Martin Hedström

Sub-attomolar detection of cholera toxin using a label-free capacitive immunosensor

A label-free immunosensor for the direct detection of cholera toxin (CT) at sub-attomolar level has been developed based on potential-step capacitance measurements. Anti-CT antibody was adsorbed on gold nanoparticles (AuNPs) incorporated on a polytyramine-modified gold electrode. The concentration of CT was determined by detecting the change of capacitance caused by the formation of antibody-antigen complexes. By using AuNPs adsorbed to the sensing surface, the signal was dramatically increased leading to a significantly more sensitive assay. In fact, under optimum conditions the immunosensor could detect CT concentration with a limit of detection of 9 x 10(-20)M or 0.09 aM, with a dynamic range between 0.1 aM and 10 pM. Good analytical reproducibility could be obtained by injecting CT up to 36 times with an RSD of 2.5%. In addition, good performance of the developed immunosensor was achieved when applied to turbid water samples collected from a local stream that were spiked with CT.

Ultrasensitive detection of HIV-1 p24 antigen using nanofunctionalized surfaces in a capacitive immunosensor.

The HIV-1 capsid protein, p24 antigen, is of considerable diagnostic interest because following HIV exposure it is detectable several days earlier than host-generated HIV antibodies (which are the target of almost all current tests used in the field) and can be used to design very sensitive assays without the need for PCR. Here, we present an ultrasensitive capacitive immunosensor that is capable of detecting subattogram per milliliter concentrations of p24 antigen, which to our knowledge is the lowest level of detection ever reported. Dilution studies using p24-spiked human plasma samples indicate that the immunosensor is robust against the interfering effects of a complex biological matrix. Moreover, the capacitive immunosensor assay is rapid (<20 min), label-free, and generates data in real-time, with a portable format in development. Additional optimization of the capture agents and/or surface chemistries may further improve performance, highlighting the potential of this platform to serve as a diagnostic tool for early detection of HIV in field settings.

A capacitive immunosensor for detection of cholera toxin.

Contamination of food with biological toxins as well as their potential use as weapons of mass destruction has created an urge for rapid and cost effective analytical techniques capable of detecting trace amounts of these toxins. This paper describes the development of a sensitive method for detection of cholera toxin (CT) using a flow-injection capacitive immunosensor based on self-assembled monolayers. The sensing surface consists of monoclonal antibodies against the B subunit of CT (anti-CT), immobilized on a gold transducer. Experimental results show that the immunosensor responded linearly to CT concentrations in the range from 1.0x10(-13) to 1.0x10(-10) M under optimized conditions. The limit of detection (LOD) was 1.0x10(-14) M. Two more analytical methods were employed for detection of CT using the same antibody namely, sandwich ELISA and surface plasmon resonance (SPR)-based immunosensor. The former had an LOD of 1.2x10(-12) M and a working range from 3.7x10(-11) to 2.9x10(-10) M whereas, the later had an LOD of 1.0x10(-11) M and a linearity ranging from 1.0x10(-9) to 1.0x10(-6) M. These results demonstrate that the developed capacitive immunosensor system has a higher sensitivity than the other two techniques. The binding affinity of CT to the immobilized anti-CT was determined using the SPR-based immunosensor and an association constant (K(A)) of 1.4x10(9) M(-1) was estimated.

A novel competitive capacitive glucose biosensor based on concanavalin A-labeled nanogold colloids assembled on a polytyramine-modified gold electrode

A highly sensitive competitive capacitive glucose biosensor was constructed based on gold nanoparticles, which were employed as a platform to immobilize concanavalin A (Con A). Gold nanoparticles were fixed on a gold electrode, on which a layer of polytyramine was preformed via electrochemical polymerization. The sensing mechanism is based on the competitive dissociation of a glucose polymer or a glycoconjugate from the glycoligand binding sites of immobilized Con A by the added glucose. To further improve the sensor response, several glucose polymers as well as a synthesized glycoconjugate using the periodate method, were screened. Consequently, dextran (MW 39 kDa) was selected and the feasibility of the proposed biosensor was evaluated for a competitive assay of glucose. Experimental results show that the biosensor responded linearly to glucose in the range from 1.0 x 10(-6) to 1.0 x 10(-2) M, corresponding to 0.18 microg mL(-1) to 1.8 mg mL(-1) of glucose with a detection limit of 1.0 x 10(-6) M under optimized conditions. The studied biosensor exhibited a response time of about 15 min and a neglectable loss in sensitivity after 10 repeated analytical cycles.

Stability of diphenylalanine peptide nanotubes in solution

Over the last couple of years, self-organizing nanotubes based on the dipeptide diphenylalanine have received much attention, mainly as possible building blocks for the next generation of biosensors and as drug delivery systems. One of the main reasons for this large interest is that these peptide nanotubes are believed to be very stable both thermally and chemically. Previously, the chemical and thermal stability of self-organizing structures has been investigated after the evaporation of the solvent. However, it was recently discovered that the stability of the structures differed significantly when the tubes were in solution. It has been shown that, in solution, the peptide nanotubes can easily be dissolved in several solvents including water. It is therefore of critical importance that the stability of the nanotubes in solution and not after solvent evaporation be investigated prior to applications in which the nanotube will be submerged in liquid. The present article reports results demonstrating the instability and suggests a possible approach to a stabilization procedure, which drastically improves the stability of the formed structures. The results presented herein provide new information regarding the stability of self-organizing diphenylalanine nanotubes in solution.

Immunochemical binding assays for detection and quantification of trace impurities in biotechnological production.

New, highly sensitive, biosensor concepts make it possible to assay biomacromolecules at concentrations that previously were far below the limit of detection. The previous generation of assays used in quality control situations during biotechnological production was designed primarily for monitoring target molecules, which typically appeared in high concentrations. Hence, novel analytical techniques with high sensitivity should become increasingly important in meeting the demands from regulatory agencies with regard to declaring levels of impurities in biopharmaceuticals. Such techniques also open up opportunities for a range of other challenging measurements, for example, in the area of biohazards. This review describes the development of immuno-based biosensors and exemplifies these by presenting analyses of common impurities in biopharmaceutical production.

A mass spectrometric investigation of native and oxidatively inactivated chloroperoxidase

The enzyme chloroperoxidase (CPO) found in Caldariomyces fumago is able to catalyze several stereoselective oxidation reactions by using a clean oxidant, usually hydrogen peroxide (H2O2), without the need for expensive cofactor generation. CPO’s lack of operational stability, however, is a major limitation for its commercial use. In the present study, a capillary‐LC on‐line trypsin‐digestion system combined with reversed‐phase chromatography and mass spectrometric detection was optimized for studying the primary sequence of CPO. Samples containing native CPO, CPO treated with H2O2, and CPO oxidatively inactivated by the use of indole and H2O2 were analyzed and compared. Three oxidized peptides were found in the samples treated with H2O2. Two additional oxidized peptides were found in the CPO samples that were completely inactivated, one of which contained an oxidized cysteine residue, Cys50, which is an essential amino acid due to its function as the axial ligand to the iron in the heme—the prosthetic group in CPO. In addition, the heme group was absent in the inactivated samples but was readily detected in other samples.

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G.

We report a flow‐injection biosensor system with a capacitive transducer for assay and quality control of human immunoglobulin G (hIgG). The sensing platform is based on self‐assembled monolayers (SAMs) of carboxylic acid terminated alkyl‐thiols with covalently attached concanavalin A. The electrochemical characteristics of the sensor surface were assessed by cyclic voltammetry using a permeable redox couple (potassium ferricyanide). The developed biosensor proved capable of performing a sensitive label‐free assay of hIgG with a detection limit of 1.0 µg mL−1. The capacitance response depended linearly on hIgG concentration over the range from 5.0 to 100 µg mL−1, in a logarithmic plot. Typical measurements were performed in 15 min and up to 18 successive assays were achieved without significant loss of sensitivity using a single electrode. In addition, the biosensor can detect hIgG aggregates with concentrations as low as 0.01% of the total hIgG content (5.0 µg mL−1). Hence, it represents a potential post‐size‐exclusion chromatography–UV (post‐SEC–UV) binding assay for in‐process quality control of hIgG, which cannot be detected by SEC–UV singly at concentrations below 0.3% of the total hIgG content. Biotechnol. Bioeng. 2009; 104: 312–320

Whole cell based microcontact imprinted capacitive biosensor for the detection of Escherichia coli

In this study, a label-free, selective and sensitive microcontact imprinted capacitive biosensor was developed for the detection of Escherichia coli. The recognition of E. coli was successfully performed by this sensor prepared with the combination of microcontact imprinting method and capacitive biosensor technology. After preparation of bacterial stamps, microcontact-E. coli imprinted gold electrodes were generated using an amino acid based recognition element, N-methacryloyl-L-histidine methylester (MAH), 2-Hydroxyethyl methacrylate (HEMA) as monomers and ethyleneglycol dimethacrylate (EGDMA) as crosslinker under UV-polymerization. Real-time E. coli detection experiments were carried out within the range of 1.0×102-1.0×107CFU/mL. The unique combination of these two techniques provides selective detection with a detection limit of 70CFU/mL. The designed capacitive sensor has high selectivity and was able to distinguish E. coli when present together with competing bacterial strains which are known to have similar shape. In addition, the prepared sensor has the ability to detect E. coli with a recovery of 81-97% in e.g. river water.

Microcontact-BSA imprinted capacitive biosensor for real-time, sensitive and selective detection of BSA

Highlights • Microcontact imprinting of proteins on a sensor chip was used.• High sensitivity was observed.• Selectivity was equal to or better than that of antibodies.• Stability of the sensor chip was very high.