A Cytochrome P450 3A4 Biosensor Based on Generation 4.0 PAMAM Dendrimers for the Detection of Caffeine

Abstract

Cytochromes P450 (CYP, P450) are a large family of heme-active-site proteins involved in many catalytic processes, including steroidogenesis. In humans, four primary enzymes are involved in the metabolism of almost all xenobiotics. Among these enzymes, CYP3A4 is responsible for the inactivation of the majority of used drugs which makes this enzyme an interesting target for many fields of research, especially pharmaceutical research. Since the late 1970s, attempts have been made to construct and develop electrochemical sensors for the determination of substrates. This paper is concerned with the establishment of such a CYP3A4-containing biosensor. The sensor was constructed by adsorption of alternating layers of sub-nanometer gold particle-modified PAMAM (poly-amido-amine) dendrimers of generation 4.0, along with the enzyme by a layer-by-layer assembly technique. Atomic force microscopy (AFM), quartz crystal microbalance (QCM), and Fourier-transformed infrared spectroscopy (FTIR) were employed to elucidate the sensor assembly. Additionally, the biosensor was tested by cyclic voltammetry using caffeine as a substrate.

Keywords: PAMAM dendrimers; biosensor; cytochrome P450; electrochemistry.

Figure 1

(A) UV-Vis scans of the Au/PAMAM G4.0 solution before and after reduction of gold by citrate. (B) Fluorescence scans with excitation at 384 nm of the supernatant (red) and resuspended pellet (black) after centrifugation.

Figure 2

Adsorption process of PAMAM G4.0-Au to Au/MPS electrode at pH 5.0 (A) and CYP3A4 adsorption to Au/MPS/G4.0-Au at pH 7.4. (B) After obtaining a stable frequency response with phosphate buffer the sample was introduced and after completion of the adsorption process a washing step was conducted to remove only deposited or non-specifically bound moieties. These recorded ∆f values were used for the calculation of ∆m by Q-Tools software.

Figure 3

Atomic force microscope images of 5 × 5 µm (2 × 2 µm for CYP3A4 layer) areas of (A) Au, (B) Au/MPS, (C) Au/MPS/PAMAM G4.0-Au and (D) Au/MPS/PAMAM G4.0-Au/CYP3A4 respectively. No significant difference can be seen between (A) and (B) which can be explained by size considerations of the MPS compounds. In C, a change in surface morphology can be seen and is in good agreement with literature data on PAMAM dendrimer adsorption to gold while protein adsorption increases surface roughness again.

Figure 4

Grazing angle FTIR spectrograms of Au/MPS (A) and Au/MPS/PAMAM G4.0-Au (B). While in A only non-specific peaks appeared which could not be attributed to the MPS molecule functional groups like S-H or C-S (A), Intense absorption bends could be observed from the amine stretching vibration at 3399 which is a cumulative one from primary surface and tertiary amines and amides. Also, absorption could be observed for amine bending vibrations at 1667 and 1599 which is in good agreement to literature data and enough for identification of the compound.

Figure 5

Cyclic voltammograms of 10mM Potassium Ferricyanide using different assembly stages of the CYP3A4 biosensor as working electrodes. The scan rate was varied between 50 and 500 mV/s. A Pt counter and an Ag/AgCl reference electrode were used. It can be seen that conductivity is reduced in all cases compared to the bare gold working electrode (A). Depending on the scan rate. Au-NP-doped PAMAM G4.0 (B) shows only reduction of conductivity between 83.97 (50 mV/s) to 68.72% (500 mV/s) while the biosensor shows no detectable current after adsorption of the CYP3A4 (C). (D) shows an overview of the collected data where the current response of the bare gold electrode was used as 100% value.

Figure 6

Cyclic voltammograms of the Au/MPS/PAMAM G4.0-Au/CYP3A4 electrode. (a) Shows the behaviour of the electrode with 0, 0.5, 1.0, 5.0 and 10.0 µM of caffeine, respectively. An increasing, reductive current can be observed at ~ −400 mV. (b) Depicts two voltammograms produced before and after treatment with CYP3A4 inhibitor eryhtromycin and under presence of substrate caffeine. An almost total disappearance of the typical P450-related reduction peak can be observed.