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Light triggered detection of aminophenyl phosphate with a quantum dot based enzyme electrode
© Khalid et al; licensee BioMed Central Ltd. 2011
Received: 18 August 2011
Accepted: 7 October 2011
Published: 7 October 2011
An electrochemical sensor for p-aminophenyl phosphate (p APP) is reported. It is based on the electrochemical conversion of 4-aminophenol (4AP) at a quantum dot (QD) modified electrode under illumination. Without illumination no electron transfer and thus no oxidation of 4AP can occur. p APP as substrate is converted by the enzyme alkaline phosphatase (ALP) to generate 4AP as a product. The QDs are coupled via 1,4-benzenedithiol (BDT) linkage to the surface of a gold electrode and thus allow potential-controlled photocurrent generation. The photocurrent is modified by the enzyme reaction providing access to the substrate detection. In order to develop a photobioelectrochemical sensor the enzyme is immobilized on top of the photo-switchable layer of the QDs. Immobilization of ALP is required for the potential possibility of spatially resolved measurements. Geometries with immobilized ALP are compared versus having the ALP in solution. Data indicate that functional immobilization with layer-by-layer assembly is possible. Enzymatic activity of ALP and thus the photocurrent can be described by Michaelis- Menten kinetics. p APP is detected as proof of principle investigation within the range of 25 μM - 1 mM.
Colloidal quantum dots (QDs), which are fluorescent semiconductor nanoparticles, have recently brought impact to various disciplines, as has been highlighted in various review articles [1–5]. QDs have been recently discussed also as new building blocks for the construction of electrochemical sensors [6–12]. Upon optical illumination (below the wavelength of the first exciton peak QDs have a a continuous absorption spectrum, with a local maximum at the exciton peak ) electron hole pairs are generated inside QDs. Due to these charge carriers electrons can be transferred to or from the QDs. QDs thus can be oxidized/reduced and can serve as light-controlled redox active element and can be integrated in electrochemical signal chains [9, 14–16]. The key advantage hereby is that the redox reaction of the QD surface can be virtually switched on and off by light. QD have been also used as elements of signal transduction of enzymatic reactions [17, 18].
In the present work we wanted to apply QDs as light-controlled redox active element for the enzymatic detection of p-aminophenyl phosphate (p APP) with alkaline phosphatase (ALP). ALP is a widely used enzyme in bioanalysis as it has a high turnover rate and broad substrate specificity . The enzyme is particularly interesting as label for immunoassays [20, 21]. Very sensitive substrate recycling schemes have been also reported [22, 23]. Four different groups of substrates are known for ALP: i) ß-glycerophosphate and hexose phosphate [24–26], ii) phenyl phosphate [27, 28] and ß-naphthyl phosphate , iii) p-nitrophenyl phosphate  and phenolphthalein diphosphate [31, 32], 4-methyl-umbellipheryl phosphate  and p-aminophenyl phosphate (p APP) , and iv) phosphoenol pyruvate . Electrochemical detection has been reported for a number of ALP substrates [36, 37], in particular for phenyl phosphate. However, p APP is claimed to be a better substrate for ALP than phenyl phosphate, as its product 4-aminophenol (4AP) is more easily oxidizable than phenol, which is the product of phenyl phosphate, as it does not foul the electrode even at higher concentrations, and as it has a rather reversible electrochemical behavior . For this reason we chose p APP as substrate in the present study. Readout of the enzymatic reaction was performed with the QD-modified electrode . We hereby put particular interest in the way of immobilization of ALP on the electrode. In previous work the enzymes were suspended in the solution above the sensor electrode [6, 9]. Here we go a step further and directly immobilize the enzyme on the QD-modified electrode. This was done in order to investigate whether a specific enzymatic reaction can be coupled with a photoinitiated reaction at a QD modified electrode in a way that the recognition element is integrated with the transducer. The potential advantage of light-triggered detection would be the possibility of spatially resolved detection [38–41]. Only at the illuminated parts of the electrode a photocurrent signal is induced. By having different enzymes immobilized at different regions of the electrode they could be selectively addressed by illumination. Thus, two key elements of this study are the following. First, instead of using enzymes in solution as in previous studies we demonstrate that enzymatic reactions can also be followed when enzymes are immobilized on the sensor surface, which is a requirement for potential spatially resolved analysis. Second, we investigate how the way of immobilization influences the sensing properties.
Materials and Methods
Materials: CdS QDs were grown via thermal decomposition of precursors under the presence of organic surfactant molecules following published procedures . 1,4-benzenedithiol (BDT) was purchased from TCI Europe, Belgium. Chloroform, toluene, methanol, acetone, ethanol, sodium sulfide (nanohydrate), alkaline phosphatase (from bovine intestinal mucosa type VII S), 4-nitrophenyl phosphate disodium hexahydrate, 4-aminophenol (4AP), phosphate buffer, sodium poly(styrene sulfonate) (PSS, Mw = 56,000), poly(allylamine hydrochloride) (PAH, Mw = 70,000), and potassium ferri/ferro cyanide were purchased from Sigma Aldrich and used without further purification. All aqueous solutions were prepared using 18 MΩ ultra purified water. The electrochemical measurement cells and electronics have been described in a previous publication  and comprised a home built potentiostat, an Ag/AgCl reference electrode (#MF 2078 RE-6 from BASi, UK), and a lock-in amplifier (EG&G Princeton Applied Research model # 5210). Illumination was done with a xenon lamp (PTI model A-1010 arc lamp housing, UXL-75XE Xenon Lamp from USHIO, powered by PTI LPS-220) modulated by an optical chopper (Scitec instruments).
Confirmation of QDs immobilization: Immobilization of CdS QDs on top of the Au electrodes was performed with current measurements. CVs were recorded before and after immobilization of BDT and QDs on top of gold electrodes with Fe3+/Fe2+ as redox couple in solution . While on bare gold electrodes the typical oxidation and reduction currents could be observed these were not visible in the case of gold electrodes coated with BDT and QDs (see Additional File 1 for data). Alternatively current at fixed bias voltage was recorded for gold electrodes before and after immobilization of BDT and QDs, while illumination was switched on and off. In the case of QDs present on top of the Au electrode a photocurrent could be measured under illumination (date are shown in Additional File 1)
Results and Discussion
The actual sensor electrode was composed out of QDs which were coupled via a 1,4-benzenedithiol (BDT) layer on top of a gold film electrode. A bias voltage U = +200 mV was applied and the corresponding current I was recorded. Upon illumination of the QDs, electron-hole pairs were generated. Electron transfer could take place in between CdS QDs and the 4AP/QI - redox couple in solution and in between the QDs and the electrode. Thus, the QDs could be used as a light-triggered interlayer to transfer electrons from the redox couple, present in solution to the electrode. The energetical situation of the electron transfer pathway is depicted in Figure 1b/c. 4AP could be only oxidized to 4QI if the two released electrons could be transferred to an energetically lower level. In case the bias U applied to a gold electrode was not positive enough (i.e. its Fermi level was above the energy of the 4AP/4QI redox couple), no oxidation of 4AP could occur (cfg. Figure 1b). However, if at the same bias illuminated QDs were used oxidation of 4AP was possible (cfg. Figure 1c). Upon illumination, electrons in the QDs were excited from the valence band (VB) to the conduction band (CB), resulting in electrons (e-) and holes (h+). The holes were trapped in defect states (DS)  at the surface of the QDs. 4AP could now be oxidized to 4QI upon transferring the electrons to the QDs where they recombined with the holes. In turn, electrons were transferred from the CB of the QDs to the gold electrode, thus creating an oxidation current I.
Oxidation currents for the different geometries
ΔImax [nA] direct detection of 4AP
ΔImax [nA] direct detection of p APP
ΔImax [nA] enzymatic reaction
Detection of p-aminophenyl phosphate: As an experimental complication it has to be pointed out that p APP has limited stability, since p APP decomposes slowly in alkaline solution . In order to be sure to test the enzyme activity on the CdS electrode, p APP has also been investigated with the 3 different geometries given in Figure 2 (without the enzyme). Only a very small response of about 1-2 nA was obtained (cfg. Table 1 and Additional File 1). This is an order of magnitude lower than the response to 4AP and ensured specific detection of the substrate p APP by the enzymatic conversion as will be shown in the following. In a first step, the enzymatic reaction of ALP with p APP causing the production of 4AP was investigated with the enzyme in solution. As has been shown above this is possible, as there is response of the photocurrent to the product 4AP, but barely to the substrate p APP. As shown in Figure 5 a-c the enzymatic reaction could be detected for all the 3 geometries in which the enzyme was free in solution, as indicated in Figure 2. However, there were significant differences in the response curves. In contrast to the detection of 4AP alone (geometry S0) the response in geometry S2 for p APP in the conversion with ALP is small, probably due to a depletion of the substrate near the electrode because of electrostatic repulsion.
In literature KM values of 0.48 mM  and 0.056 mM  have been reported, which are in the same order of magnitude as the values detected in our work with the enzyme in solution. For the sensor configuration developed (I1) a larger value can be derived from the experiments. It has to be pointed out that in the case of the polyelectrolyte -fixed enzyme the KM value has to be considered as apparent KM value since here the concentration of half maximum conversion rate is influenced by the immobilization . Comparison of the ΔImax values as obtained for direct detection of 4AP (Figure 4) and detection of 4AP after enzymatic degradation of p APP to 4AP shows that both oxidation signals (detected at the same geometry and provided abundance of enzyme) are quite similar. This is in good agreement with the detection principle proposed.
In summary the developed sensor as illustrated in Figure 2d by immobilizing the ALP via the polyelectrolyte PAH, provides the proof of principle for a detection system for the enzyme substrate p APP. The analytical performance with a detection regime within the concentration range from 0.025 to 1 mM is relatively poor, so that the here presented device has to be seen as a proof of principle demonstrator rather than as an applicable sensor.
A light controlled bioelectrochemical sensor for p APP has been demonstrated. By using QDs as interlayer on gold, 4AP could be oxidized and thus detected via a corresponding photocurrent in case the QDs were illuminated. Enzymes could be functionally immobilized on the sensor surface. This provides the basis for future spatially resolved measurements  by selectively illuminating and reading-out only the area of interest of an electrode which is non-structured, but modified with different immobilized enzyme systems. The approach presented here allows for observing enzymatic reactions which yield 4AP as product. We have demonstrated this for the substrate p APP and the enzyme ALP. A crucial point for such measurements is to ensure high local enzyme concentration and specificity for the detection of the enzymatic product. By using a polyelectrolyte layer of PAH, the enzyme ALP could be immobilized on the electrode surface, retaining enzymatic activity. However, polyelectrolyte layers can also hinder diffusion of the molecule to be detected 4AP to the QD surface, thus hindering detection. For this reason permeability of the polyelectrolyte layers has been studied here for the respective molecule.
This work was supported by the German Research Foundation (DFG, grants PA 794/3-1, LI706/2-1).
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