Stepwise surface regeneration of electrochemical immunosensors working on biocatalyzed precipitation

A new strategy of stepwise surface regeneration for electrochemical immunosensors, working on a biocatalyzed precipitation reaction, has been developed. The strategy is based on the combination of deposited product thin-film dissolution and bound-protein displacement reactions from the modified sensor surfaces. As a model system, surfaces functionalized with biotin groups and their affinity recognition/ displacement reactions with antibiotin antibody molecules were chosen and investigated for affinity-sensing and stepwise regeneration reactions.     

The impact of antibody/epitope affinity strength on the sensitivity of electrochemical immunosensors for detecting small molecules

A displacement immunoassay involves having a labelled analogue of the analyte (the epitope) already bound to the antibody. The presence of the analyte causes a competition for antibodies, and some of the antibodies dissociates from the epitope so that it can bind with the analyte. Herein, the influence of the affinity of the surface-bound epitope for the antibody on the sensitivity and selectivity of a displacement immunosensor is explored both theoretically and experimentally. An electrochemical immunosensor described previously [1], where the dissociation of antibodies from an electrode surface causes an increase in current from surface-bound ferrocene species, is used for this purpose. As expected, the ease and effectiveness of the bound antibody being displaced is inversely related to the affinity of the antibody to the surface-bound epitope relative to the analyte in solution as expected. However, if the affinity constant is too low, selectivity and/or sensitivity are compromised. Experimental results are qualitatively compared with a simple mass-action model.

A disposable electrochemical immunosensor for prolactin involving affinity reaction on streptavidin-functionalized magnetic particles

A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL(-1) with a slope value of 7.0 nA mL ng(-1). The limit of detection was 3.74 ng mL(-1). Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.     

The immunosensors for measurement of 2,4-dichlorophenoxyacetic acid based on electrochemical impedance spectroscopy

Electrochemical impedance spectroscopy (EIS) was evaluated for the direct determination of herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Specific antibody against 2,4-D was immobilised onto different gold electrodes. Several methods of antibody immobilisation by covalent linkage to modified surface were studied. Self-assembled monolayers formed using thiocompounds as cystamine, 4-aminothiophenol (ATPh), 3,3'-dithiopropionic acid di-(N-succinimidyl ester) (DTSP) and 11-mercaptoundecanoic acid (MUA) were chosen for the sensing surface activation. Three different sensor types were tested: screen-printed disc and finger-like structures and interdigitated array (IDA) electrodes produced by lithography. The measurements were carried out in a stationary arrangement, and the reaction between hapten and the immobilised antibody was observed online. Changes of impedance parameters were evaluated, and the best immobilisation technique (using 4-aminothiophenol) was chosen for further measurements. Impedance changes due to immunocomplex formation were evaluated, and the possibility of direct monitoring of 2,4-D binding to the antibody was demonstrated at a fixed frequency. For the strip sensor, the calibration curves were constructed in concentration range from 45 nmol l(-1) to 0.45 mmol l(-1) of 2,4-D.     

Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand

Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.     

Tuning the electron affinity and secondary electron emission of diamond (100) surfaces by Diels-Alder reaction

The tuning of electron affinity and secondary electron emission on diamond (100) surfaces due to cycloaddition with 1,3-butadiene is investigated by photoemission experiments and density functional theory (DFT) calculations. A significant reduction in electron affinity up to 0.7 eV and enhancement of secondary electron emission were observed after 1,3-butadiene adsorption. The lowering of vacuum level via 1,3-butadiene adsorption is supported by DFT calculations. The C-H bonds in the covalently bonded organics on diamond contribute to the enhanced secondary electron emission and reduced electron affinity in a mechanism similar to that of C-H bonds on hydrogenated diamond surfaces. This combination of strong secondary emission and low electron affinity by the organic functionalization of diamond has potential applications in diamond-based molecular electronic devices.     

Strategy for purifying maltose binding protein fusion proteins by affinity precipitation

The maltose binding protein (MBP) affinity tag has been extensively used for protein purification. A commercial grade cationic starch could precipitate MBP or an MBP-tagged protein quantitatively by simultaneous addition of 10% (w/v) polyethylene glycol (PEG) and 50 mM calcium chloride. The precipitated MBP or MBP-tagged protein could be selectively dissociated by suspending the precipitate in 1 M NaCl. In the case of a soluble MBP fusion with a fragment of human immunodeficiency virus protein gp120, 38% of the contaminating proteins could be removed by precipitation with PEG/CaCl(2) and 100% of the fusion protein was recovered. In all cases, the purified proteins showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expected changes in fluorescence emission spectra upon binding to maltose.     

Biotinylated polypyrrole films: an easy electrochemical approach for the reagentless immobilization of bacteria on electrode surfaces

Biotinylated bacteria were immobilized onto biotin/avidin modified electrode surfaces. Firstly, an electrospotting deposition method, followed by fluorescence microscopy, showed that bacteria were specifically grafted onto a gold surface. Fluorescence intensity versus the quantity of bacteria deposited on the surface was correlated, allowing determination of the microbial saturation point. Secondly, biotinylated bacteria were immobilized onto a glassy carbon macro-electrode in order to assess immobilized bacterial denitrification activity. During a 7-day trial, the modified electrode completely denitrified 5 mM nitrate, with a rate of 1.66 mM/day over the first 3 days. When the same electrode was placed in fresh nitrate solution, the denitrification rate dropped to 0.80 mM/day. Crucially, the immobilized bacteria did not become detached from the electrode during the study.     

Surfactant bilayers for the direct electrochemical detection of affinity interactions

Simple methods of preparing the direct affinity sensors are proposed. Due to the self-consistent introduction of a hydrocarbon chain bound with oligonucleotide pentadecathymidylate (dT(15)) into the hydrophobic region of surfactant bilayer or the adsorption of antibodies on the bilayer surface, the immobilizations of oligonucleotide or antibodies were carried out correspondingly. The responses were detected by impedance spectroscopy. Whereas the specific DNA-coupling caused the decrease of real part of impedance, the antibody-antigen interaction caused the increase of real part. The obtained results give an opportunity for the development of impedimetric affinity sensors for clinical analysis or for the detection of various environmental pollutants.     

基于适配体亲和毛细管电泳-激光诱导荧光检测的凝血酶分析

以一种高亲和力适配体作为亲和荧光探针,以自建的毛细管电泳-激光诱导荧光(CE-LIF)检测装置为基础,建立了一种高灵敏、快速测定人凝血酶的方法。荧光标记的凝血酶适配体特异性地与凝血酶结合并形成稳定的凝血酶-适配体复合物,采用CE-LIF对复合物进行分离检测,从而测定凝血酶浓度。探讨了盐离子种类及浓度对适配体与凝血酶结合的影响,并在选定的电泳条件下对凝血酶检测的线性范围、检出限和重现性进行了测定。结果表明,盐离子存在的条件下适配体与凝血酶的亲和力降低,不利于两者的结合;人血清溶液中,凝血酶浓度在0.25～10 nmol/L范围内与复合物峰面积具有良好的线性相关性(r20.991),检出限(S/N3)为55.6 pmol/L;精密度和回收率测定结果均能满足分析的要求。     

诱导自催化法制备Ni-P超细非晶合金的动力学研究

加入诱导剂(如KBH~4)可使Ni^2+与H~2PO~2^-反应在常温下进行, 便于测量反应中放出的H~2体积, 有利于研究反应的动力学过程及其与反应条件下关系。反应为二级自催化反应, 反应活化能约60kj.mol^-1, pH值不影响反应速率常数, 但pH值越大, 反应诱导期越长, 一般可在40min内完成。由于加入诱导剂能一次成核,生成的Ni-P非晶合金为均匀的球形颗粒。     

Transferring complementary target DNA from aqueous solutions onto solid surfaces by using affinity microcontact printing

In this paper, we report a method of transferring complementary target DNA from an aqueous solution onto a solid surface by using affinity microcontact printing. In this approach, the probe DNA is first immobilized on the surface of an aminated poly(dimethylsiloxane) (PDMS) stamp. After a complementary target DNA hybridizes with the probe DNA on the stamp surface, the PDMS stamp is printed on an aminated glass slide. By using fluorescent microscopy, we show that only complementary target DNA, but not noncomplementary DNA, can be captured onto the surface of the stamp and then transferred to the aminated glass slide. The transfer of DNA can be attributed to the stronger electrostatic attraction between DNA and amine groups compared to the hydrogen bonds between the hybridized DNA molecules. We also investigate several factors that may influence the transfer of DNA, such as the surface density of amine groups, hybridization conditions, and contamination from unreacted PDMS monomers.     

Studies on the nef reaction induced by organic bases

Studies of the Nef reaction in the presence of primary, secondary and tertiary amines have been described. A convenient synthesis of N-substituted pyrroles and 3,6-dimethyl-4-hydrazinocarbonyl-5-(4-amino-3-nitrophenyl)-pyridazine 16 has been developed.     

Stamp transfer electrodes for electrochemical sensing on non-planar and oversized surfaces

This article describes a new alternative approach to the fabrication of printed electrochemical sensors and biosensors based on the transfer of electrode patterns comprising common conductive and insulating inks from elastomeric stamps to a wide variety of rigid and flexible substrates. This simple, low cost, yet robust methodology is demonstrated to be well-suited for the formation of electrochemical sensors on non-planar substrates and large objects/structures, which have traditionally been off-limits to conventional screen printing techniques. Furthermore, the stamped electrode devices are shown to exhibit electrochemical performance that rivals that of their screen printed counterparts and display resilience against severe mechanical deformation. The stamp transfer approach is further extended to the demonstration of epidermal electrochemical sensors through the transfer of the electrode patterns directly onto the skin. The resulting sensors demonstrate a wide range of usability, from the detection of various physiological analytes, including uric acid on the skin, to the identification of residues originating from the handling of munitions and explosives. The migration of printable electrochemical sensors to non-conventional (non-planar and/or oversized) surfaces provides new opportunities within the personal healthcare, fitness, forensics, homeland security, and environmental monitoring domains.     

Optimised layer systems for immunosensors based on the RIFS transducer

Several approaches to direct affinity sensing are currently under investigation. Prominent approaches based on optical transducers are surface plasmon resonance (SPR), grating couplers and interferometers. In this paper, investigations on the reflectometric interference spectroscopy (RIFS), a transducer based on thin dielectric films, are presented and discussed. The main advantages of the RIFS transducer are its ruggedness, the small active area and simple construction. However limits of detection so far achieved are only in the medium range. One way to improve detection is to increase the signal-to-noise ratio (s/n) in the primary signal (i.e. the reflectance spectra of the transducing film). The influence of the s/n-parameter was investigated by systematic variation of measurement parameters in a given system. A linear relationship between baseline noise of the transducer and s/n ratio of the primary signal was found with an offset value of about 1 pm rms. As the s/n value in the primary signal depends strongly on the optical properties of the transducing layer system, several single and two layer systems have been investigated for theoretical and experimental performance. An up to five-fold increase in signal intensity was achieved as compared to conventional single layer systems. In addition the improved layer systems can be prepared on low refractive index substrates (float glass), producing disposable transducers and giving improved optical systems. The experimental results indicate a thickness resolution of as low as 1 pm rms corresponding to protein coverages below 10 pg/mm². In applications where only moderate detectivity is required, these results offer the possibility for further miniaturisation of the device.     

Development of an electrochemical method for the rapid determination of microbial concentration and evidence for the reaction mechanism

A rapid amperometric method for the determination of microbial concentration is described. The micro-organisms reduce the redox mediator, potassium hexacyanoferrate(III), which is reoxidized to yield a current proportional to the microbial concentration. Escherichia coli gave a linear response (standard error (S_YX)=0.17, r=0.9999, n=5, P>99.9%) over the concentration range 10⁶–10⁸ cells ml^?1 with a response time of less than 1 min. The effect of varying temperature showed that the electrochemical response of the bacteria was linked to respiration rate. Studies with respiratory inhibitors suggested that hexacyanoferrate (III) is reduced by the segment of the respiratory chain between nicotinamide adenine dinucleotide (NADH) dehydrogenase and the terminal oxidases.     

Controlled rearrangement of adsorbed undecanol films on mica surfaces induced by an atomic force microscopy tip

An undecanol film adsorbed on a mica surface was found to rearrange and spread in a position-controlled way induced by a tapping mode atomic force microscopy (AFM) probe. AFM images of varying scanning times showed that before forming an ordered monolayer the undecanol molecules were adsorbed on the mica surface in the disordered and disorganized status. With the proceeding of scanning, these undecanol molecules gradually formed an ordered and flat film. Such behavior was caused by the formation of a stable film and had never been reported for other alcohols.     

Edge effects in an electrochemical reaction: HCOOH oxidation on a Pt ribbon

The use of a ribbon-shaped Pt electrode gives rise to edge effects of the interfacial potential, as is predicted from the potential theory in the form of the corresponding reaction-migration equation. They are studied in the bistable region of formic acid oxidation. Essentially, the edges tend to be more passive than the bulk of the electrode, which also causes a passivation (activation) transition to originate from the edges (center) of the ribbon. The experimental results are in agreement with simulations of the reaction-migration system.     

Ferrocenyl-doped silica nanoparticles as an immobilized affinity support for electrochemical immunoassay of cancer antigen 15-3

The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0-240 U mL⁻¹ with a coefficient 0.9986 and a detection limit of 0.64 U mL⁻¹ at S/N = 3.