3. Results
3.1 Biochemical characterization of human guanylate kinase
3.1.5 Electrochemical detection of guanosine monophosphate with a
3.1.5 Electrochemical detection of guanosine monophosphate with a quantum dot-based biosensor modified with hGMPK
A new approach has been developed to detect kinase-catalyzed reactions based on the light-triggered electrochemical sensing of NADH in a three-step coupled-enzyme assay as described in section 2.2.7. We demonstrated the proof of concept of using a photochemical sensor for the
HEK293 stable
cell line
6-TG EC50 ± SD (µM)
EGFP-hGMPK ~10 ± 3
EGFP ~62 ± 7
a.
c.
b.
Fig. 3.13. Sensitivity of EGFP-hGMPK and EGFP-expressing HEK293 stable cell lines to 6-thioguanine. (a) & (b), 6-thioguanine (6-TG) dose-response curves of EGFP and EGFP-hGMPK-expressing stable cell lines, respectively. Cell survival was measured by the MTT assay after 48 h incubation with 6-TG. Untreated samples (0 µM 6-TG) were set at 100% cell survival. Each data point (mean ± SD, n=3) is indicated as percentage of the value for control wells with no 6-TG. Sigmoidal curves were fitted to data points of stable cell lines over a 106 -fold range of 6-TG concentrations (0.001-1000 µM) using Gnuplot 5.0 software (nonlinear regression fitting). (c) Determination of 6-TG EC50 for EGFP-hGMPK and EGFP HEK293 stable cell lines. EC50 values are shown as mean ± SD.
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electrochemical detection of a nucleotide kinase-catalyzed reaction. This novel technique senses the change in photocurrent in a GMP concentration-dependent way by a CdS/ZnS quantum dot (QD)-based electrode [87, 88].
First, we tested the validity of our approach for the control reaction between pyruvate (the product of pyruvate kinase in step 2) and NADH catalyzed by lactate dehydrogenase (LDH). The sensor electrode was composed of CdS/ZnS QDs which were layered on a gold electrode via stilbenedithiol (StDT). Varied concentrations of pyruvate substrate were used in the reaction buffer (100 mM HEPES pH 7.5, 100 mM KCl and 20 mM MgCl2) in a 1 ml electrochemical cell container. Electron hole pairs were generated upon illumination of QDs. The electron transfer takes place in between the CdS/ZnS QDs electrode and the NADH/NAD+ redox pair in solution.
The current “I” was recorded before and after the reaction was completed at a constant bias voltage, U = +50 mV. It was found that the CdS/ZnS-modified gold electrode was acting as a transducer for the analysis of pyruvate which depletes NADH by oxidizing it into NAD+ in the LDH-catalyzed reaction. The change in pyruvate concentration was directly proportional to the change in photocurrent under pulsed illumination at a constant biased potential of +50 mV against Ag/AgCl (Fig. 3.14a & b).
a.
I (nA)
[Pyruvate] (mM)
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A linear relationship was observed between pyruvate concentration and the change in photocurrent. We coupled this control reaction to two other steps in the coupled assay as depicted in Materials and Methods section 2.2.7. In this case, the concentration of GMP (substrate of hGMPK) was altered in the range of 50 µM-1600 µM at constant concentrations of hGMPK and all other constituents of the assay. Each time, 5-10 min were provided for the reaction to complete. The change in photocurrent (∆I) was measured upon illumination of CdS/ZnS QDs before and after adding the GMP substrate to the reaction mixture. It was plotted against the respective GMP concentrations using OriginLab 8.1 software (Fig. 3.15a & b). The data showed
Fig. 3.14. Dose-response curve for the detection of pyruvate in the control reaction. (a) Photocurrent detected before and after adding pyruvate (Py) to the reaction mixture. (b) The control reaction as shown below was catalyzed by lactate dehydrogenase (LDH) in a buffer (100 mM HEPES pH 7.5, 100 mM KCl and 20 mM MgCl2) containing 1.2 mM NADH at a constant bias potential of +50 mV and 25 oC. The concentration of pyruvate was varied from 50 µM to 1600 µM. Before adding pyruvate to the reaction mixture, the current was recorded.
Upon addition of pyruvate, the reaction was allowed to complete for 5-10 min. The current was detected again and the change in current “∆I” in nA was plotted against the respective pyruvate concentrations “c” in µM using the OriginLab software (version 8.1). Data points are represented as means ± standard deviation of triplicate measurements.
b.
[Pyruvate] (µM)
∆I (nA)
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that GMP can be detected from a very low concentration like 50 µM to as high as 1200 µM under the specified experimental conditions.
a.
Fig. 3.15. Dose-response curve for the detection of GMP in the electrochemical detection assay (see section 2.2.7). (a) Photocurrent detected before and after adding GMP to the reaction mixture. (b) The first reaction in the three-step coupled assay was catalyzed by hGMPK in the buffer (100 mM HEPES pH 7.5, 100 mM KCl and 20 mM MgCl2) at a constant bias potential of +50 mV and 25 oC. The current was recorded before adding the GMP substrate (50 µM-1600 µM) to the reaction mixture. The reaction was allowed to complete for 5-10 min and the current was detected again. The change in current “∆I” in nA was plotted against the respective GMP concentration in µM using the OriginLab 8.1 software. Data points are represented as means ± standard deviation of triplicate measurements.
b.
GMP
I (nA)
[GMP] (µM)
∆I (nA)
[GMP] (µM)
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To shape our electrochemical detection system into a photosensor format, we immobilized the enzymes on the CdS/ZnS QDs electrode using layer-by-layer assembly of the polyelectrolytes, PSS (poly (styrene sulfonate)) and PAH (poly (allylamine hydrochloride)), as shown in Fig. 3.16 [114, 115].
Human GMPK and two helper enzymes (PK and LDH) were deposited on the QDs electrode by two consecutive bilayers of PSS/PAH, with PAH forming the outermost layer. The overall structure of the assembled electrode can be expressed as (PSS/PAH)2/hGMPK;PK;LDH/QDs/
StDT/Au/Si. This geometry was used for the detection of the GMP-dependent oxidation current as shown in the dose-response curve in Fig. 3.17.
Fig 3.16. Human GMPK immobilized on QDs/StDT/Au using polyelectrolyte bilayers.
CdS/ZnS QDs were attached to the Au (gold) electrode surface via StDT (stilbenedithiol). The hGMPK and two helper enzymes (PK and LDH) were immobilized on the electrode surface by two bilayers of PSS (poly (styrene sulfonate)) and PAH (poly (allylamine hydrochloride)) polyelectrolytes using the layer-by-layer (lbl) assembly technique.
.
PAH
Enzymes QDs
StDT
Au electrode
PSS
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Fig 3.17. Dose-response curve for the detection of GMP by human GMPK immobilized on QDs/StDT/Au electrode surface. (a) Photocurrent detected before and after adding GMP to the reaction mixture. (b) The hGMPK enzyme was immobilized on the gold electrode surface at the bottom of the electrochemical cell using PSS and PAH polyelectrolytes bilayers (the specific geometry is shown in Fig. 3.16). The first reaction in the three-step electrochemical detection assay (see section 2.2.7) was catalyzed by hGMPK in the buffer (100 mM HEPES pH 7.5, 100 mM KCl, 20 mM MgCl2), and detected at a constant bias potential of +50 mV and 25 oC. The current was recorded before adding GMP to the reaction mixture. Various amounts of GMP (50 µM-1600 µM) were added to the reaction cell. The reaction was allowed to complete for 5-10 min, and the current was detected again. The change in current “∆I” in nA was plotted against the GMP concentration in µM using the OriginLab 8.1. Data points are represented as means ± standard deviation of triplicate measurements.
a.
b.
I (nA)
[GMP] (µM)
[GMP] (µM)
∆I (nA)
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The data revealed that the concentration of GMP can be detected within the range of 50 μM to 800 µM by using this biosensor. Above 800 µM, the photocurrent response was saturated. The deposition of hGMPK in the above-mentioned setup successfully showed activity which is an important precondition for the sensor. However, the maximum current detected (37 nA) was comparatively lower than that of free enzyme in solution (45 nA). It may be due to the electrostatic interactions of the nucleotide substrates with the negatively (PSS) and positively (PAH) charged polyelectrolytes. The three dose-response curves mentioned above are compared in Fig. 3.18.
Fig. 3.18. Comparison of the dose-response curves for the detection of GMP in the three-step electrochemical detection assay and pyruvate in the one-step control reaction. The change in current ∆I (nA) was plotted against the respective concentrations “C” of the substrate in µM: pyruvate as “Cpyruvate” and GMP as “CGMP” using the OriginLab 8.1 software. The control reaction is catalyzed by pyruvate kinase (PK).
∆I (nA)
C (µM)
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3.1.6 Human GMPK-catalyzed reactions in polyelectrolyte containers of different shapes