1. Why is this potentiometric measurement superior to amperometric measurements?
Potentiometric sensing is inherently more sensitive than amperometry. It does not require a potential to be applied to the system, which can cause problems with interferents being oxidized or reduced as well as the mediator. Potentiometry is a passive measurement in which no external charge is exchanged with the sensor. The change in sensor potential is measured by a high impedance buffer-amplifier circuit such as is used with a pH electrode. Potentiometric sensing produces less potential noise and a greater change in signal for a given response. (large signal to noise ratio) compared to amperometry. However in the past reproducibility was a problem with potentiometric systems and so other than ISFETs and other ion selective sensors work was stopped. In reality we have cracked the reproducibility problem and we are able to apply our system to measure potentiometrically traditionally amperometric biosystems as well as to biosystems that can not be measured by amperometry.
We do not use anything to mediate or amplify the signal. That means that in contrast to amperometric systems our technology has a potential for further development and improvement not only in terms of polymerisation procedures, but also by other means.
2. Is this sensor system more applicable to a certain type of immunoassay, that is, is it a better design for a competitive assay or non-competitive sandwich type assay?
The sensors perform equally as well with either competitive or sandwich assays. In the future we would like to overcome one of the general problems for immunoassays – sample dilutions. Many systems are designed to measure the least possible concentration of an analyte. A sample must be re-measured, if the result is out of range. With UTS it is possible to use more than one sensor with different levels of sensitivity to cover the measuring range completely from ng/ml to ug/ml or even mg/ml. We have also used software and two different measurement times to increase the dynamic range of the assay on a single sensor.
3. Is there any activation time for the sensors?
There is a short activation time required in some cases but not enough to significantly affect the sample incubation time of 5-15minutes depending on required sensitivity and bioreagent affinity dynamics.
4. How is the analysis time divided up between sample incubation and measurement ? Is the "charge step" portion a chemical change induced by the "measurement solution"?
The incubation with a sample (e.g. blood) usually dictates the timing for an assay. It takes from 5 to 15 min and depends on the concentration to be targeted. The diffusion process dictates some limitations; a finite time is required to form the minimum number of complexes to produce the signal to be measured. It takes longer for pg/ml than for ng/ml. The sensitivity in UTS is higher, than, for example, in ELISA, so shorter incubation with a sample is required. Usually10 min incubation with a sample in UTS gives the same or better signal than 1hr incubation for ELISA, in some cases, UTS signal is more than an order of magnitude higher. This can be further enhanced in a fluidics system. Feasibility studies indicate a total time of <2 minutes from sample to response is possible with a fluidics system.
The detection time is made up of two steps, the equilibration (0.5 – 2min) and background level (20sec) (this can be performed physical in a buffer or electronically), followed by the measurement step where the enzyme turnover drives the sensor to the final potential. This is done by releasing a set potential or by addition of the enzyme substrate to the buffer solution (60sec).
5. It appears that it is electron transfer in the polypyrrole membrane which is ultimately responsible for the change in measure potential? How good is the reproducibility of the formation and electrochemical properties of the polypyrrole films from chip to chip (sensor to sensor), if this is the key importance for a measured change in potential?
This appears to be very good. We are getting CV’s of less than 10% for measurements of 0.1ng/ml in our QC assay. We are able to control the polymerisation process closely with programmed electrochemical growth profiles and by using freshly distilled pyrrole in the polymerisation solution. The surface properties of the base carbon electrode is one of the key factors for maintaining reproducibility of the polymer layer. We have found that screen-printed sensors can provide this reproducible surface. The other equally important factor is the reproducibility and stability of the reference electrode which is highly dependent on the formulation.
6. Is the polypyrrole system tuned to have pH-sensing capabilities? If yes, what sensitivity, selectivity?
The polypyrrole system is pH sensitive, but it is not tuned to measure pH. The polypyrrole layer was developed to measure the change caused by the enzyme redox reaction (HRP converting OPD into 2, 3-diaminophenazine (DAP) in the presence of H2O2). It is likely that it is the combination of the redox, pH and ionic events which change the physical (porosity, density, thickness) and electrochemical properties (conductivity, charge) of the polypyrrole layer leading to the observed shift in potential of the sensor.
7. What is the meaning of "charge-step"? Has it something to do with the "ion-step"-technique of P.Bergveld ?
The name "charge-step" does not fully reflect the process itself. This is an electro-physiochemical phenomenon that may be described as an induced change in potential due in part to electron depletion of the overoxidized polypyrrole layer. Changes in pH or ionic strength of the solution immediately adjacent to the surface could also explain the change in potential. The process is passively induced by electrochemical activity at the polypyrrole surface provided by the enzyme HRP converting OPD into 2, 3-diaminophenazine (DAP) in the presence of H2O2.
We think that the key difference from "ion-step" is that we perform our assay at a fixed pH, only two measurements taken within a 90s period are used. ISFET-based assays are performed in a continuously changing pH environment over quite a large pH range. The ISFET method is based on detecting a shift of isoelectric point of the ISFET membrane, whereas UTS measures the change of electrode potential.
The "ion-step" procedure has only been previously described in connection with use of an ISFET. Bergveld at al are convinced that the ion step procedure is applicable only to ISFET’s (Bergveld P., A critical evaluation of direct protein detection methods. Biosensors and Bioelectronics, 6, 55-72, 1991).
8. If the transduction principle is a pure redox-potential measurement, why is there a need for charged enzyme products ? why not neutral products ? or ionic mixture ?
There needs to be a permanent transfer of charge from the polypyyrole to something, this happens to be the product in the case of HRP. We have not investigated the mechanism of the system as yet so we cannot answer this question properly. But it is well known that many things affect the conductivity and overpotential of the polypoyrrole and as these are controlled we keep a tight grip on the causes of the changes and can be confident on the result.
9. What kind of enzymes are used; standard ELISA- ones like AP or urease, peroxidases ...?
Any of the enzymes mentioned above can be used, as all enzymatic (redox) reactions involve the electron transfer to a charged product, which change the potential of the electrode. AP and urease were used in UTS at the early stage of the development, but later we chose peroxidase. There is a wide range of bioreagents labelled with peroxidase, which make it easier to construct different models of assays for UTS.
We can also use other enzymes, which cannot be used in direct optical measurements, such as catalase. No coloured product is produced, but it is possible to measure the shift in potential caused by the redox reaction. We cannot however detect all enzymatic reactions at the required sensitivities. They need to relatively high turnover active enzymes. In addition we can do enzyme substrate assays such as glucose, urea or creatinine in our system.
10. Depending on exchange-current density, redox-sensors can be very high-ohmic and therefore very noise-sensitive; what is the electrical set-up?
The present electrical setup is specifically designed to avoid problems associated with high ohmic sensors. We use electronics with a high input impedance of 100MOhms or more and a low leakage current of <1pA. We also filter any high frequency components
The sensor are buffered and as close as possible to the active electronics. This reduces any chance of extraneous pick-up from the high-impedance connection. This reduces the background noise. Any leakage currents drawn by the buffer amplifier are reduced to a practical minimum, this reduces any tendency to artificially polarise the active parts of the sensor.
The software averages a number of samples further reducing the effects of noise.
