Biosensors for polyphenols and other analytes. A novel label-free electro- chemical scheme for probing the electronic properties of DNA binding with small molecules was recently described by our group [ 67 ]. The strategy, called metal-enhanced electrochemical detection (MED) involved the electrooxidation of silver ions, deposited as a monolayer onto a gold electrode surface in the presence of dsDNA molecules. The results indicated that by oxidizing the silver monolayer, highly reactive oxides of silver ions are generated, causing a change in the electronic properties of the immobilized dsDNA. Thus, in the presence of low molecular weight organic DNA binding molecules, structural change in the DNA occurs, generating corresponding change in the redox properties of the silver monolayer. These variations are proportional to the concentration of DNA binding molecules and can be easily quantifi ed by voltammetric techniques. The approach off ers an alternative route for the detection of DNA hybridization reactions, DNA-protein interactions, and gene detection. It was demonstrated that the performance of the DNA biosensors are strongly dependent on hybridization reactions at the transducer-solution interface. Thus, control of the hybridization conditions such as temperature and hybridization time can considerably extend the dynamic range and lower the sensitivity [ 67 ].
The strategy for MED using the reactivity of silver ions is illustrated in Fig. 27.6 . A monolayer of silver is deposited on a gold electrode or other conducting substrates (e.g., platinum, or glassy carbon). The silver deposition can also be achieved by incubating silver compounds for approximately 5 minutes in the dark at room temperature. The electrochemical oxidation of silver produces silver ions and electrons accompanied by a reversible redox signal ( Fig. 27.6(a) ). If dsDNA is present at the surface, the silver ions are dispersed and are held electrostatically. Upon reduction, the silver ions return to the surface and a reduction current is measured ( Fig. 27.6(b) ). If a DNA binding low molecular weight organic molecule is introduced into the solution, structural change in the DNA molecule occurs, which is signifi ed by a
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simultaneous change in the redox currents from silver, and a decrease in current is measured ( Fig. 27.6(c) ). This decrease is proportional to the concentration of the DNA binding molecule. The underlying signal transformations produced here could result from the DNA conformational or structural changes in the presence of the analyte molecules, which ultimately hinder the fl ow of electrons.
Typical results for MED are shown in Fig. 27.7 using diff erential pulse voltammetry.
Using the proposed MED concept and un-optimized assay format, a detection limit of 4 ppt has been recorded for Bisphenol (BPA), an endocrine disrupting chemical ( Table 27.1 ), whereas the limit of detection for the same analyte
Figure 27.6 Metal-enhanced electrochemical detection (MED) concept for electrochemical amplifi cation using Ago/Ag+ couple (a) in buff er only, (b) at an immobilized dsDNA electrode in buff er, or (c) at an immobilized dsDNA electrode in buff er and analytes.
Sensor response
Sensor response (a)
(a)
(c)
buffer
Intercalator
Sensor response Ag
Au
Ag++ e-
Ag Au
Ag++ e–
Ag Au
Ag++ e– Ag+
Ag+
Ag+
Ag+
Ag+
Ag+ Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
Ag+
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using ELISA was 228 ppb. This represents a 1,000-fold improvement over the gold standard. In addition, the time required for the MED biosensor was about 20 minutes, compared to between 24 hours and 3 days necessary for the ELISA techniques. Apart from small organics, we have tested the utility of the MED concept for other molecules with molar masses ranging from 200 to 150,000 amu. The range of analytes tested include nucleic acids, PCBs, nonylphenols, cisplatin, and other naturally occurring isofl avonoids [ 64 , 65 ].
Biosensors for endocrine disrupting chemicals. In recent years, it has become evident that many environmental chemicals, including synthetic and endogenous estrogens, can mimic, block, or alter the action of endogenous steroid hormones and can interfere with hormone regulated physiological processes [ 15 , 68–72 ].
These industrial and environmental chemicals are known as endocrine
Figure 27.7 Typical metal-enhanced electrochemical detection (MED) signals at (a) diff erent analyte concentrations of DNA(b) calibration curve for the MED-based assays.
0 20 40 60 80 100 120
0 1 2 3 4 5 6
Log [pM]
Current (uA)
0 -100 -200 -300
Current Density (uA/cm2)
Potential (mV) -400
-500 -600
0 25 50
(a) (b)
75
(iV) (iii) (ii) (i) 100 125 150 175 200
Table 27.1 Comparison of MED-based Biosensor Performance with Conventional Techniques
Analytes Limit of Detectiona,b,c
ELISA Electrochemical
Fe 2+/Fe3+
Detection Ag0/Ag+ Couple
BPA 228 ppb 10 nM 4 ppt
NP 37.5 ppb ND 14 ppt
Cisplatin 1 nM 10 pM
PCBs ND 5 ppt
BPA: Bisphenol A; ND: None Detected; nM: Nanomolar; PCB: Polychlorinated biphenyls;
pM: Picomolar; ppt: Parts per trillion.
aSadik et al, JACS, 123, 2001, 11335; bNgundi M., PhD Binghamton University, 2003,
cKowino I., PhD Binghamton University 2006.
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disrupting chemicals (EDCs) and structurally resemble endogenous estrogens.
They have been analyzed for many decades in numerous biological and medical investigations. Screening and confi rmatory strategies for these steroids involve chemical or immunochemical methods followed by the complete instrumental confi rmation of steroids by mass spectrometry. Until recently, the standard technique for analyses has been GC/MS. Moreover, the limits of detection were not suffi cient to analyze steroids at low levels in urine and environmental samples. Also, these techniques typically require sample pretreatment, expensive apparatus, and skilled personnel.
Synthetic estrogens are characterized by the presence of phenolic functional groups, a common structural feature that is also found in natural estrogens.
This structural feature could facilitate binding to estrogen receptor [ 68–72 ] and possibly generate receptor-induced transformations. Sadik et al. presented a summary of diff erent approaches reported for EDCs [ 15 ] and also demonstrated the feasibility of in situ monitoring of the interaction between bisphenol-A (BPA) and dsDNA [ 65 ]. In another report, Ngundi et al. demonstrated the comparative electrochemical behavior of β-estradiol and selected EDCs, specifi cally alkylphenols, and proposed a possible link between the structure and their estrogenic activity [ 72 ]. More recently, we have reported the isolation and complete structural characterization of quercetin and other isofl avonoids [ 74 ].
We have also developed and optimized a fully autonomous electrochemical biosensor for studying the role of alkylphenols on A549 lung adenocarcinoma cell line. This advanced biosensor uses a prototype 96-electrode (DOX-96) well-type device that allows the measurement of cell respiratory activity via the consumption of dissolved oxygen. The system provides a continuous, real-time monitoring of cell activity upon exposure to naturally occurring polyphenols, specifi cally resveratrol (RES), genistein (GEN), and quercetin (QRC). The system is equipped with a multipotentiostat, a 96-electrode well for measurements and cell culturing with 3 disposable electrodes fi tted into each well. A comparison with classical “cell-culture” techniques indicates that the biosensor provides real-time measurement with no added reagents. A detection limit of 1x10 4 was recorded versus 200 and 6x10 3 cells/well for MTT and fl uorescence assays, respectively. This method was optimized with respect to cell stability, reproducibility, applied potential, cell density per well, volume/
composition of cell culture medium per well, and incubation. Others include total measuring time, temperature, and sterilization procedure. This study represents a basic research tool that may allow researchers to study the type, level, and specifi c infl uence of isofl avonoids on cells.
The DOX approach utilizes a prototype multi-channel system that enables simultaneous, quantitative, and continuous measurement of dissolved oxygen using a 96-well electrode biosensor prototype shown in Fig. 27.8 known as the DOX device. DOX is fully automated, portable, equipped with a multipotentiostat, and can be connected to a computer. The latter enables external control of the instrument, on line recording of experimental parameters, graphical presentation and data storage. The instrument software plots current intensity versus time
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for each well while data are simultaneously processed for 12 channels, each corresponding to 8 sensors. Experimental set-up involves the use of 96 disposable electrodes in a three-electrode format (reference, working, and auxiliary).
These 96 sensors are placed in a conventional 96 well plate in which the cells are cultured. Respiration of cells generates a reduction in the concentration of dissolved oxygen, which is determined using electrical current produced [ 21 , 22 ] according to the following reaction:
O2 + 4H+ + 4e–→ 2H2O (27.3) Cytotoxicity measurements involve the application of cells into the optimum growth medium containing a selected isofl avonoid. This is followed by a continuous monitoring of the oxygen consumed by the cells with time. Basically, if an inhibition of cell proliferation occurred in the presence of PPh this will generate higher oxygen content, proportional with the inactivation level induced by PPh. This could be indirectly correlated to the concentration of PPh introduced in the medium.
Figure 27.8 Schematic diagram of the multi-channel DOX oxygen sensor system used for measuring cytotoxicity. Two confi gurations of the 96 electrodes are available disposed in top or bottom of the well plate. Each sensor consists of three electrodes: reference (RE), auxiliary (CE), and working (WE).
Top electrodes 96 plate
Cell culture Electrode system
Electric contact PC
Current
Time 12 Channels x 8 electrodes
Bottom electrodes Temperature control: 37°C
Multipotentiostat
RE WE AE
External control On-line recording Data storage and treatment
Graphical presentation
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