The enzyme is of sufficient purity for use in electrochemical measurements.
3. This sensing system using a nanoparticle-modified probe has the ability to detect 10 pM genomic DNA from MRSA without amplification by PCR. Current responses are linearly related to the amount of genomic DNA in the range of 10–166 pM (Fig.4b). Selectivity is confirmed by showing that this sensing system could distinguish MRSA from SA DNA (Table1).
y = 0.0173x + 0.102 R2= 0.99011
1.0 2.0 3.0
ΔI (nA)
0 50 100 150 200
DNA concentration (pM)
A B
1 nA
50 s ΔI
Fig. 4(a) Chronoamperometry of hybridization products. Thearrowed lineindicates whenL-proDH andL-proline were injected, after which oxidation currents were immediately observed. (b) Quantification of the DNA from MRSA based on current response. The error bars show standard deviations of triplicate experiments (nẳ3)
Table 1
Purification of LPDH from recombinant cells
Steps
Total
activity (units)
Total protein (mg)
Specific activity (units/mg)
Fold purification
Yield (%)
Crude extract 65.1 651 0.100 – –
Heat treatment 63.2 121 0.524 5.24 97.1
Ni-NTA Superflow 54.0 60.4 0.894 8.94 82.9
Sephacryl S-300 55.2 59.1 0.934 9.34 84.8
Acknowledgment
This is the author’s version of a work accepted for publication by Elsevier. Changes resulting from the publishing process, including peer review, editing, corrections, structural formatting, and other quality control mechanisms, may not be reflected in this document.
Changes may have been made to this work since it was submitted for publication. The definitive version has been published in Bio- sensor and Bioelectronics, VOLUME 67, SPECIAL ISSUE BIO- SENSORS 2014, May 15, DOI: 10.1016/j.bios.2014.08.075
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Electrochemical Lateral Flow Paper Strip for
Oxidative-Stress Induced DNA Damage Assessment
Jared Leichner, Mehenur Sarwar, Amirali Nilchian, Xuena Zhu, Hongyun Liu, Shaomin Shuang, and Chen-zhong Li
Abstract
The phrase “oxidative-stress induced DNA damage” is commonly used in both the scientific literature and common media outlets, and is frequently linked to detrimental elements of aging as well as the onset of illnesses. Due to the growing focus on this topic, a clear need has emerged to develop a quantitative, low- cost methodology to allow for periodic monitoring of oxidative-stress induced DNA damage within individuals. Recent literature examining the link between oxidative stress and the onset of various cancers has made monitoring an even more pressing need. The mechanism of oxidative-stress induced DNA damage originates in chronic inflammation, which in turn activates various transcription factors and diseases that influence the onset of tumor development, chemoresistance, radioresistance, and other harmful cellular processes. While current technologies that aim to provide quantitative metrics require extremely expensive equipment and significant technical expertise, our laboratory has designed a low-cost methodology utiliz- ing a combination of carbon nanotubes, paper electrodes, and immunochromatographic strips.
Key words ROS, DNA oxidative damage, Cancer, 8-OHdG, Lateral flow immunosensor, Paper strip, Carbon nanotubes, Chronoamperometric, Colorimetric
1 Introduction
1.1 Oxidative Stress- Induced DNA Damage
To understand the origin of oxidative stress-induced DNA Dam- age, it is essential to begin with a discussion of oxidative stress.
When natural antioxidants within the body fail to maintain the balance between production and neutralization of the reactive oxy- gen species (ROS), the resultant condition is called oxidative stress [1]. ROS are identified as both necessary and harmful molecules, with prototypical examples of these molecules being superoxide anion, hydroxyl radical, and hypochlorite ion. These species are utilized by the thyroid gland to synthesize a hormone called thy- roxine. Inside the immune system, cells such as macrophages and neutrophils use ROS to kill bacteria by phagocytosis. Under normal
Ben Prickril and Avraham Rasooly (eds.),Biosensors and Biodetection: Methods and Protocols, Volume 2: Electrochemical, Bioelectronic, Piezoelectric, Cellular and Molecular Biosensors, Methods in Molecular Biology, vol. 1572,
DOI 10.1007/978-1-4939-6911-1_3,©Springer ScienceþBusiness Media LLC 2017
23
physiological conditions ROS are produced and controlled by enzymes such as superoxide dismutase, catalase and vitamins such as vitamin E and C. Overproduction of these ROS can occur through numerous mechanisms such as exposure to ultraviolet or ionizing radiation or through ingestion of inflammatory agents such as through tobacco smoking. It is clear that while these reactive species have a positive effect in the human body, they can be easily overexpressed and underregulated under certain conditions.
Elevated levels of ROS can lead to diseases such as diabetes, cancer, or neurodegenerative diseases [2]. The figure below demonstrates ways in which potential damaging agents can interact with cells to create cell-specific mutations(Fig.1). These agents, such as X-rays, oxygen radicals, and UV light can lead to the development of disease conditions and directly damage DNA.
Our immune system, with the help of antioxidants and DNA repair mechanisms, helps balance the damage caused by the agents previously described. Examples of antioxidants include vitamin C, vitamin A, and vitamin E. The various DNA mechanisms that exist to help detect and repair any problems caused by the damaging agents include base excision repair (BER) for single stranded breaks, recombination repair (RR) for double strand breaks, nucleotide-excision repair (NER) for when bulky adducts cause the formation of abnormal DNA, and finally by mismatch repair, to handle when adenine and guanine bond instead of the typical Fig. 1This is an example of the mechanism by which oxidative damage can cause accumulative stresses that lead to cancer and other forms of cellular damage. We demonstrate how this molecular mechanism can contribute to the 8-OHdG production
adenine–thymine pair. When the body fails to maintain this balance due to high concentrations of damaging agents, various problems can occur including cell cycle arrest, apoptosis, and mutation in chromosomes. These mutations may also activate certain genes and can push the cell cycle towards uncontrolled cell division and hence cancer.
The mechanisms that connect ROS and cancer onset originate in the process of chronic inflammation [3]. The inflammation of affected tissue activates a number of transcription factors, including Nrf2, β-catenin/Wnt, p53, and AP-1. The activation of these factors upregulates the activity of numerous genes, such as those for growth factors, cell-cycle regulatory molecules and chemokines.
This pattern can be witnessed in pathologies such as Crohn’s disease, where chronic inflammatory bowel disease puts patients at a far higher risk of colon adenocarcinoma. Due to the powerful and scientifically validated downstream effect of ROS on cancer, our laboratory has decided to focus on cancer detection through analysis of oxygen radicals.
Measuring the presence and danger imposed by these free oxygen radicals can conveniently be performed through the detec- tion of 8-oxo-20-deoxyguanosine (8-OHdG). Until now, the mea- surement of 8-OHdG has been done solely through complicated analytical techniques. The experimental diagram below demon- strates the production of 8-OHdG from 20-deoxyguanosine (20- dG) through the application of oxygen radicals.
During the process of DNA repair discussed previously, endo- nuclease and glycosylase enzymes participate to cleave oxidized guanine. This cleaved molecule is water soluble in nature, and hence can be excreted into the urine without being metabolized any further. Thus, urinary 8-OHdG is considered to be an impor- tant biomarker of generalized, cellular oxidative stress and reflects the ‘whole body’ repair capacity. Specifically, 8-OHdG formation from 20-dG can be used to measure and monitor ROS levels, and there is a great need to find quantitative detection methods that are faster, less costly, and less labor intensive than currently available techniques. Quantitative assessment of ROS levels can be extremely useful in providing pre-symptomatic indications of disease.
1.2 Typical Analytical Detection Techniques
The measurement of 8-OHdG up until this point has been done solely through complicated analytical techniques such as high per- formance liquid chromatography (HPLC), electrochemical detec- tion (ECD)/(HPLC-ECD) [3], mass spectrometry (MS)/(HPLC- MS) [4], gas chromatography (GC)/(GC-MS), capillary electro- phoresis (CE)/(CE-ECD), and enzyme-linked immunosorbent assay (ELISA). The combination of HPLC and ECD provides an accurate quantification method for electrochemically active com- pounds. ECD has been shown to have three orders of magnitude of sensitivity in excess of UV detection [5]. MS works through
ionization of chemical compounds and generates resultant charged molecules of either positive or negative charges. Experimental results have demonstrated a wide range of detection between 0.5 and 25 ng/mL using HPLC tandem MS [6,7]. However, its cost and technical expertise requirements make it difficult for smaller facilities to own. GC is often combined with MS, but together they are a lengthy and expensive process [8]. Capillary electrophoresis is also a very effective method for separating mixtures, and can some- times be combined with ECD to improve the sensitivity of the analysis [9]. Finally, ELISA is an extremely sensitive test which is used to detect specific antigens. ELISA can also be combined with other analytical measurement techniques such as MS to improve its sensitivity [10,11].
1.3 Reducing the Cost of Detection:
Electrochemical Biosensors
The invention of the first enzymatic electrochemical biosensor is by Sir Leland C. Clark, who used immobilized glucose oxidase for the fabrication of an enzyme electrode and presented it at the New York Academy of Sciences Symposium in 1962. Since then, many bio- sensors have been fabricated and commercialized. These biosensors use various electrical characteristics in their design, and can be broadly classified as potentiometric, amperometric, and impedi- metric, as well as combinations of these techniques.
Typically, three types of electrodes are used in an electrochemi- cal cell, the working electrode (WE), the reference electrode (RE), and the counter electrode (CE). Gain or loss of electrons (Redox) takes place at the site of the WE. Usually, this is the electrode which remains in contact with the working solution. An important feature of this WE is that it must be electrochemically inert over a wide range of potentials. REs, typically made of silver/silver chloride, maintains stability in potential against the potential of WE. The third type of electrode, CE, is usually in the form of a platinum wire, and acts as a separate platform for a redox reaction to occur.
This separate redox reaction balances the redox reaction occurring at the surface of the WE. The main advantage of this electrode is that in the presence of the CE a sufficient current into the solution can be produced without requiring excessive voltage or creating a nonuniform current distribution on the WE.
Our laboratory has studied the detection of 8-OHdG exten- sively in recent years and we have developed novel platforms to optimize the quality and sensitivity of our detection system [12–14]. Utilizing activated carbon fiber microelectrodes within a lateral flow immunoassay, we can now measure cellular 8-OHdG in real-time, utilizing single wall carbon nanotubes (SWCNTs) from the surface of a single cell. Our substrate of interest, 8-OHdG, has redox properties and can be detected by an electrochemical method since it is formed by an oxidation reaction via a two-electron two-proton charge transfer reaction (Fig.2). This electrochemical property has been well characterized for quantitative measurement.
Figure3demonstrates the basic principle behind the quantita- tive detection of 8-OHdG. The assay begins by applying a known concentration of 8-OHdG to the application pad (Fig. 3a). The capillary action helps the sample to move forward and to rehydrate the area where AuNP-anti-8-OHdG is sequestered. An immunor- eaction takes place, forming AuNP-anti-8-OHdG-8-OHdG com- plex. These complexes further move along the strip (Fig.3b) and reach the test zone. When AuNPs accumulate on the test zone, a color change of the strip test zone can be noticed. A positive test is demonstrated by red color formation, which can be seen by the naked eye (Fig.3c). AuNPs which are bound only to antibodies and not with the 8-OHdG get eliminated by the BSA-8-hydroxyguanosine and is immobilized in the control line, never reaching the test zone (Fig.3d). A current vs time plot can be achieved by using a chron- oamperometric setup with an electrochemical analyzer at exactly 10 min after application of the sample (Fig.3e). Qualitative data can also be obtained by using a scanner, which can detect signals of color intensities. The color intensity is proportional to the amount of AuNPs present in the test zone. This numerical value is inversely proportional to the concentration of 8-OHdG in the sample solution.
1.4 Combined Methodologies:
Electrode Integrated Lateral Flow Immunosensor
Electrode integrated lateral flow sensors and lateral flow immuno- sensors are both extremely useful for rapid biological measurement assays due to their ease of use and small sample size requirements.
However, combining these two methodologies presents additional benefits. Our protocol provides two separate measurements, color- imetric and chronoamperometric, which aid in improving the con- fidence of the final result. This is akin to the use of two separate fluorescent tags during immunofluorescent staining when attempt- ing to identify a cell type. Since the two methods differ in their signal transduction and detection strategies, we can avoid the pos- sibility of false positive or false negative results originating from a single method. Finally, the placement of these two sophisticated Fig. 2Formation of 8-OHdG by oxygen radicals
Fig. 3Mechanism of quantitative detection of 8-OHdG
techniques on a paper strip allows their use in point of care (POC) settings, where expensive scientific equipment is unaffordable and where simplicity, low cost and portability are needed.
To describe the advantages and disadvantages of this combina- tion in further detail, it is important to understand the limitations of the immunosensor and the electrode approach to 8-OHdG detection. Our immunosensors utilize gold nanoparticles to assist in signal amplification, and while the test can be performed using a machine such as a reflectometer or even the human eye, it can be prone to inaccurate results depending on the sample environment and is thus far more difficult to utilize on real patient samples than with stock solutions. Additionally, the precision of the assay depends on the availability of antibodies to adsorb onto the surface of the gold nanoparticles, which itself relies on hydrophobic and ionic interactions. A sample which would react poorly with our lateral flow immunosensor would have a high ionic strength, a high urea concentration or a low pH, which would accelerate the resultant dissociation of the antibody-gold nanoparticle complex and thus interrupt the measurement procedure. A patient who is simply dehydrated can invalidate the entire protocol.
To overcome these drawbacks of immunostrip assays, electro- chemical methods are used. To achieve this, we have integrated carbon nanotubes (CNTs), paper electrodes, and immunochroma- tographic strips in the format demonstrated in Fig.3. The structure of the new design is similar to that of the traditional lateral flow strip. Two electrodes are integrated into the original strip design to carry out the electrochemical detection function. CNT-embedded conductive paper is used as the working electrode. The CNTs paper is placed on the control line with the sensing surface facing down, and its extension beyond the strip is laminated with a silver plated copper wire and connected to an electrochemical analyzer. Ag/
AgCl ink painted copper paper is placed at a 1 mm distance from the CNTs paper on the side farthest from the absorption pad to serve as the reference/counter electrode. Finally, the entire strip is laminated in order to ensure complete contact between the sample, membrane, and electrodes.
Electrochemistry is an appealing methodology because the precision of the signal transduction process does not rely on the conjugation of antibodies and is therefore still reliable when envi- ronmental conditions alter the binding and dissociation rates of nanoparticle–antibody complexes. Electrochemistry also has a drawbacks, though, ironically due to its high specificity. While the electrochemical method does responds only to 8-OHdG mole- cules, the ability of antibodies to bind to specific molecular epitopes allows them to respond not only to the 8-OHdG molecule but also to short nucleotide sequences carrying 8-OHdG. This capability allows the antibody method to be slightly more sensitive in detect- ing 8-OHdG due to the added capability of detecting these small nucleotide sequences.
8-OHdG levels through this platform are thus determined through a combination of chronoamperimetric and colorimetric measurement techniques [8]. The chronoamperimetric measure- ments are performed using a CHI 660C electrochemical analyzer.
10 min after the 8-OHdG-spiked urine samples are applied to the sensor strip, simultaneous chronoamperimetric measurements (at a fixed voltage of 0.42 V for 20 s) and photographic measurements (utilizing a digital scanner) are performed. ImageJ software is used to quantify the color intensity of the test lines and chronoamperi- metric measurements are expressed based on the mean and stan- dard deviation of three trials.
The combined methodology is able to provide a detection limit of 2.07 ng/mL with only 10 min to complete the assay. Our methodology has demonstrated that it is possible to take the expensive machinery used in analytical electrical or immunological detection strategies and miniaturize it into a convenient paper strip apparatus. Additionally, we have proven that following miniaturiza- tion, it is possible to combine the methodologies to provide a more thorough snapshot of biological processes. Over the past 5 years, our technique has evolved into a multimodal measurement method that is not only low cost but also robust. Future work may include an integrated wireless platform to allow for remote bio-sensing, storage, and tracking.
2 Materials
2.1 Preparation of the Immunochromato- graphic Strip
2.1.1 Formation of the AuNPs
AuNPs ae produced by a modified citrate production method utilizing:
l Zetasizer, Nano series (Malvern Instruments, Woodstock, GA).
l Analytical balance is bought from Amazon vendor.
l 220 Mini Hotplate/Magnetic Stirrer is bought from Amazon vendor.
l Thermometer and stand/holder is bought from Amazon vendor.
l 10 mL graduated cylinder is bought from Thermo Fisher scientific.
l 250 mL graduated cylinder is bought from Thermo Fisher scientific.
l 250 mL Erlenmeyer flask is bought from Thermo Fisher scientific.
l 1 L dark bottle is bought from Thermo Fisher scientific.
l HAuCl4is bought from Sigma-Aldrich (St. Louis, MO).
l Trisodium citrate dihydrate is bought from VWR (West Chester, PA).