No abstract

An amperometric biosensor for the detection of trypsin was developed. The latter was based on a two-layer configuration, namely, a polymer-glucose oxidase inner layer and a gelatin outer layer. In the presence of glucose, the enzyme layer produces H2O2 and hence an amperometric signal due to H2O2 electrooxidation was generated by potentiostating the electrode at 0.6 V. The biosensor detects the change in the increase in the maximum current caused by the proteolytic digestion of gelatin, which covers the platinum electrodes, thereby facilitating a speedier access for the glucose substrate to the electrode modified with both poly(pyrrole-alkylammonium) and glucose oxidase molecules. Our biosensor detected low trypsin concentrations down to 42 pM with a response time of approximately 10 min, making it a very sensitive device in the detection of lower trypsin levels with such future putative applications as the diagnosis of pancreatic diseases.

A flow-injection impedimetric immunosensor for the sensitive, direct and label-free detection of cholera toxin is reported. A limit of detection smaller than 10 pM was achieved, a value thousands of times lower than the lethal dose. The developed chips fulfil the requirement of low cost and quick reply of the assay and are expected to enable field screening, prompt diagnosis and medical intervention without the need of specialized personnel and expensive equipment, a perspective of special relevance for use in developing countries. Since the chip layout includes two sensing areas each one with a 2 × 2 sensor array, our biochips can allow statistical or (alternatively) multiplex analysis of biorecognition events between antibodies immobilized on each working electrode and different antigens flowing into the chamber.

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split‐intein‐mediated SICLOPPS technology provides a generic access to fully genetically encoded head‐to‐tail cyclized peptides and large libraries thereof (SICLOPPS=split‐intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light‐dependent intein using the photocaged tyrosine derivative ortho‐nitrobenzyltyrosine incorporated at an internal, non‐catalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.

The construction of amperometric immunosensors to cholera antitoxin immunoglobulins were shown to have improved sensitivity when the cholera toxin B subunit biorecognition entity was linked to an electrogenerated biotinylated polypyrrole film copolymerized with pyrrole-lactobionamide monomer. The copolymer exhibits greater film permeability than biotinylated polypyrrolic or polyphenolic films for the permeation of electroactive species. Hence, when the presence of the HRP marker of the immunoassay was determined using hydroquinone, the production of electroactive quinone was shown to permeate faster to the electrode, thus providing a faster response time.

No abstract

N-(3-Aminopropyl)pyrrole was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a pyrrole-alginate conjugate for subsequent use in biosensor applications. The pyrrole-alginate conjugate was quantified by UV spectroscopy at 230 nm, by an HPSEC-MALLS analytical method, as well as by FTIR and 13C NMR spectroscopies. The new pyrrole-alginate conjugate was used for the immobilization of polyphenol oxidase (PPO) onto an electrode surface by physical entrapment resulting from the gellification process and electrochemical polymerization of the pyrrole groups. The efficiency of this cross-linking approach (chemical and electrochemical) was investigated by comparing the amount of enzyme released from polypyrrole-alginate and regular alginate. In addition, biosensors were prepared by entrapment of the PPO in polypyrrole-alginate and regular alginate matrixes and their performance for the amperometric determination of catechol chosen as a model analyte was examined, yielding a sensitivity of 350 and 80 microA M(-1) cm(-2), respectively, for polypyrrole-alginate and alginate biosensors.

In the present work, the standard monometallic localized surface plasmon resonance (LSPR) biosensing sensitivity is highly improved when using a new system based on glass substrates modified with high-temperature annealed gold/silver bimetallic nanoparticles (Au/Ag bimetallic NPs) coated with polydopamine films before biomolecule specific immobilization. Thus, different zones of bimetallic NPs are spatially created onto a glass support thanks to a commercial transmission electron microscopy (TEM) grid marker in combination with two sequential evaporations of continuous films of gold (4 nm) and silver (2 nm) and followed by annealing at 500 °C for 8 h. By using the scanning electron microscopy (SEM), it is found that annealed Au/Ag bimetallic NPs have uniform size and shape distribution that exhibited a sharper well-defined LSPR resonant peak when compared with that of monometallic Au NPs and thereby contributing to an improved sensitivity in LSPR biosensor application. The controlled micropatterns consisting of bimetallic particles are used in the construction of LSPR biochips for high-throughput detection of different concentrations of a model antigen named bovine serum albumin (BSA) on a single glass sample, with a lower limit of detection of 0.01 ng/mL under the optimized conditions.