Roy M. Pemberton

Some Recent Designs and Developments of Screen‐Printed Carbon Electrochemical Sensors/Biosensors for Biomedical, Environmental, and Industrial Analyses

Abstract This review describes the design and fabrication of electrochemical sensors/biosensors based on screen‐printing technology and their applications in pharmaceutical, biomedical, environmental, and industrial analyses. Specific emphasis is placed on naturally‐occurring biomolecules, drugs, and potential environmental and industrial pollutants or toxins.

An electrochemical immunosensor for milk progesterone using a continuous flow system.

An electrochemical biosensor for cows milk progesterone has been developed and used in a competitive immunoassay under thin-layer, continuous-flow conditions. Single-use biosensors were fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs). Three operational steps could be identified: (1) Competitive binding of sample/conjugate (alkaline-phosphatase-labelled progesterone, AP-prog) mixture, (2) establishment of a steady-state amperometric baseline current and (3), measurement of an amperometric signal in the presence of enzyme substrate (1-naphthyl phosphate, 1-NP). In the thin-layer cell, the enzyme product, 1-naphthol, showed electrochemical behaviour consistent with bulk conditions and gave a linear amperometric response under continuous-flow conditions (E(app)=+0.3 V vs. Ag/AgCl) over the range 0.1-1.0 microg/ml. After pre-incubating biosensors with progesterone standards, signal generation within the cell (substrate concentration=5 mM) was recorded amperometrically as rate (nA/s) or maximum current (i(max), nA). Response values for milk standards were approximately 50% of those prepared in buffer. In both cases, calibration plots over the range 0-50 ng/ml progesterone were obtained. By conducting sample binding under flowing conditions, only 7% of the previous response was obtained, even at a substrate concentration of 50 mM, resulting in low signal:noise ratio. Using a stop-flow arrangement (i.e. quiescent sample binding, followed by continuous flow), low-noise amperograms were obtained at [1-NP]=5 mM. Calibration plots were obtained over the range 0-25 ng/ml, with a coefficient of variation of 12.5% for five replicate real milk samples.

A comparison of 1-naphthyl phosphate and 4 aminophenyl phosphate as enzyme substrates for use with a screen-printed amperometric immunosensor for progesterone in cows' milk.

4-Aminophenyl phosphate (4-APP) and 1-naphthyl phosphate (1-NP) were compared as enzyme substrates for an amperometric milk progesterone biosensor utilising progesterone-conjugated alkaline phosphatase in a competitive immunoassay format. Cyclic voltammetry of the corresponding hydrolysis products, 4-aminophenol and 1-naphthol, at the surface of screen-printed carbon base transducers, uncoated or coated with anti-progesterone monoclonal antibody (mAb) showed well-defined anodic responses for both species, with the more sensitive being 4-aminophenol. Scan rate studies produced evidence that surface mAb could impede the diffusion of 4-aminophenol, but not 1-naphthol, toward the electrode surface. This was supported by computer simulation for the electrochemical rate constant (khet) using 4-aminophenol, which gave values at uncoated and mAb-coated electrodes of 6.5 x 10(-4) and 3.0 x 10(-4) cm s-1, respectively. The applied potential for oxidation of 4-aminophenol was 230 mV lower than for 1-naphthol. Nevertheless, by operating below +400 mV versus a saturated calomel reference electrode, it was possible to obtain a chronoamperometric signal for 1-naphthol in the absence of electrochemical interference from milk. Using mAb-coated SPCEs, calibration curves were obtained for progesterone in oestrus whole cows milk spiked with standard concentrations over the range 0-50 ng/ml, using either 4-APP or 1NP as enzyme substrate. Precision values for triplicate sensors were 5.3-18.3% for 4-APP and 4.1-12.4% for 1-NP. An assay of real whole milk samples from different cows at various stages of the oestrus cycle produced correlations against a commercial EIA of r = 0.840 and 0.946 for 4-APP and 1-NP, respectively, 1-NP possesses the advantages over 4-APP of being inexpensive, easy to obtain and soluble (1-naphthol cf. 4-aminophenol) at high pH. From these observations, it is concluded that 1-NP is the preferred substrate for use with our proposed milk progesterone biosensor.

Studies towards a disposable screen-printed amperometric biosensor for progesterone

A screen-printed carbon electrode (SPCE) has been investigated as the base transducer for a disposable amperometric progesterone biosensor. The biorecognition element was a monoclonal sheep anti-progesterone antibody (mAb). This was immobilized onto the transducer by interaction with a layer of rabbit IgG which had been previously coated onto the SPCE; optimum conditions for these loadings were deduced experimentally. The device was employed in a competitive assay using alkaline phosphatase-labelled progester-one. Three possible substrates for the enzyme were considered, namely, phenyl phosphate, phenolphthalein phosphate and 4-aminophenol phosphate. Cyclic voltammetry and amperometry were carried out on the corresponding aromatic phenols and phenol itself was found to give the best electrochemical characteristics; consequently, phenyl phosphate was employed as the substrate. Chronoamperometry was used to measure the phenol produced by the reaction of bound enzyme-labelled progesterone and substrate. The chronoamperometric response was dependent on unlabelled progesterone over at least three orders of magnitude with a detection limit of about 1 x 10(-9) mol/dm3. This suggests that the device may have applications for the analysis of biological fluids.

A microband lactate biosensor fabricated using a water-based screen-printed carbon ink

The present study demonstrated for the first time that screen-printed carbon microband electrodes fabricated from water-based ink can readily detect H(2)O(2) and that the same ink, with the addition of lactate oxidase, can be used to construct microband biosensors to measure lactate. These microband devices were fabricated by a simple cutting procedure using conventional sized screen-printed carbon electrodes (SPCEs) containing the electrocatalyst cobalt phthalocyanine (CoPC). These devices were characterised with H(2)O(2) using several electrochemical techniques. Cyclic voltammograms were found to be sigmoidal; a current density value of 4.2 mA cm(-2) was obtained. A scan rate study revealed that the mass transport mechanism was a mixture of radial and planar diffusion. However, a further amperometric study under quiescent and hydrodynamic conditions indicated that radial diffusion predominated. A chronoamperometric study indicated that steady-state currents were obtained with these devices for a variety of H(2)O(2) concentrations and that the currents were proportional to the analyte concentration. Lactate microband biosensors were then fabricated by incorporating lactate oxidase into the water-based formulation prior to printing and then cutting as described. Voltammograms demonstrated that lactate oxidase did not compromise the integrity of the electrode for H(2)O(2) detection. A potential of +400 mV was selected for a calibration study, which showed that lactate could be measured over a dynamic range of 1-10mM which was linear up to 6mM; a calculated lower limit of detection of 289 microM was ascertained. This study provides a platform for monitoring cell metabolism in-vitro by measuring lactate electrochemically via a microband biosensor.

Fabrication of microband glucose biosensors using a screen-printing water-based carbon ink and their application in serum analysis

Microband glucose biosensors were fabricated by screen-printing a water-based carbon ink formulation containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, then insulating and sectioning through the thick (20mum) film to expose a 3mm-long working electrode edge. The performance of these biosensors for glucose analysis was investigated at 25 degrees C. Voltammetry in glucose-containing buffer solutions established that an operating potential of +0.4V vs. Ag/AgCl was suitable for analysis under both stirring and quiescent conditions. The influence of pH on biosensor performance was established and an operational pH of 8.0 was selected. Steady-state responses were obtained under quiescent conditions, suggesting a mixed mechanism predominated by radial diffusion, indicative of microelectrode behaviour. Calibration studies obtained with these biosensors showed steady-state currents that were linearly dependent on glucose concentration from the limit of detection (0.27mM) up to 2.0mM, with a precision for replicate biosensors of 6.2-10.7%. When applied to the determination of glucose in human serum, the concentration compared favourably to that determined by a spectroscopic method. These results have demonstrated a simple means of fabricating biosensors for glucose measurement and determination in situations where low-current real-time monitoring under quiescent conditions would be desirable.

Development of a sandwich format, amperometric screen-printed uric acid biosensor for urine analysis

A screen-printed carbon electrode (SPCE) incorporating the electrocatalyst cobalt phthalocyanine (CoPC), fabricated using a water-based ink formulation, has been investigated as the base transducer for a uric acid biosensor. A sandwich biosensor was fabricated by first depositing cellulose acetate (CA) onto this transducer (CoPC-SPCE), followed by uricase (UOX) and finally a polycarbonate (PC) membrane; this device is designated PC-UOX-CA-CoPC-SPCE. This biosensor was used in conjunction with chronoamperometry to optimize the conditions for the analysis of urine: temperature, 35°C; buffer, pH 9.2; ionic strength, 50 mM; uricase, 0.6 U; incubation time, 180 s. The proposed biosensor was applied to urine from a healthy subject. The precision determined on unspiked urine (n=6) was 5.82%. Urine was fortified with 0.225 mM UA, and the resulting precision and recovery were 4.21 and 97.3%, respectively. The linear working range of the biosensor was found to be 0.015 to 0.25 mM (the former represents the detection limit), and the sensitivity was calculated to be 2.10 μA/mM.

Application of screen-printed microband biosensors to end-point measurements of glucose and cell numbers in HepG2 cell culture.

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 degrees C, at an operating potential of +0.4V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol(-1) was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n=4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R(2)=0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20mM, with those determined spectrophotometrically (R(2)=0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10(6) cells min) based on a 24-h period in culture.

Development of an amperometric screen-printed galactose biosensor for serum analysis

The development of a disposable amperometric biosensor for the measurement of circulating galactose in serum is described. The biosensor comprises a screen-printed carbon electrode (SPCE), incorporating the electrocatalyst cobalt phthalocyanine (CoPC), which is covered by a permselective cellulose acetate (CA) membrane and a layer of immobilized galactose oxidase (GALOX). The optimal response of the biosensor, designated as GALOX-CA-CoPC-SPCE, was obtained by systematically examining the effects of enzyme loading, temperature, pH, and buffer strength. The optimal performance of the biosensor occurred with 2U of GALOX, at 35°C, using 50mM phosphate buffer solution (pH 7.0). The sensitivity was 7.00μAmM(-1)cm(-2) and the linear range from 0.1 to 25mM with a calculated limit of detection (LOD) of 0.02mM; this concentration range and LOD are appropriate to diagnose galactosemia, i.e., concentrations >1.1mM in infants. When the biosensor was used in conjunction with amperometry in stirred solution for the analysis of serum, the precision values obtained on unspiked (endogenous level of 0.153mM) and spiked serum (1mM added) (n=6) were 1.10% and 0.11%, respectively, with a calculated recovery of 99.9%.

Recent Advances in the Fabrication and Application of Screen-Printed Electrochemical (Bio)Sensors Based on Carbon Materials for Biomedical, Agri-Food and Environmental Analyses

This review describes recent advances in the fabrication of electrochemical (bio)sensors based on screen-printing technology involving carbon materials and their application in biomedical, agri-food and environmental analyses. It will focus on the various strategies employed in the fabrication of screen-printed (bio)sensors, together with their performance characteristics; the application of these devices for the measurement of selected naturally occurring biomolecules, environmental pollutants and toxins will be discussed.