Porphyrins sensors

Brovkovych et al. [38] applied the electrochemical porphyrinic sensor technique for the direct measurement of NO concentrations in the single endothelial cell. It was found that NO concentration was the highest at the cell membrane (about 1 pmoll-1) and decreased exponentially with distance from the cell, becoming undetectable at the distance of 50 pm. Now we will consider the principal reactions of nitric oxide relevant to real biological systems. 

Currently available amperometric and voltammetric porphyrinic sensors for detection of electroactive analytes are based on their electrochemical oxidation or reduction on polymeric conductive films of metalloporphyrins. If the current generated during the process is linearly proportional to the concentration of an andyte, the current can be used as an analytic signal. This current can be measured in either the... 

Figure 5. Schematic diagram of the porphyrinic sensor for measurement of nickel (a), pH deperKlence of demetalation of polymeric (TMHPP)Ni" (b), Film thickness, 20 equivalent monolayers time, 60 s and Ni(ll)...

Table 1. Optimum Conditions for Preparation of Porphyrinic Sensors for Nickel and Nickel (II)...

Figure 10. Schematic diagram of porphyrinic sensor for detection of NO released from cells grown directly on its surface.

Figure 11. Schematic diagram of intravenous catheter-protected porphyrinic sensor for in vivo measurement of NO.

Single-fiber sensors and catheter-protected sensors can operate in an amperometric or voltammetric mode. In both methods a current proportional to NO concentration is measured. Of the several voltammetric methods available, differential pulse voltammetry (DPV) is most suitable for the measurement of NO. In DPV, a potential modulated with rectangular pulses is linearly scanned from 0.4 to 0.8 V. The resulting voltammogram (alternating current versus voltage plot) contains a peak due to NO oxidation. The peak current should be observed at a potential of 0.63-0.67 V which depends on the pulse amplitude. This potential is the characteristic potential for NO oxidation on Nafion coated porphyrinic sensor. 

A tissue culture can be grown directly on a porphyrinic sensor and measurements of NO can be made by using voltammetric or amperometric methods. - This macrosize sensor is advantageous for screening large numbers of cells for NO relea.se without using more tedious microelectrode techniques. Sensor production consi.sts of deposition of conductive polymeric film on the surface of the conductive solid support recticulated vitrous carbon (RVC). 

Figure 12. Photograph of endothelial cells (porcine aorta) grown directly on a porphyrinic sensor (deposited on recticulated vitrous carbon (RVC)).

A standard calibration curve is prepared by subsequent addition of small volumes of standard aqueous saturated NO solution (concentration 1.74 mM) into a constant volume of freshly boiled and deoxygenated buffer (pH 7.4 at 37 C). -The current generated after each addition of NO is measured, and the resulting plot of current versus concentration should be linear. The detection limit of a correctly prepared sensor should be about 10 nM for a single-fiber sensor, and 1 to 5 nM for multifiber catheter-protected sensors for in vivo measurement. The detection limit of die cell-culture macrosensor is 5 nM. The response time (time for signal increase from 10-75%) of porphyrinic. sensors is 0.1 ms for micromolar NO concentrations, and 10 ms for nanomolar concentrations. 

By use of a manual or motorized computer-controlled micromanipulator with 0.2 pm x-y-z-re,solution, the porphyrinic sensor can be implanted into a single cell, or placed on the surface of the cell membrane, or kept at a controlled di.stance (0.2-10 pm) from a NO-generating cell. 

Figure 13. Amperogram of NO recorded with porphyrinic sensor (diameter 1 urn) placed 5 2 pm from the surface of an isolated single endothelial cell (a) and endothelial cell in cell culture (b). NO release was stimulated from rabbit aorta endothelial cells with calcium ionophore.

The height of the pe of NO release depends on the distance from a membrane surface (lugure 14). The highest NO concentration is observed on the cell membrane (650 30 nM), with the concentration decreasing exponentially with the distance from the cell membrane. At about 50 pm from the cell membrane, the NO concentration was 15 nM (single isolated cell) and at a distance greater than 50 pm from the cell membrane, NO is not detectable by the porphyrinic sensor. The decrease of NO concentration from the cell surface is not so rapid when the cell is surrounded by another cell from the cell culture. However, at a distance greater than 150 pm from the cell membrane (in cell culture), NO is also not detectable by the porphyrinic sensor. 

Flgure 14. Exponential decrease of NO concentration with Increasing distance of porphyrinic sensor from the membrane of single Isolated endothelial cell (dashed line) and single endothelial cell in cell culture (solid line). Rabbit aorta endothelial cells were stimulated with calcium ionophore. 

Figure 15. T)rpical amperograms showing changes of NO concentration near the surface of the endothelium of aorta of normotensive (WKY rat) (a) and spontaneously hypertensive rats (SHR rat) (b). NO was stimulated with calcium iono( ore and its concentration was measured with a porphyrinic sensor (diameter 7pm) in the presence (dotted line) and absence (solid line) of PEC-SOD.

This is a typical example of the unique application of the electrochemical sensor for detection of NO release ftom normotensive and hypertensive rats." - It has been reported based on spectroscopic measurements, that endothelium of hypertensive rats produced more NO2/NO3 than endothelium of normotensive rats. However these reports were in contradiction to data obtained on smooth-muscle relaxation, hindered in hypertensive rats. This means that the endothelium of hypertensive rats should produce less NO. Electrochemical measurements with porphyrinic sensor clearly show that net concentration of NO produced by endothelium of SHR rats is lower than that produced by the endothelium of WKY rats. These results correlate well with previously reported smooth-muscle relaxation data. Total production of NO by the endothelium of hypertensive rats is slightly higher than in normotensive rats. However, the endothelium of SHR rats also generated... 

Figure 16a shows the amperometric curve measured in vivo during endotoxemia with a porphyrinic sensor placed in the... 

Figure 16. In vivo measurement of nitric oxide release with porphyrinic sensor in lung (rat) during the first 100 min after administration of LPS (20 mg/kg) (a) NO2 and NOJ measured in blood (UV-Visible spectroscopy, Criess reagent) after administration of LPS (b).

Figure 17. Response of the catheter-protected porphyrinic sensor to changing fluid pressure (a), to mechanically bending the active tip of the sensor at a frequency of 3Hz (b), and to the ECG signal (c). The vertical bars represent the equivalent current that would be generated by the porphyrinic sensor in the 100 nM NO ((a), lower tracing) or 50 nM NO (b).

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