Pankaj Vadgama

Polypyrrole-based conducting polymers and interactions with biological tissues

Polypyrrole (PPy) is a conjugated polymer that displays particular electronic properties including conductivity. In biomedical applications, it is usually electrochemically generated with the incorporation of any anionic species including also negatively charged biological macromolecules such as proteins and polysaccharides to give composite materials. In biomedical research, it has mainly been assessed for its role as a reporting interface in biosensors. However, there is an increasing literature on the application of PPy as a potentially electrically addressable tissue/cell support substrate. Here, we review studies that have considered such PPy based conducting polymers in direct contact with biological tissues and conclude that due to its versatile functional properties, it could contribute to a new generation of biomaterials.

Glucose enzyme electrode with extended linearity: Application to undiluted blood measurements

Abstract An enzyme electrodes is described for glucose determination in unstirred, undiluted whole blood. The system comprises an H2O2-detecting electrode upon which is placed a membrane laminate incorporating glucose oxidase. The external membrane was pretreated with methyltrichlorosilane. The electrode response was linearly dependent on glucose concentration up to 50 mmol l−1 glucose, it had a decreased dependence on dissolved oxygen concentrations and gave response times of 30–90 s. Whole blood glucose measurements correlated well with a routine spectrophotometric method.

Analytical aspects of biosensors

The aims of simplifying biochemical measurement and of extending assay reliability outside the con®nes of a central laboratory are present in many applied biology sectors, not just clinical chemistry. An increasing range of desktop analysers are commercially available that are economical both of sample and of operator time. A common theme running through many approaches is the exploitation of biological reagents, with the ultimate in simpli®cation being integration of biological and measurement elements into a simple, monolithic device. This is the basic concept of the biosensor, a biological sample-interactive phase in close contact with a physical or chemical transducer. A typical biosensor construct has three features ± a recognition element, a signal transducing structure and an ampli®cation/processing element (see Fig. 1). Various transduction mechanisms have been used: electrochemical, electrical, optical, thermal and piezoelectric, as summarized in Table 1. Most commonly, in a biosensor, a biorecognition phase (e.g. enzyme, antibody, receptor, single-stranded DNA) interacts with the analyte of interest to produce some chargebased or optical change at the local sensor± transducer interface. Through signal processing this interaction is converted into digital values that relate to the build-up of concentration or activity of the analyte in the vicinity of the device, which in turn relates to the ambient levels in the bulk sample under investigation. A biosensor is not necessarily a stand-alone entity, but should be considered as part of a general development in instrumentation, designed to address generic medical and non-medical measurement science problems. Biosensors, when deployed in a clinical setting, offer the advantage of extra-laboratory analysis of a variety of relevant substances, including hormones, drugs of abuse and metabolites (both in vivo and in vitro). Continuous realtime monitoring of analytes is also a possibility; for example, monitoring of metabolites in blood (where the sample matrix is inevitably optically opaque) in the critical care situation. Generally, biosensors permit the use of low cost, `clean’ technologywith reduced requirements for sample pre-treatment and large sample volume; ultimately, the user can be someone without prior laboratory skills. The `niche’ application is therefore extra-laboratory testing, as realized with conventional dry reagent dipsticks. This review provides basic descriptions of the main subtypes of biosensorwith an indication of their operational capability from a clinical chemistry perspective. Owing to a greater appreciation of the capabilities and limitations of biosensors and new input from the microfabrication and materials science ®elds, the direction of biosensor research is undergoing rapid change. The descriptions that follow are intended to re ect this change and illustrate the shift in emphasis from a preoccupation with bioreagent immobilization and chemistry to a renewed effort towards total system integration. A functionally ef®cient juxtaposition of sample and sensor remains essential for proper function, and the descriptions given provide some relevant examples.

A study of macromolecular diffusion through native porcine mucus

A diffusion chamber technique based on time-lag analysis for the estimation of effective diffusion coefficients of radiolabelled macromolecules of varying molecular weights through native mucus gel is reported. For all solutes studied, a reduction in effective diffusion coefficients was observed with a retardation of solute flux in both aqueus and mucus layers. Over the molecular weight range of solutes investigated (126–186 000 Daltons), a consistent effect of molecular weight was evident with regard to the retarding effect of mucus. No apparent or absolute molecular weight cut-off for macromolecular transfer was exhibited. However, at high molecular weights (>30 000 Daltons) the retardation was greatly enhanced. The results confirm that mucus can be regarded as a gel with finite pores, but that it does not constitute an absolute barrier to even high molecular weight solutes.

Determination of urate in undiluted whole blood by enzyme electrode

An amperometric enzyme electrode is described for the assay of urate in undiluted, unstirred whole blood. The electrode used Aspergillus flavus uricase (EC.1.7.3.3) cross-linked to bovine serum albumin by means of glutaraldehyde, sandwiched between a dimethyldichlorosilane-treated microporous polycarbonate membrane and an inner cellulosic H2O2-selective membrane. The resulting device had a low pH dependence, was capable of repeated use in blood, and gave an acceptable correlation with a standard spectrophotometric method. Electrode steady state and dynamic response were found to be dependent upon the amount of enzyme loading, and could be further optimised by the incorporation of catalase in the enzyme layer.

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).

Plasticized poly(vinyl chloride) as a permselective barrier membrane for high-selectivity amperometric sensors and biosensors

Abstract A poly(vinyl chloride) (PVC) membrane system is described for use as a high-selectivity barrier in amperometric sensors and biosensors. Membrane casting and electrode fabrication techniques are presented. The membrane properties, both physical and with respect to permselectivity, are outlined. This new form of homogeneous membrane barrier shows very much greater selectivity for H 2 O 2 in oxidase-based enzyme electrodes than any previously reported barrier. Selectivity for the phenolic compounds catechol, hydrocaffeic acid, 4-aminophenol and paracetamol, as model electrochemically active compounds, is described and related to interference from ascorbate and urate; the responses of equimolar catechol: ascrobate and catechol: urate are both 61 500:1, and that of 1 mM catechol: undiluted serum is 60 000:1. Comparative results for a low molecular weight cut-off cellulose acetate layer, commonly employed in amperometric sensors and biosensors for clinical monitoring, demonstrates the superior selectivity of PVC, showing the PVC to have 7 and 180 times greater selectivity for H 2 O 2 and paracetamol, respectively, against ascorbate. Biocompatibility is also excellent, with no loss of signal after prolonged exposure to serum.

3D Printed Microfluidic Device with Integrated Biosensors for Online Analysis of Subcutaneous Human Microdialysate

This work presents the design, fabrication, and characterization of a robust 3D printed microfluidic analysis system that integrates with FDA-approved clinical microdialysis probes for continuous monitoring of human tissue metabolite levels. The microfluidic device incorporates removable needle type integrated biosensors for glucose and lactate, which are optimized for high tissue concentrations, housed in novel 3D printed electrode holders. A soft compressible 3D printed elastomer at the base of the holder ensures a good seal with the microfluidic chip. Optimization of the channel size significantly improves the response time of the sensor. As a proof-of-concept study, our microfluidic device was coupled to lab-built wireless potentiostats and used to monitor real-time subcutaneous glucose and lactate levels in cyclists undergoing a training regime.

Modification of electrode surfaces with oxidised phenols to confer selectivity to amperometric biosensors for glucose determination

Abstract The preparation of electrochemical coatings on a platinum anode, using phenol and dopamine solutions, are described. In the amperometric mode, the modified electrode displayed a high degree of selectivity to H2O2 and against ascorbate, urate, serum and paracetamol. Such selectivity is superior to that of conventional cellulose acetate membranes used in oxidase based biosensors. Most significantly, with a coated electrode surface, the decrease in paracetamol response while a large H2O2 signal is retained, is shown to alleviate the problem of paracetamol interference in a glucose sensor. This glucose sensor based on glucose oxidase is free from any significant paracetamol interference and represents a major advance over existing cellulose acetate membrane devices. The influence of both the phenolic used, and the degree of coating, on the linear response range, response time and lifetime are discussed, and compared with a cellulose acetate membrane.

Influence of nanopatterns on endothelial cell adhesion: Enhanced cell retention under shear stress.

In this study, nanopatterned crosslinked films of collagen Type I were seeded with human microvascular endothelial cells and tested for their suitability for vascular tissue engineering. Since the films will be rolled into tubes with concentric layers of collagen, nutrient transfer through the collagen films is quite crucial. Molecular diffusivity through the collagen films, cell viability, cell proliferation and cell retention following shear stress were studied. Cells were seeded onto linearly nanogrooved films (groove widths of 332.5, 500 and 650nm), with the grooves aligned in the direction of flow. The nanopatterns did not affect cell proliferation or initial cell alignment; however, they significantly affected cell retention under fluid flow. While cell retention on unpatterned films was 35+/-10%, it was 75+/-4% on 332.5nm patterned films and even higher, 91+/-5%, on 650nm patterned films. The films were found to have diffusion coefficients of ca. 10(-6)cm(2)s(-1) for O(2) and 4-acetaminophenol, which is comparable to that observed in natural tissues. This constitutes another positive asset of these films for consideration as a scaffold material for vascular tissue engineering.