fiber optic biosensor for detection of dna damage

Analytica Chimica Acta 444 (2001) 51–60

Fiber optic biosensor for detection of DNA damage

Kim R. Rogers a,∗

, Alma Apostol a, Steen, J. Madsen

b, Charles W. Spencer

c

a US Environmental Protection Agency, National Exposure Research Laboratory Las Vegas, Las Vegas, NV 89193, USA b Department of Health Physics, University of Nevada-Las Vegas, Las Vegas, NV 89154, USA

c Nevada Radiation Oncology Centers, Las Vegas, NV 89106, USA

Received 26 April 2000; received in revised form 19 September 2000; accepted 25 September 2000

Abstract

This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The hybridization pair consists of a biotin labeled 38 mer oligonucleotide immobilized to a streptavidin-coated optical fiber and a fluorescently-labeled near-complementary (two base mismatch) oligonucleotide re-porter sequence. The hybridization-based assay detected 50 nM labeled probe and could be run up to 10 times on the same fiber. Melting profiles were sensitive to high energy radiation and to 3-morpholinosydnonimine (SIN-1)-generated reactive decomposition products. The dynamic range of the assay for ionizing radiation extends from 20 to 1000 cGy. Oxidative damage induced by SIN-1 was measured over a concentration range of 250 M to 3 mM. Published by Elsevier Science B.V.

Keywords: Fiber optic biosensor; DNA; Hybridization; Chemical DNA damage; Oxidative DNA damage

1. Introduction

One of the approaches for reducing uncertainties in the assessment of human and ecosystem exposures is to better characterize hazardous pollutants that may contaminate these environments. A significant limi-tation to this approach, however, is that sampling and laboratory analysis of contaminated environmental and biological samples can be slow and expensive, thus, limiting the number of samples that can be analyzed within time and budget constraints. Rapid and inexpensive indicator assays that can be used to assess the potential for exposure and which can be

∗ Corresponding author. Present address: US EPA, 944E

Harmon Avenue, Las Vegas, NV 89119, USA. Tel.: +1-702-798-2299; fax: +1-702-798-2107.E-mail address: [email protected] (K.R. Rogers).

0003-2670/01/$ – see front matter. Published by Elsevier Science B.V. PII: S0003 - 2670(01)01150 - 3

related to biological targets such as DNA could be of great benefit to the exposure assessment process.

Oxidative damage to nucleic acids can have serious effects in eucaryotic cells, e.g. resulting in apopto-sis or malignant transformation [1]. A wide variety of chemical and radiation sources cause this type of damage to cellular DNA. A number of the chemical compounds are known or suspected environmental pollutants that may contaminate matrices relevant to human or ecosystem exposure such as groundwater, soil, and sediments [2]. As a result, screening assays for chemically-induced oxidative DNA damage can be of considerable value to certain aspects of the exposure assessment process.

A wide variety of methods have been used to assess the genotoxic potential of chemical substances. More specifically, methods that have been used to measure oxidative DNA damage caused by ionizing radiation

52 K.R. Rogers et al. / Analytica Chimica Acta 444 (2001) 51–60

or chemical exposures include gel electrophoresis [3], capillary electrophoresis [4], and HPLC [5], as well as a number of optical [6], acoustic [7] and electrochem-ical [8] techniques. These techniques are typically sensitive and well suited to laboratory analysis of oxidative damage (particularly single-strand breaks) in naked DNA or DNA isolated from exposed organ-isms. Nevertheless, because these methods typically require alkaline denaturation followed by the sepa-ration of double- and single-stranded forms, they are often time consuming and expensive. Consequently, there is a need for rapid and cost-effective methods for screening environmental samples for the potential to cause genotoxic effects. In this paper, we report a rapid and sensitive biosensor method for detection of radiation-induced or chemically-induced oxidative DNA damage.

2. Experimental

2.1. Materials and buffers

Buffers were composed as follows:

PBS (Na2HPO4, 10 mM; NaCl, 100

mM; pH 7.4); 2× SSC (Na3C6H5 O7, 300 mM; NaCl 30 mM; pH 7.0).

PerfectHybTM

buffer from Sigma (St. Louis, MO) and was diluted 1:1 (HB). We obtained oligonucleotides from Life Technologies. The capture oligonucleotide sequence was as

follows: (5_–3_) biotin-GGG GAT

CGA AGA CGA TCA GAT ACC GTC GTA GTC TTA AC. The fluorescently labeled reporter sequence was as

follows: (5_–3_) Bodipy

TM-GTT AAG

ACT TCG ACG GTA TCT GAT CGT GTT CGA TCC CC. Note that the capture and reporter sequences are complementary with the exception of two single base mismatches at bases 10 and 28. Streptavidin (SA), succinimidyl-4-maleimidobutyrate (GMBS), mercap-topropyltriethoxysilane (MTS) and 3-morpholinosy-dnonimine (SIN-1) were obtained from Sigma.

2.2. Instrumentation

The fiber optic fluorimeter used in these experi-ments was from Research International (Woodinville, WA). A 635 nm diode laser and photodiode detector provided excitation and detection, respectively. The general schematic of the system is shown in Fig. 1. The

flow cell was placed in the heating chamber of a PCR System 9700 (Perkin-Elmer, Foster City, CA), and the temperature was calibrated using a temperature probe and monitor system from Cole-Parmer (Vernon Hills, IL) interfaced to a strip chart recorder. The bifurcated fiber bundle was interfaced to tapered quartz fibers obtained from the Naval Research Laboratory (NRL), Washington, DC and prepared as previously described [9]. Data were collected using the Analyte 2000 inte-grated software and transferred to an

ExcelTM

spread-sheet for further calculations.

The optical principle used for this assay involves the total internal reflectance fluorescence using a tapered fiber format and has been described previously in de-tail [10]. In short, as light is propagated down the fiber, an evanescent wave excites fluorescent tracers bound to the fiber surface. This occurs because the evanes-cent wave decays exponentially with the distance from the fiber surface such that the excitation radius extends only about 100 nm into the buffer medium. A por-tion of the probe (BDP 635/650) emission is captured and propagated back through the fiber to the detector. Thus, with the exception of the nonspecific binding of the labeled oligonucleotide, only the reporter se-quence that is hybridized to the immobilized capture sequence is detected.

2.3. Immobilization of the capture oligonucleotide

The preparation of the fibers and immobilization of protein to the fiber surface has been described pre-viously [9]. In short, the tapered fibers were silanized using MTS. SA was then immobilized using the het-erobifunctional crosslinker GMBS. The SA-coated fibers were then

stored at 4◦

C in PBS buffer.

2.4. Damage to probe oligonucleotide

Radiation damage resulted from exposure of the BDP-labeled oligonucleotide probe to high-energy emissions (i.e. X-rays of 6 meV) from a linear ac-celerator (Siemens, Mevitron, New York, NY). The samples containing BDP-probe in PBS buffer were placed beneath 1.5 cm thick water-equivalent build-up material and irradiated at an X-ray source-to-sample distance of 100 cm. The dose was varied between 20 and 1000 cGy. The fluorescence spectra of the BDP-labeled reporter in solution were recorded

K.R. Rogers et al. / Analytica Chimica Acta 444 (2001) 51–60 53


before and after exposure to ionizing radiation using a Perkin-Elmer LS-50 spectrofluorometer. For the chemically-induced oxidative damage, the BDP-labeled reporter oligonucleotide was exposed to SIN-1 for 60 min at concentrations ranging from 250 M to 3.0 mM. The reaction was stopped by addition of mannitol (45 mM).

2.5. Hybridization

The SA-coated fibers were incubated for 2 h in the presence of the biotinylated capture oligonu-cleotide at 50 nM in 2× SSC buffer. After immobi-lization of the biotinylated capture oligonucleotide to the streptavidin-coated fiber, the optical fiber was mounted in a temperature-controlled flow cell. Un-bound biotinylated capture oligonucleotide was then washed away using 15 ml of 2× SSC buffer. The flu-orescently labeled complementary strand (50 nM in HB) was then introduced in a stopped-flow procedure and incubated for 10 min at room temperature. After a stable hybridization response had been recorded, the reporter-containing hybridization buffer was ex-changed with 2× SSC buffer. The fiber was again monitored until a stable response was obtained (about 2 min). In the case of the melting curve analysis, the reporter-containing hybridization buffer remained in the flow cell. The temperature of the cell

was then increased from 25 to 74◦C,

at a rate of 3.6◦C/min, then decreased

to 30◦C. The labeled reporter was

then stripped away from the fiber surface by exchanging the 2× SSC for 10 mM NaOH (incubation for 2 min) followed by 15 ml of 2× SSC buffer. The reporter oligonucleotide could again be hybridized to the cap-ture oligonucleotide-coated fiber. Using this stripping protocol, 10 samples could be run on the same fiber. Positive controls (i.e. no DNA damage) were run on each fiber. In addition to a number of positive con-trols, negative controls were also conducted. For these controls, the biotinylated capture DNA was elimi-nated from the protocol so that nonspecific binding of the BDP-labeled probe to the SA-coated fiber could be determined.

2.6. Data analysis

Although the initial signal intensity for control ex-periments varied somewhat among separate runs for

the same fiber or between initial runs for separate fibers, the temperature-induced dissociation curves were very similar. To account for the differences in the signal intensities between experiments, normal-ized signal values were calculated using the following equation:

NS = S0 − St

where NS is the normalized signal, S0

the signal at time 0, and St the signal at time t. Slopes were deter-mined using least square regression and converted to a percent of the slope for the first or control experiment for each fiber.

3. Results and discussion

3.1. Hybridization of reporter sequence to the immobilized capture sequence

Addition of the Bodipy-labeled reporter oligonu-cleotide (613 ng/ml) to the capture oligonucleotide-coated fibers resulted in a significant increase in fluorescence (Fig. 2A). The increased signal, indica-tive of hybridization, reached steady state within about 5 min. The real-time hybridization response curves were similar to those previously observed for hybridization of complementary 40 mer oligonu-cleotides using a resonant mirror device [11]. Ex-change of the reporter-containing HB buffer with wash buffer (2× SSC containing no reporter) resulted in a transient decrease in the fluorescence followed by a steady state signal level similar to that prior to the buffer exchange. The transient decrease in the signal was likely due to removal of nonspecifically bound reporter from the fiber surface (see Fig. 2B). The new steady state signal was most likely due to changes in pH and ionic strength between the HB buffer and wash buffer. Changes in buffer conditions are known to affect both hybridization and quantum yield of the fluorescent dye [11].

The addition of NaOH (10 mM) resulted in disso-ciation of the capture and reporter hybridization pairs and subsequent decrease in signal. Although treatment with NaOH disrupted hybridization, it did not appear to disrupt the avidin–biotin linkage that attached the capture oligonucleotide to the fiber. After exchange


Fig. 2. Hybridization of the Bodipy-labeled reporter oligonucleotide (50 nM in HB buffer) to the SA-coated fiber that had been previously(A) treated with biotinylated capture oligonucleotide-coated fiber or (B) exposed to buffer alone. The addition of 2× SSC buffer and NaOH are as described in the Section 2. Fluorescence values were recorded every second in a spreadsheet format.

with wash buffer (2× SSC), the regenerated fiber could be used for up to nine additional hybridization cycles.

The addition of reporter oligonucleotide to the streptavidin-coated fiber that had not been exposed to biotinylated capture oligonucleotide resulted in a small and rapid increase in fluorescence (Fig. 2B). The addition of wash buffer (not containing reporter) resulted in a decrease in the signal to near back-ground levels. We attribute this signal to nonspecific binding of the reporter to the fiber surface within the evanescence zone.

3.2. Melting and annealing profiles

After steady state hybridization had been reached in the presence of reporter oligonucleotide, the tempera-

ture of the flow cell was increased linearly to 70◦C then

decreased to 30◦C. Indicative of temperature-induced

strand separation (i.e. melting), the fluorescence signal decreased with increasing temperatures to near back-

ground levels at temperatures of about 60◦C. Then, as

the temperature was lowered, the signal increased indicative of the rehybridization (annealing) of the

reporter sequence to the immobilized capture se-quence (Fig. 3A). Again, the absence of capture oligonucleotide immobilized on the fiber surface showed no hybridization and resulted in low back-ground fluorescence levels. In addition, the fluores-cence due to nonspecific binding was not effected by the temperature changes (Fig. 3B). To determine the effect of the temperature alone on the fluorescence of the dye-labeled reporter, fluorescence spectra of the

reporter were recorded in solution at 25 and 80◦C

(spectra not shown). Temperature showed no effect on the spectra.

Reported hybridization-based biosensor techniques employ a broad range of transducer and reporter mechanisms. As part of the characterization of sev-eral of these biosensors, melting profiles for the hybridization of oligonucleotides have been reported [12–14]. One of these reports involved a microelec-trode biosensor that used an enzyme-labeled reporter oligonucleotide [12]. Although there are some obvi-ous experimental differences between our method and the microelectrode technique such as the use of 25 mer oligonucleotides for the microelectrode and a slower


Fig. 3. Melting and annealing profiles. After steady state hybridization had been reached, the temperature was increased from 30 to 70◦C then

back to 30◦C. Pre-treatment of the fiber with (A) or without (B) capture oligonucleotide are described in Fig. 2.

temperature transition (i.e. 0.25◦C/min compared to

3.6◦C/min for the herein-reported method), the basic

shape of the melting profiles, including the slight upward bow in the melting curve, are quite similar. Fiber optic fluorosensors have also been reported that use either intercalation probes, as an indicator of hy-bridization [13], or a fluorescently labeled reporter sequence [14]. Although these hybridization biosen-sor methods have reported melting profiles, complete melting and annealing curves have not, been previ-ously reported.

3.3. Effect of ionizing radiation on melting profiles

The initial fluorescence signals measured af-ter steady state hybridization varied among sepa-rate assays using a single fiber as well as between fibers. The normalized slopes of the melting curves, however, were not systematically dependent on experiment-to-experiment or fiber-to-fiber variations. Consequently, we examined the effect of oxidative damage on the melting curves. Although the basic shape of the melting profiles were similar between the controls and radiation treated or chemically treated

samples, the damaged DNA showed flattening of the melting profiles. This was reflected in the normalized slopes of the linear portions of the melting curves (Fig. 4). We observed that the normalized slope values were a sensitive indicator of exposure of the reporter oligonucleotide to increasing doses of ionizing radia-tion. Ionizing radiation is known to produce various types of damage to DNA including base alterations and strand breaks. These changes would be expected to result in lower transition (melting) temperatures.

To determine any direct effect of radiation on the fluorescence characteristics of the dye-labeled oligonucleotide, fluorescence spectra of the control and highest-dose sample were recorded in solution. Both spectra were identical within experimental error indicating that the observed effect was due to changes in hybridization caused by oxidative damage to the DNA portion of the reporter oligonucleotide.

The calibration plot for the biosensor response slopes versus the log of the radiation dose was fit using a second-order exponential decay function (Fig. 5). Measured doses ranged from 20 to 1000 cGy. The “no dose” controls and “no capture oligonucleotide” controls are placed on the (broken) log dose axis at about two-orders of magnitude below the lowest


Fig. 4. Effect of ionizing radiation on normalized slopes of melting profiles. Melting profiles were recorded after exposure of the reporter oligonucleotide to ionizing radiation. The profile slopes were normalized as described in the Section 2.

Fig. 5. Dose response for ionizing radiation. Experimental conditions were as described in Fig. 4. Relative response slope values were

determined from least squares fit of the normalized signal data for; ( ) radiation exposed reporter oligonucleotide; ( ) no dose controls and

(_) no capture oligonucleotide controls as described in Fig. 3.


measured dose and essentially bracket the dynamic range of the assay.

Although a number of biosensor techniques have been reported for hybridization applications, few of these methods have been explored for the measurement of DNA damage and potential applica-tion for the detection of chemical genotoxins. Notable exceptions include several examples of rapid and inex-pensive electrochemical biosensor methods that have been reported for detection of chemical- [15], UV-[16], and ionizing radiation-induced DNA damage. Although these techniques can be extremely sensitive to oligonucleotide complementarity (i.e. can detect a single mismatch in a 15 mer hybridization pair [17]), they are only moderately sensitive to ionizing radiation (i.e. detection limit of 1000 cGy doses to supercoiled plasmid DNA [8,18]). By contrast, the most sensitive methods for detecting radiation-induced DNA damage include capillary electrophoresis [4], differential fluo-rescence assay [6], and single-cell gel electrophoresis (comet assay) [19], some of which show detection limits <1 cGy. These methods, however, tend to be complex, time consuming and are not well suited for development as field analytical methods.

3.4. Effect of chemically-induced damage on melting profiles

The hybridization-based biosensor was also sen-sitive to the effects of chemically-induced oxidative damage to the reporter oligonucleotide. Fig. 6 shows the calibration plot for the biosensor response slope as a function of the SIN-1 concentration. The data were fit to an exponential decay. The lowest measured dose was 250 M.

The SIN-1 spontaneously decomposes in aqueous systems to form nitric oxide and superoxide which further interact to form peroxynitrite and hydroxyl radicals [20]. These radical species are thought to be responsible for the SIN-1-induced oxidative damage to isolated DNA [5]. Concentration ranges for SIN-1 of 100 M to 1 mM have been reported to result in damage to calf thymus DNA including single- and double-strand breaks as well as the formation of 8-hydroxyguanosine (8-OH-dG). The SIN-1-induced damage to the reporter oligonucleotide is expected to be similar to the type of oxidative damage caused by the radiation exposure. As in the case of the ion-izing radiation, the expected lowering of the melting

Fig. 6. Dose response for SIN-1 treatment of the reporter oligonucleotide. The reporter oligonucleotide was exposed to SIN-1 for 1 h followed by the addition of mannitol (45 mM). Data analysis is as described in Fig. 5


temperatures was also observed for SIN-1 exposure. The concentration profile reported here is also simi-lar to that recently reported for optical detection of SIN-1-induced single-strand breaks in calf thymus DNA [21].

Due to the potential applicability as a general tool for molecular biology, the interface of biosen-sor technology to DNA hybridization techniques has generated reports describing a wide variety of DNA-hybridization-based biosensors. These biosen-sors have not typically been used to detect or measure DNA damage, however, some general similarities in operational characteristics among these techniques and with the herein-reported method are apparent. Target and reporter sequences typically vary in size from 40 mer [11] to 15 mer [17] oligonucleotides and vary in composition from bacterial pathogen se-quences [15] to single base repeating primers [22]. Our method uses hybridization sequences of inter-mediate size (i.e. 38 mer oligonucleotides) and a random-base sequence. Detection limits among these methods also vary widely (e.g. from 10 g/ml [17] to 300 fg/ml [13] of target DNA) depending pri-marily on the signal transducer and transduction mechanism. Again, for the herein-reported tech-nique, we use an intermediate concentration of re-porter oligonucleotide (i.e. 613 ng/ml). There were significant similarities among these hybridization biosensors with respect to oligonucleotide size and assay sensitivity as well as the capability of tem-perature control for some. Consequently, it is con-ceivable that the ability to measure DNA damage reported for our biosensor technique could be appli-cable to many of the previously described biosensor techniques.

4. Conclusions

A hybridization-based biosensor for detection of radiation-induced and chemically-induced oxidative DNA damage is described. The assay is relatively rapid (about 2 h) and simple compared to current electrophoresis-based [3,4] or chromatography-based [5] techniques for measuring oxidative damage. This biosensor assay is also significantly more sensitive to ionizing radiation than other reported biosensor assays [8].

Potential applications for this biosensor technique include the use of various reporter sequences as sur-rogate DNA to develop screening assays to detect the potential of contaminated environmental samples to cause oxidative damage. It is expected that a screen-ing method developed using these concepts could be employed among a battery of methods specific for var-ious aspects of chemical genotoxicity.

Acknowledgements

The US Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research through a competitive internal grant (to K.R. Rogers). It has been subject to the EPA’s peer and administrative review has been approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use.

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