Tibor Hianik

Immunosensors based on supported lipid membranes, protein films and liposomes modified by antibodies

Abstract The comparison of physical properties, sensitivity and reproducibility of various detection parameters of several newly developed affinity biosensors for determination of human IgE and herbicide 2,4-D is presented. The protein film based biosensors were composed of antibody (swine anti-human IgE (Q-SwaHU/IgE) or monoclonal antibody (MAb) against herbicide 2,4-D) attached to thin gold support through cysteamine or cysteamine–bovine serum albumin. The antigen (Ag)–antibody (Ab) interaction was detected by measurement of conductivity. The detection limit for human IgE was ∼1 nM, however, the sign of response depends on the method of antibody attachment to the gold support. This type of design and detection method was not appropriate for detecting small molecules, like 2,4-D. In the case of metal (stainless steel or gold) supported lipid films (s-BLM), the sensor was constructed by means of binding of avidin-modified antibody to s-BLM contained biotinylated phospholipids. Measurement of electrical capacitance, C , and elasticity modulus in direction perpendicular to the membrane plane, E ⊥ , allowed to detect the Ag–Ab reaction, that resulted in decrease of C and increase of E ⊥ . Most reproducible results have been obtained with lipid films supported on thin gold layers with detection limit of determination of 2,4-D∼1 μM. The best reproducibility and sensitivity (0.1–1 nM) have been obtained in liposome immunoassay. The Ag–Ab reaction was monitored by means of measurement the changes of ultrasound velocity in liposome suspension.

Biosensors Based on Thin Lipid Films and Liposomes

This article reports the theory and analytical applications of thin lipid films. Recent advances of electrochemical devices based on lipid membranes have lead to reports of construction of biosensors for environmental and food applications, and may provide opportunities for commercial fabrication. The methods of formation of lipid membranes on various supports including metals (silver, gold, stainless steel), agar, conducting polymers and ultrafiltration membranes have provided stabilization of lipid films with a diversity of analytical applications in real samples. Methods of immobilization and incorporation of various functional macromolecules are summarized. Several examples of the application of various methods for study of physical properties of supported bilayer lipid membranes are described. Applications of lipid-based biosensors in analytical chemistry for determination of compounds are demonstrated, including a diversity of chemical compounds such as environmental pollutants (ammonia and carbon dioxide, cyanide ions, etc.) and food toxins (aflatoxin M1and direct detection of toxin in real samples such as milk and milk preparations). Methods for application of liposomes as a sensing system are also summarized.

Cholesterol-Induced Variations in the Volume and Enthalpy Fluctuations of Lipid Bilayers

The sound velocity and density of suspensions of large unilamellar liposomes from dimyristoylphosphatidylcholine with admixed cholesterol have been measured as a function of temperature around the chain melting temperature of the phospholipid. The cholesterol-to-phospholipid molar ratio xc has been varied over a wide range (0 </= xc </= 0.5). The temperature dependence of the sound velocity number, of the apparent specific partial volume of the phospholipid, and of the apparent specific adiabatic compressibility have been derived from the measured data. These data are particularly discussed with respect to the volume fluctuations within the samples. A theoretical relation between the compressibility and the excess heat capacity of the bilayer system has been derived. Comparison of the compressibilities (and sound velocity numbers) with heat capacity traces display the close correlation between these quantities for bilayer systems. This correlation appears to be very useful as it allows some of the mechanical properties of membrane systems to be calculated from the specific heat capacity data and vice versa.

Electrochemical Aptasensor of Human Cellular Prion Based on Multiwalled Carbon Nanotubes Modified with Dendrimers: A Platform for Connecting Redox Markers and Aptamers

The present work aims to develop an electrochemical biosensor based on aptamer able to detect human cellular prions PrP(C) as a model biomarker of prion disease with high sensitivity. We designed the biosensor using multiwalled carbon nanotubes (MWCNTs) modified with polyamidoamine dendrimers of fourth generation (PAMAM G4) which in turn were coupled to DNA aptamers used as bioreceptors. Electrochemical signal was detected by a ferrocenyl redox marker incorporated between the dendrimers and aptamers interlayer. MWCNTs, thanks to their nanostructure organization and electrical properties, allow the distribution of aptamers and redox markers over the electrode surface. We demonstrated that the interaction between aptamers and prion proteins leads to variation in the electrochemical signal of the ferrocenyl group. High sensitivity with a detection limit of 0.5 pM and a wide linear range of detection from 1 pM to 10 μM has been demonstrated. Detection of PrP(C) in spiked blood plasma has been achieved in the same range of concentrations as for detection of PrP(C) in buffer. The sensor demonstrated a recovery of minimum 85% corresponding to 1 nM PrP(C) and a maximum of 127% corresponding to 1 pM PrP(C).

Reagentless biosensing using electrochemical impedance spectroscopy

The use of electrochemical impedance spectroscopy (EIS) and the conducting polymer, poly (pyrrole), as an integrated recognition and transduction system for reagentless biosensor systems was demonstrated with two different systems. The first system being an immunoassay for detection of luteinising hormone (LH) with the antibody being entrapped with in the poly (pyrrole) matrix and the second, a construct for DNA hybridisation discrimination able to differentiate single- and double-stranded DNA based on the interaction of the DNA with poly (pyrrole).

Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance

We report a multichannel surface plasmon resonance (SPR) sensor for detection of thrombin via DNA aptamers immobilized on the SPR sensor surface. A detailed investigation of the effect of the immobilisation method on the interaction between thrombin and DNA aptamers is presented. Three basic approaches to the immobilisation of aptamers on the surface of the SPR sensor are examined: (i) immobilisation based on chemisorption of aptamers modified with SH groups, (ii) immobilisation of biotin-tagged aptamers via previously immobilized avidin, neutravidin or streptavidin molecular linkers, and (iii) immobilisation employing dendrimers as a support layer for subsequent immobilisation of aptamers. A level of nonspecific binding of thrombin to immobilized human serum albumin (HSA) for each of the immobilisation methods is determined. Immobilisation of aptamers by means of the streptavidin–biotin system yields the best results both in terms of sensor specificity and sensitivity.

Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%.

Giga-seal solvent-free bilayer lipid membranes: from single nanopores to nanopore arrays

A robust platform providing a fluid lipid bilayer is in great demand not only for specific basic research on membrane proteins, but also for related applications. Here we present electrically sealing solvent-free bilayer lipid membranes spanned over arrays of cylindrical nanopores. The nanopores are milled through thin Si3N4 diaphragms using a focused ion beam (FIB). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) reveal pores with regular shapes and inter-pore spacing. Nanopore-spanning bilayer lipid membranes (npsBLMs) are formed reproducibly by directed fusion of giant unilamellar vesicles (GUVs) to the pore-containing diaphragms. The arrays of npsBLMs exhibit electrical resistances in the GΩ range, lifetimes of up to several days, and breakdown voltages above 250 mV. Perfusion robustness of the npsBLMs and low aspect ratio of the nanopores allow easy access to both sides of the bilayers. npsBLM conductance in the presence of the pore-forming toxin gramicidin D increases depending on the concentration levels. Peptide-to-lipid molar ratios can reach as high as 1 : 23. Recordings of ionic currents through alamethicin channels are possible with single-channel resolution after dielectric passivation of the substrates. This demonstrates the applicability of the platform to biophysical research of membrane proteins as well as pharmaceutical drug screening assays.

Thin-film microsystem applicable in (bio)chemical sensors

Abstract A strip thin-film microsystem based on the silicon diaphragm or glass substrate, thin-film heater and planar interdigital array of electrodes has been developed. The used thin-film and silicon micromachining technologies are compatible with microelectronics IC technologies which allow a further development towards smart sensors. Description of some possible applications of thin-film microsystem is presented: (i) gas-resistance sensor; (ii) electrochemical voltammetric sensor, and (iii) biosensor based on the supported bilayer lipid membranes. The last one is presented in more details because the lipid bilayers seem to be the attractive candidates for biosensors applications.

Electrostriction of lipid bilayers on a solid support. Influence of hydrocarbon solvent and d.c. voltage

Abstract A new method for forming bilayer lipid membranes on solid substrates (s-BLMs) was recently developed by Tien and Salamon. They showed that s-BLMs could be used in practical applications for the development of molecular electronic devices and biosensors. Using the electrostriction method, we have studied the elasticity modulus perpendicular to the membrane plane ( E ⊥ ), dynamic viscosity coefficient (η), electrical capacitance ( C ) and membrane potential (ΔΦ m ) of s-BLMs formed from soybean phosphatidylcholine as a function of length of hydrocarbon chain of the solvent, cholesterol concentration and d.c. voltage applied to the membrane. We found that E ⊥ of s-BLMs is one order of magnitude less than that for conventional BLMs formed in the aqueous phase. Unlike that for BLMs, E ⊥ of s-BLMs did not depend on the length of hydrocarbon chain of the solvent or the cholesterol concentration in the lipid solution. The parameters E ⊥ , η and C of s-BLMs showed a complicated behaviour as a function of the amplitude, polarity and rate of change of applied d.c. voltage. In addition, s-BLMs are considerably more stable than BLMs: their electrical breakdown voltage can reach 1.5 V. Significant differences between s-BLMs and BLMs are very probably due to differences in bilayer structure. A model of s-BLM structure and compressibility explaining these differences is presented.