt processibility of PANI doped by generic sulphonic acid, such as methanesulphonic acid or alkylbenzenesulphonic acid. Molecular recognition is also offered as an explanation for the previously observed high solubility of camphorsulphonic acid (CSA) doped PANI in phenols.     

Molecular recognition in carnitine acyltransferases

We are designing and synthesizing rigid guests to probe the topography of the carnitine acyltransferases, regulatory enzymes in lipid metabolism. Our designs are based on structural studies of substrates and possible molecular mechanisms of enzymatic activity. Recent X-ray,¹H NMR, and force-field computational studies on carnitine and acetylcarnitine, coupled with the known stereospecificity for activity in carnitine acyltransferases, have led us to propose a molecular mechanism for acyl transfer in these enzymes. The folded conformation of an acylcarnitine is most populated and should be preferred for binding to these enzymes, because, in this conformation, the acyloxy is the most sterically accessible. There are four key recognition sites on the enzymes: I, carboxylate; II, trimethylammonium; III, coenzyme A; IV, acyl. Sites, I, II and III serve as the three loci required to create a chiral environment on the enzymes for carnitine. An addition-elimination reaction involving the formation of a tetrahedral intermediate is suggested as the mechanism for O-to-S acyl transfer. This proposed tetrahedral intermediate is chiral and the enzymes should prefer theR configuration at this center. Based on this proposal, conformationally rigid tetrahedral-intermediate analogues have been designed, synthesized and assayed. Morpholinium and 2-hydroxymorpholinium derivatives inhibit carnitine acetyltransferase and palmitoyltransferase. Because of rigidity at their two chiral centers, these inhibitors serve as probes of molecular topography of recognition sites, I, II, and IV.     

Molecular recognition and conductance in crown ethers

Crown ethers have the remarkable property of recognizing and binding specific metal cations in complex mixtures. We propose to combine molecular recognition with molecular electric conductance. The question we address is: can the event of binding a cation be sensed by a change in conductance? Specifically, we study a short molecular wire (MW) containing a crown-6 molecule connected via sulfur atoms to two gold atomic wires acting as metallic leads. Upon binding a cation, the density of states of the system is only slightly affected. This reflects the fact that the cation binding is largely electrostatic in nature and is accompanied by little electronic reorganization. Yet, the cationic binding does significantly lower conductance. We also identify strong interference affecting the conductance. A striking feature is the insensitivity of conductance to the type of ligand with the exception of the proton.     

Molecular recognition via base-pairing

Hydrogen-bonding interactions in DNA/RNA systems are a defining feature of double helical systems. They also play a critical role in stabilizing other higher-order structures, such as hairpin loops, and thus in the broadest sense can be considered as key requisites to the successful translation and replication of genetic information. This importance, coupled with the aesthetic appeal of nucleic acid base (nucleobase) hydrogen-bond interactions, has inspired the use of such motifs to stabilize a range of synthetic structures. This, in turn, has led to the formation of a number of novel ensembles. This tutorial review will discuss these structures, both from a synthetic perspective and in terms of their potential application in areas that include, but are not limited to, self-assembled macrocyclic and high-order ensemble synthesis, supramolecular polymer preparation, molecular cage construction, and energy and electron transfer modeling.     

HREELS signal processing via wavelets

In spectroscopy, the recorded spectra can often be modelled as the noisy convolution product of an instrumental function with the ‘true’ signal to be estimated. Such models have often been used for high‐resolution electron energy‐loss spectroscopy (HREELS). In this article, a new method is suggested to estimate the ‘true’ HREELS signal, i.e. the original electronic diffusion function with ‘true’ peak intensities. Our method relies upon the use of wavelets that, because they exhibit simultaneous time and frequency localization, are well‐suited for signal analysis. Firstly, a wavelet shrinkage algorithm is used to filter the noise. This is achieved by decomposing the noisy signal into an appropriate wavelet basis and then thresholding the wavelet coefficients that contain noise. This algorithm has a particular threshold related to frequency and time. Secondly, the broadening due to the instrumental response is eliminated through a deconvolution process. This step mainly rests on the existing relation between the Lipschitz regularity of the signal and the decay with scale of its wavelet coefficients and on least squares. The efficiency of this technique is highlighted by comparing the results obtained with those provided by other published methods. This work is the second in a series of three papers in this issue. The first one presents background knowledge on the wavelets required to understand the estimation methods. The third paper explores the application of wavelet filtering and deconvolution techniques to x‐ray photoelectron spectroscopy. Copyright © 2004 John Wiley & Sons, Ltd.     

Molecular recognition of amidines in water

[structure: see text] Tetraphosphonates of the general structure shown above are biomimetic hosts for bisamidinium cations in drugs such as pentamidine and DAPI. Similar to their insertion into DNA's minor groove, these drugs are often sandwiched by two tetraphosphonate hosts (2:1). The alternative binding mode (1:2) produces extremely high association constants in water of approximately 10(8) M(-)(2) ( approximately 12 kcal/mol), which can compete with the biological process.     

Data and signal processing using photochromic molecules

Photochromes are chromophores that are reversibly isomerized between two metastable forms using light, or light and heat. When photochromes are covalently linked to other chromophores, they can act as molecular photonic analogues of electronic transistors. As bistable switches, they can be incorporated into the design of molecules capable of binary arithmetic and both combinatorial and sequential digital logic operations. Small ensembles of such molecules can perform analogue signal modulation similar to that carried out by transistor amplifiers. Examples of molecules that perform multiple logic functions, act as control elements for fluorescent reporters, and mimic natural photoregulatory functions are presented.     

Molecular recognition phenomena in polyelectrolyte systems

The term “molecular recognition” is commonly used to describe various specific interactions, sometimes being not well defined. Let's assume that some species A can separately interact with either species B₁, or B₂, (or B₃, B₄ etc.) forming A·B₁, or A·B₂, (or A·B₃, or A·B₄ etc.) complexes. Here we say “recognition” assuming selective complexation of A with B₁ in the mixture containing also B₂ (B₃, B₄… etc.). At the same time it means discrimination of all other B species except B₁.     

Molecular recognition in a thermoplastic elastomer

Selective incorporation of bisurea guests in thermoplastic elastomers with poly(tetrahydrofuran) soft blocks and bisurea containing hard blocks is observed when the distances between the urea groups of host and guest match. The incorporation leads to significant modulation of mechanical properties. With bisurea-functionalized dyes as guests, a strong difference in extractability by detergent solution was shown between dyes differing by just one methylene unit between urea groups. Upon elongation of elastomer films, strong differences in alignability of matching and nonmatching dyes were observed.     

Gold nanoparticle based signal enhancement liquid crystal biosensors for DNA hybridization assays

A novel signal enhanced liquid crystal biosensor based on using AuNPs for highly sensitive DNA detection has been developed. This biosensor not only significantly decreases the detection limit, but also offers a simple detection process and shows a good selectivity to distinguish perfectly matched target DNA from two-base mismatched DNA.