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Real-time detection of Escherichia coli O157 : H7 sequences using a circulating-flow system of quartz crystal microbalance

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Real-time detection of Escherichia coli O157:H7 sequences using a

circulating-flow system of quartz crystal microbalance

Vivian C.H. Wu

a

, Sz-Hau Chen

b

, Chih-Sheng Lin

b,

aDepartment of Food Science and Human Nutrition, University of Maine, Orono, ME 04469-5735, USA

bDepartment of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu 30050, Taiwan

Received 7 August 2006; received in revised form 28 November 2006; accepted 7 December 2006 Available online 16 January 2007

Abstract

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5-monophosphate (12-dT)] for the detection of E.

coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA

gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.

© 2006 Elsevier B.V. All rights reserved.

Keywords: E. coli O157:H7; Quartz crystal microbalance; DNA biosensor; Foodborne pathogens

1. Introduction

Escherichia coli O157:H7 has been an important foodborne

pathogen in a variety of foods worldwide in the last 20 years. It is classified as an enterohemorrhagic E. coli with the ability to cause hemorrhagic colitis, which includes symptoms, such as bloody diarrhea, hemolytic uremic syndrome and thrombotic

thrombocytopenic purpurea (Doyle et al., 1997). Centers for

Disease Control and Prevention (CDC, 2005) reported that an

estimated 73,000 cases of E. coli O157:H7 infection and 61 resultant deaths occur in the United States each year. Consump-tion of as few as 10 of E. coli O157:H7 cells can result in growth of the pathogen in the intestine and production of shiga toxins in susceptible patients. These toxins can kill the cells of the intesti-nal lining, destroy the kidneys and cause blood clots in the brain,

Corresponding author. Tel.: +886 3 5131338; fax: +886 3 5729288. E-mail address:lincs.biotech@msa.hinet.net(C.-S. Lin).

as well as seizures, paralysis and respiratory failure (FDA, 2001;

CDC, 2005). Therefore, it is desirable to develop efficient ana-lytical methods with a high degree of sensitivity and specificity to detect this organism in food.

Detecting E. coli O157:H7 with conventional procedures can

take several days (Meng et al., 2001). A variety of rapid methods

have been proposed to detect and screen many microorganisms, including E. coli O157:H7. These methods include antibody-based methods (immunofluorescence, immunoimmobilization, enzyme-linked immunosorbent assay, immunomagnetic sepa-ration, etc.), nucleic acid-based methods (hybridization and polymerase chain reaction [PCR]), biochemical and enzymatic methods (miniaturized microbiological methods and commer-cial miniaturized diagnostic kits) and membrane filtration

methods (hydrophobic grid membrane filter) (Wu et al., 2004).

In recent years, modern bio-techniques, such as real-time PCR (Yoshitomi et al., 2003; Fu et al., 2005), nanoparticles (Zhao et al., 2004; Mao et al., 2006) and biosensing systems (biosensors) (Campbell and Mutharasan, 2005; Simpson and Lim, 2005; Mao

0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.12.016

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et al., 2006) have been developed for detection of pathogenic microorganisms. Biosensors are devices that detect biological or chemical complexes in the form of antigen–antibody, nucleic acids, enzyme-substrate or receptor-ligand compounds. Interest in using biosensors to detect foodborne pathogens is on the rise (Hall, 2002; Patel, 2006; Rasooly and Herold, 2006).

DNA probes (i.e. an oligonucleotide sequence immobilized on a fixed support able to hybridize the complementary strand present in solution) are powerful molecular tools for monitoring and detecting specific microorganisms in the environment or in

food (Marx, 2003; Mann and Krull, 2004). The standard method

of detecting nucleic acid hybrids is by labeling the probe with radioactive nucleotides, chemiluminescent dye or various hapten molecules, such as biotin. Piezoelectric mass-sensing devices based on quartz crystal microbalance (QCM) enable the label free detection of molecules, such as ligands, peptides and nucleic

acids (Marx, 2003).

The core of the QCM is a specifically manufactured quartz plate with a fundamental resonance frequency in the range of 5–30 MHz. The crystal is excited to resonance and the effect of

molecular absorption monitored (Sauerbrey, 1959). The QCM is

comprised of thin film electrodes, usually gold (Au), deposited on each face of a crystal. Voltage is applied across these elec-trodes to deform the crystal plate, producing relative motion between the two parallel crystal surfaces. The crystal is induced to oscillate at a specific resonant frequency. Changes in the mass of the material on the surface will alter the resonance

frequency of the crystal (Marx, 2003). A linear relationship

exists between deposited mass and frequency response for quartz crystals. The resonance frequency decreases linearly with the increase of mass on the QCM electrode at the nanogram level or less. This characteristic of QCM can be exploited to develop

bioanalytical tools on a 10−10 to 10−12g scale (Zhou et al.,

2000). QCM-based immunoassay has been designed and applied

in several different areas (Su et al., 2000; Kurosawa et al., 2003;

Su and Li, 2004). Using QCM sensing, with an appropriate DNA probe immobilized on the sensor surface, it is possible to detect a specific target sequence without additional label-ing procedures. The DNA-based sensors can be coupled with the PCR to increase the sensitivity of the systems and offer a viable alternative to gel electrophoresis and other traditional DNA sequences detection methods that require labeled probes. Currently, some QCM devices are capable of detecting label-free oligonucleotides and/or PCR-amplified DNA fragments (Deisingh and Thompson, 2001; Mo et al., 2002; Mannelli et al., 2003; Mao et al., 2006). However, a DNA biosensing system for real-time detection of pathogens, such as E. coli O157:H7 in food has yet to be fully developed.

The objective of the study was to develop a DNA piezoelec-tric biosensor, QCM, in a circulating-flow system for real-time detection and identification of E. coli O157:H7 sequences. We evaluated the enhancement of hybridization efficacy by adding

an oligonucleotide spacer to the 5-end of a 30 mer probe.

The specificity of the QCM system for synthesized targets of varying lengths (30 and 104 mer) and PCR-amplified DNA fragments (104 bp) from real samples of E. coli O157:H7 was explored.

2. Materials and methods

2.1. Reagents and oligonucleotide primers, probes and targets

All oligonucleotides were designed using Primer Express software (Applied Biosystems, Foster, CA) and synthesized by Applied Biosystems (Applied Biosystems). They are specified in Table 1, which includes single-strand DNA (ssDNA) of probes, targets and PCR primers. In the sequence detection for

the E. coli O157:H7 gene eaeA (Genbank U32312) (Call et

al., 2001), the specific probe (P) sequences{probe eaeA;

thiol-linkered tag modification [HS-(CH2)6, (–SH)] at 5 terminus with

or without additional 12 deoxythymidine 5-monophosphate

[12-dT (12T)]: P-30-SH (30 mer) and P-30/12T-SH (42 mer); non-thiolated and with or without additional 12-dT: P-30

(30 mer) and P-30/12T (42 mer)} used in the experiments were

designated. The target (T) sequences included complementary target oligonucleotides, T-30AS (30 mer; AS, the anti-sense strand to the probe sequences) and T-104AS (104 mer), and

Table 1

Sequences of the oligonucleotide probes, targets, and primers used in this study Probe sequences for E. coli O157:H7 eaeA

P-30-SH HS-(CH2)6-5-AGC TCA AGA GTT GCC CAT

CCT GCA GCA ATG-3(30 mer)

P-30/12T-SH HS-(CH2)6-5-TTT TTT TTT TTT AGC TCA AGA

GTT GCC CAT CCT GCA GCA ATG -3(42 mer) P-30 5-AGC TCA AGA GTT GCC CAT CCT GCA

GCA ATG-3(30 mer)

P-30/12T 5-TTT TTT TTT TTT AGC TCA AGA GTT GCC CAT CCT GCA GCA ATG -3(42 mer)

Short target sequences (30 mer) for E. coli O157:H7 eaeA

T-30S 5-AGC TCA AGA GTT GCC CAT CCT GCA GCA ATG -3

T-30AS 5-CAT TGC TGC AGG ATG GGC AAC TCT TGA GCT-3

Long target sequences (104 mer) for E. coli O157:H7 eaeA

T-104S 5-AAA GTT CAG ATC TTG ATG ACA TTG TAT TTT CTC TTA ATT AAA TTT ATA TTT ACA GAA GCT CAA GAG TTG CCC ATC CTG CAG

CAA TGT TAT TCC CTG AAA AAT TG -3

T-104AS 5-CAA TTT TTC AGG GAA TAA CAT TGC TGC

AGG ATG GGC AAC TCT TGA GCT TCT GTA

AAT ATA AAT TTA ATT AAG AGA AAA TAC AAT GTC ATC AAG ATC TGA ACT TT-3 PCR primers

E157eae/F 5-CAA TTT TTC AGG GAA TAA CAT TGC-3

E157eae/R 5-AAA GTT CAG ATC TTG ATG ACA TTG-3

Probe (P) sequences were designed according to the E. coli O157:H7 eaeA gene and used to detect the sequence of E. coli O157:H7. –SH, thiol-linkered tag [HS-(CH2)6] modification at 5 terminus of probe;/12T, additional 12 mer

of dT oligonucleotides to the probes; P-30 (30 mer) and P-30/12T (42 mer), non-thiolated probes with or without additional 12-dT; P-30-SH (30 mer) and P-30/12T-SH (42 mer), thiolated probes with or without additional 12-dT. Target (T) sequences include complementary target oligonucleotides, T-30AS (30 mer; AS, anti-sense strand to the probe sequence) and T-104AS (104 mer), and non-complementary target oligonucleotides, T-30S (30 mer; S, sense strand to the probe sequence) and T-104S (104 mer). The primer pair was used to amplify the 104 bp DNA fragment of eaeA gene from E. coli O157:H7 genomic DNA.

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non-complementary target oligonucleotides, T-30S (30 mer; S, the sense strand to the probe sequences) and T-104S (104 mer). For PCR-amplified DNA from real samples, the primer pair

(E157eae/F and E157eae/R) specific for the E. coli O157:H7

eaeA gene was used for the amplification of target DNA

fragments (104 bp).

The buffer and reagents used in the experiments were pur-chased from Merck (Darmstadt, Germany) and Sigma–Aldrich

(St. Louis, MO). These include 30% hydrogen peroxide (H2O2,

Merck), 98% sulfuric acid (H2SO4, Sigma–Aldrich), sodium

chloride (NaCl, Sigma–Aldrich), sodium phosphate (Na2HPO4,

Merck), hydrogen chloride (HCl, Merck) and sodium hydroxide (NaOH, Sigma).

2.2. Culture preparation

The target bacteria, E. coli O157:H7 (ATCC 43894), were obtained from American Type Culture Collection (Rockville, MD). The culture was grown in brain heart infusion (BHI) broth

(Difco Laboratories, Detroit, MI) at 37◦C for 24 h before use and

the bacterial counts were determined by conventional spread-plating method using tryptic soy agar (TSA, Difco).

2.3. DNA extraction

Total genomic DNA was extracted from E. coli O157:H7

using the WizardTMGenomic DNA Purification Kit (Promega,

Madison, WI). For each DNA preparation, a pellet containing

approximately 1× 106CFU/ml cells was resuspended in 600␮l

of Nuclei Lysis Solution and incubated at 80◦C for 5 min to lyse

the cells. Three microliters of RNase Solution was added to the cell lysate and treated for 30 min. Two hundred microliters of Protein Precipitation Solution was added to the RNase-treated cell lysate and the DNA extraction was carried out as according to the manufacturer’s protocol for bacteria (Promega). The puri-fied DNA was examined by gel electrophoresis (0.8% agarose)

and quantified by determining A260 (OD260) with a SpectraMax

190 spectrophotometer (Molecular Devices Corp., CA).

2.4. PCR conditions

For detection of real samples of E. coli O157:H7, a 104-bp DNA fragment within the E. coli O157:H7 eaeA gene was

amplified by the synthetic primers (E157eae/F and E157eae/R)

indicated inTable 1. A PTC-100TMthermal cycler (MJ Research

Inc., NV) was used with 10× reaction buffer, dNTP (deoxynu-cleoside triphosphate) concentrated set solution and Super Taq DNA polymerase (all obtained from HT Biotechnology Ltd., Cambridge, England).

PCR reactions contained 3␮l of genomic DNA (10 ng), 1 ␮l

of each primer (10␮M), 5 ␮l of 10× PCR buffer [100 mM

Tris–HCl (pH 9.0), 15 mM MgCl2, 500 mM KCl, Triton X-100

(1%, v/v), gelatin (0.1%, w/v)], 2␮l of 10 mM dNTP, 1 ␮l of

0.5 U Super Taq DNA polymerase and 38␮l of distilled water

resulting in a total volume of 50␮l. Thermal cycler

(MiniCy-cler; MJ Research, Waltham, MA) conditions were as follows:

initial denaturation at 94◦C for 5 min; then 32 cycles of 30 s

denaturation at 94◦C, annealing at 55◦C for 30 s, elongation

at 72◦C for 30 s and final extension at 72◦C for 3 min. The

PCR products were purified using WizardTM PCR Preps DNA Purification System (Promega) according to the manufacturer’s protocol and then visualized on 2.5% agarose gels stained with ethidium bromide under UV light. The concentration of DNA in the samples was measured by a UV-absorption spectropho-tometer at a wavelength of 260 nm.

The PCR-amplified DNA was sequenced using E157eae/F and

E157eae/R as sequencing primers. Both strands of the DNA

frag-ment were sequenced using a Taq DyeDeoxy Terminator Cycle Sequencing Kit and 373A DNA Sequencer (Applied Biosys-tems).

2.5. The circulating-flow QCM system

The piezoelectric quartz crystals, which consist of a 9 MHZ

AT cut quartz crystal slab with a layer of a gold electrode on each

side were (0.091 cm2in area on each side; the detection limit

of the QCM instrument in liquid = 1 Hz) obtained from ANT Technology Co., Ltd. (Taipei, Taiwan). The flow injection and continuous frequency variation recording were operated using Affinity Detection System (ADS; ANT Technology Co., Ltd.). The system has five main components including electronic oscil-lation circuit, frequency counter, piezoelectric quartz of fixed biosensor molecule (p-chip), circulating-flow system and a

com-puter to demonstrate the curve of frequency change (F) in

real time (Fig. 1A). The experimental data were analyzed by

P-Sensor software in real time. The sensor unit was composed of resolution: 0.1 Hz, sampling period: 1 s, frequency range:

2–16 MHz, temperature range: 4–60◦C and voltage: 110 V,

50–60 Hz. The reaction cell was one sensor signal channel with

30␮l of reaction cell volume. The circulating-flow system

con-sisted of a temperature controller, sample tubes, pipelines and

one tubing pump with a flow rate: 10–200␮l/min and a sample

loop volume of 150␮l.

2.6. Gold-QCM device preparations

The gold electrode surface of the QCM device was cleaned with distilled water for 2–3 min. The water droplets on the sur-face of the electrode were then blown dry using an air gun. The gold electrode surface was subsequently cleaned with a

piranha solution consisting of H2O2(30%) and H2SO4in a 1:3

ratio. It was then thoroughly washed with distilled water, dried with the air gun and used immediately afterwards. The cleaning procedures with piranha solution and distilled water removed organic compounds adhering to the gold surface and enhanced the efficiency of immobilization when the thiolated DNA probes

covalently attached to the gold surface (Cho et al., 2004; Su and

Li, 2004).

2.7. Immobilization of the oligonucleotide probes and hybridization with oligonucleotide targets

The gold-QCM device was inserted into the flow injection ADS. The importing and exporting pipeline ends were placed

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Fig. 1. The real-time and circulating-flow QCM system. (A) A schematic illus-tration of the real-time and circulating-flow QCM system. The gold films on the quartz crystal oscillator are 3.4 mm in diameter and 0.2 cm2in the area of both sides. The volume of the sample tube is 1.5 ml. The volume of the reaction cell is 30␮l and the total volume of the pipeline loop, including flow in and flow out, is 100␮l. (B) An example of real-time detection of Escherichia coli O157:H7 sequences performed in the study using the circulating-flow QCM system.

in the sample tube to create a circulating-flow system (Fig. 1A).

The phosphate buffer (1 M NaCl and 1 mM Na2HPO4, pH

7.4) was flushed through the system at the speed of 50␮l/min.

With the frequency of the chip steady at 300 s, F within

±1 Hz/100 s, the solution containing specific probe sequences

(5-thiolated: P-30-SH, P-30/12T-SH or non-thiolated: P-30,

P-30/12T;Table 1) for detecting the E. coli O157:H7 gene eaeA

was added to the sample tube (total volume: 500␮l). The probes

were self-assembly immobilized on the gold electrode through the flow-circulation for 30 min. After immobilization of the probes, the sample tube containing the probes was removed. The circulating pipeline was cleaned by flowing through the phosphate buffer for 15 min to remove unbound probes on the gold surface and probe residues in the pipeline. The gold-QCM device was then exposed with the sample flow (total volume:

500␮l) containing target sequence (T-30S, T-30AS, T-104S

or T-104AS; Table 1). Hybridization of probes and targets in

the QCM device was at a flow speed of 50␮l/min for 30 min.

The temperature of the QCM system was maintained at 30◦C

except in the study of temperature effects on the hybridization. TheF during the hybridization were recorded in a real-time observation.

In these experiments, different concentrations of the probe

oligonucleotides (0, 0.25, 0.5, 1.0 and 2.0␮M) were evaluated

for the efficiency of immobilization on the gold surface of the QCM device. The influence of varying concentrations of

the target oligonucleotids onF and hybridization efficiency

(H%) were also compared. TheF was observed in a real-time

continuous reading and reported as the difference between the final value and the value before the hybridization or immobi-lization. The H% was calculated by dividing the hybridized

target coverage by the immobilized probe coverage (Su et

al., 2005). The mass of immobilized probes as well as hybrid DNA of probes and targets on the QCM device was

calcu-lated using Sauerbrey’s equation (Kanazawa and Gordon II,

1985) F = −F3/2 0  ρ1η1 πρqμq 1/2 (1)

whereF is the measured frequency shift, F0 a resonant

fre-quency of the unloaded crystal, ρl the density of liquid in

contact with the crystal,ηlthe viscosity of liquid in contact with

the crystal,ρq the density of quartz (2.648 g cm−3) andμqis

the shear modulus of quartz, 2.947× 1011g cm−1s−2. The

fre-quency change of 1 Hz corresponds to a mass change of 0.883 ng. For detection of PCR-amplified DNA of E. coli O157:H7

gene eaeA, the thiolated probe, P-30/12T-SH (1␮M), was used.

The double-strand sequences of the PCR-amplified DNA are the same as the synthetic sequences of 104 mer targets, T-104AS and

T-104S (Table 1). Before the PCR-amplified DNA was applied

into the circulating-flow QCM system, the DNA was treated in

a denaturing solution (TE and 0.5 M NaCl) at 90◦C for 5 min.

The denatured DNA was then applied to the circulating-flow QCM system following the same procedure used for synthetic target oligonucleotides. Temperature effects (20, 30, 40, 50 and

60◦C) on the DNA hybridization (hybridization buffer: 1.0 mM

Na2HPO4and 1.0 M NaCl) during the circulating-flow detection

were evaluated. In each hybridization, 1␮M of PCR-amplified

DNA (equivalent to 1␮M of the target T-104AS) was used to

hybridize with the thiolated probes immobilized on the QCM device.

2.8. Data analysis

The experimental data were analyzed by P-Sensor soft-ware of the ADS system in real time. Each experiment was repeated five times using five different QCM devices to test the reproducibility of the QCM sensor. All data were presented

as the mean± standard deviation (S.D.). Differences between

groups were evaluated by a two-tailed Student’s t-test. P-values less than 0.05 were considered to be statistically significant differences.

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3. Results and discussion

3.1. The QCM system and detection

In the circulating-flow QCM system, real-time frequency shift was recorded. The frequency decreased gradually with addition of oligonucleotides, reflecting immobilization of the probes or hybridization of probes and targets on the

gold surface of QCM. Fig. 1B shows an example of the

real-time detection of the QCM system performed in our study. There was a 58 Hz decrease in series resonant frequency for immobilization of probes P-30-SH (30 mer), while probes P-30/12T-SH (42 mer) yielded a decrease of 75 Hz. There were decreases in series resonant frequency of approximately 42 and 51 Hz when complementary strands (T-30AS) were introduced and hybridized with P-30-SH and P-30/12T-SH, respectively.

In our circulating-flow QCM system, the thiolated probes were circulated constantly to ensure continuous interaction with the gold surface on QCM device thereby enhancing the efficiency of probe immobilization and the hybridiza-tion of probes and targets. The flow circulahybridiza-tion also limits non-specific binding, recirculating unattached probes for immobilization.

3.2. Immobilization of synthesized oligonucleotide probes

In fabricating a DNA sensor, maximizing the immobilization of DNA on the sensor’s surface is crucial. Therefore, various

concentrations (0, 0.25, 0.5, 1.0 and 2.0␮M) of thiolated

probes, with or without the addition of 12-dT [P-30-SH (30 mer) and P-30/12T-SH (42 mer)], were used to evaluate immobilization efficiency on the gold surface of QCM devices.

The results showed thatF increased almost linearly with the

increase of probe concentrations up to concentrations of 1.0

and 2.0␮M. Probe concentrations of 1.0 and 2.0 ␮M yielded

the greatest covalent attachment to the gold surface compared

with other concentrations (P < 0.01) (Fig. 2A). There was no

significant difference betweenF at concentrations of 1.0 and

2.0␮M (P > 0.05), indicating saturation of the immobilization

sites on the gold surface of the QCM device. Therefore, the

probe concentration of 1.0␮M was selected for the following

experiments.

In general, the thiolated probes with additional 12-dT

(P-30/12T-SH) showed greaterF (P < 0.05) than thiolated probes

without additional 12T (P-30-SH) among the various

con-centrations tested (Fig. 2A). This was because the weight of

P-30/12T-SH per single molecule (1.31× 104g mol−1) is larger

than that of P-30-SH (9.42× 103g mol−1). According to the

calculation by Sauerbrey’s equation, the molecule densities of P-30-SH and P-30/12T-SH immobilized onto the gold surface of

the QCM device were 1.7± 0.1 and 2.1 ± 0.2 (ssDNA/10 nm2),

respectively, when 1.0␮M of probes was used in the

immo-bilization. These molecule density values show no significant differences (P > 0.05). Therefore, the efficiency of immobiliza-tion of P-30-SH and P-30/12T-SH onto the gold surface of the QCM was similar.

Fig. 2. Immobilization efficiency of oligonucleotide probes on the gold surface of the QCM device and detection of the short target oligonucleotides, T-30-AS, hybridized with the thiolated probes (1.0␮M) immobilized onto the gold surface of the QCM device. Frequency change (F) after immobilization (A), frequency change (B) and hybridization efficiency (C) after hybridization were measured and calculated. Values derived from five independent detections, error bars mean standard deviation (S.D.). Asterisk (*) and (**) indicate P < 0.05 and P < 0.01, respectively, vs. P-30-SH.

3.3. Detection of the short (30 mer) synthesized target oligonucleotides

Different concentrations (0, 0.25, 0.5 and 1.0␮M) of the short

synthesized target oligonucleotides (T-30AS, 30 mer) comple-mentary to the probes immobilized on the gold surface of QCM

device were compared for theF and H% in the

circulating-flow QCM system. TheF due to hybridization increased with

increasing concentrations of the targets up to 0.5␮M (P < 0.01)

(Fig. 2B). At concentrations≥1 ␮M, the frequency shift was less sensitive, indicating the saturation of the probe

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with increasing concentrations of the targets (Fig. 2C). The H%

of target T-30AS at 0.25 and 0.5␮M hybridized with the probe

P-30/12T-SH were significantly higher (P < 0.01) than those of the target hybridized with probe P-30-SH. Results indicate the addition of 12-dT to the probes increased the H% in the QCM system.

According to theF results, the probes were able to hybridize

with their complementary sequences. The probes with the spacer (12-dT) influenced the hybridization with the target

oligonucleotides (Shchepinov et al., 1997), and increased the

hybridization efficiency in our circulating-flow QCM system. During DNA hybridization, spacers have been shown to reduce steric interference, making the probe end closet to the surface of

the device more accessible (Shchepinov et al., 1997; Southern et

al., 1999). Spacers, such as 12-dT also reduce steric hindrance in three-dimensional space and increase of molecule collision to increase H%. The use of spacers, such as poly dT or dA were

reported for other biosensor development (Bassil et al., 2003;

Wolf et al., 2004; Mukumoto et al., 2006).

3.4. Specificity of the QCM detection

We evaluated the DNA hybridizations among four probes

(P-30-SH, P-30/12T-SH, P-30 and P-30/12T; 1␮M) and two targets

(T-30AS and T-30S; 0.5␮M) (Table 1). We compared the probes

with or without thiol-linkered tag modification on the 5-end for

influencing the efficiency of probe immobilization on the gold surface of the QCM device and subsequent hybridization. The results indicate that non-thiolated probes (P-30 and P-30/12T)

fail to attach covalently to the gold surface (F < 2 Hz for

30 min), preventing the subsequent hybridization of probes and

targets (Fig. 3A). Non-specific binding was investigated using

T-30S (a sense strand to the probe sequences, non-complimentary strand). The thiolated probes (P-30-SH, P-30/12T-SH) were covalently immobilized on the gold surface and specifically hybridized to the complementary targets (T-30AS), but not to

the non-complementary targets (T-30S) (Fig. 3). The target

T-30S, as the negative control of hybridization in our QCM system did not yield a measurable frequency shift when applied to the probe-immobilized QCM device. Hybridization of surface-bound ssDNA is dependent on surface coverage and materials. The thiolated ssDNA has a more profound effect on surface cov-erage, such as with the gold in our system, than non-thiolated

ssDNA (Herne and Tarlov, 1997; Levicky et al., 1998). The

hybridization of P-30/12T-SH and T-30AS showed greaterF

(P < 0.01) (Fig. 3A) and higher H% (Fig. 3B) than the

hybridiza-tion of P-30/SH and T-30AS, reflecting the reduchybridiza-tion of steric hindrance in the three-dimensional space and the increase in molecule collisions caused by the additional 12-dT.

3.5. Detection of the long (104 mer) synthesized target oligonucleotides

The 104 mer synthesized targets (T-104AS and T-104S) were

applied in the QCM system and theF and H% were

evalu-ated. Within the target of T-104AS, only the 30 mer sequences are complementary to the probes, P-30-SH and P-30/12T-SH

Fig. 3. Detection specificity of the circulating-flow QCM system. Four probes (P-30-SH, P-30/12T-SH, P-30 and P-30/12T; 1␮M) and two targets (T-30AS and T-30S; 0.5␮M) were applied to test the efficiencies of probe immobilization and hybridization of the target to the probe immobilized QCM device. In each treatment, frequency change (A) and hybridization efficiency (B) were measured and calculated. Values derived from five independent detections, error bars mean S.D. Asterisk (**) indicates P < 0.01 vs. T-30S and‡ indicates P < 0.01 vs. P-30-SH.

(Table 1). As shown previously, no measurable frequency shift was detected when the targets were applied to the immobilized probes without thiol-linkered tag modification. Using T-104S, a non-complementary strand to the probes, non-specific binding

was investigated. No measurableF was detected when T-104S

was applied to the probe-immobilized QCM. TheF (due to

the hybridization), and the H% significantly increased (P < 0.05

or P < 0.01) with increasing concentrations (0.5 and 1␮M) of

the target T-104AS (Fig. 4A and B). However, the frequency

shift decreased when a relatively high concentration of T-104AS

(2␮M) was applied for the hybridization with P-30/12T-SH,

indicating the upper limit of hybridization and saturation of the probe hybridization sites.

InFig. 2, the results show that the 12-dT spacer enhanced the F and H% by approximately 1.4-fold when the probes

hybridized with the 30 mer target T-30AS (0.5␮M). InFig. 4,

the F had a two-fold increase when the 104 mer target T-104AS hybridized to the probe P-30/12T-SH instead of to the probe P-30-SH (P < 0.01). This phenomenon indicates that the 12-dT spacer had greater effects on the 104 mer targets than on the 30 mer targets in reducing steric interference during DNA hybridization. This result is confirmed by previous reports, which showed that the addition of spacers to probes has a greater increase in hybridization efficacy when the probes hybridized to

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Fig. 4. Detection of the long oligonucleotide targets, T-104AS and T-104S, hybridized with the probes (1.0␮M) immobilized onto the gold surface of the QCM device. Frequency change (A) and hybridization efficiency (B) were mea-sured and calculated. Values derived from five independent detections, error bars mean S.D. Asterisk (*) and (**) indicate P < 0.05 and P < 0.01, respectively, vs. target T-104AS (0.5␮M). † and ‡ indicate P < 0.05 and P < 0.01, respectively, vs. probe P-30-SH.

a longer rather than shorter target sequence (Herne and Tarlov,

1997; Levicky et al., 1998). We infer that it is easier to form a bent sequence and generate surface inhibition on the gold sur-face of the QCM device when the target T-104AS molecules

hybridize to the probe P-30-SH than to the probe P-30/12T-SH. This occurrence may obstruct entrance of other target sequences

for hybridization (Scheme 1).

Interestingly, the values of H% (20–25%) in the hybridiza-tion of the probe P-30/12T-SH with the 104 mer targets were significantly lower (P < 0.05) than those in the hybridization of

the 30 mer targets at optimal conditions (85–95%) (Figs. 2C

and4B). This indicates that free fragments extending from the

complementary region of the target obstructed the hybridization of the target sequences to their probes. In addition, long target oligonucleotides may produce the secondary structures due to hairpin formation in the hybridization conditions we used. Even though H% was reduced to 20–25% in the hybridization of the

probe P-30/12T-SH and the 104 mer targets, the detectableF

reached 70–80 Hz, indicating that the sensitivity of our QCM system is sufficient to detect sequences, such as the 104 mer targets that hybridized to the 30 mer probes.

3.6. Detection of PCR-amplified DNA of E. coli O157:H7 gene eaeA

The PCR-amplified DNA fragment is 104 bp and is located within the respective region of E. coli O157:H7 eaeA gene (Fig. 5A). The PCR products were detected in real-time by the circulating-flow QCM system. The temperature effects on the hybridization of P-30/12T-SH and PCR-amplified DNA are

indi-cated inFig. 5B. The results show that theF equalled 70 ± 4 Hz

when the system temperature was maintained at 30◦C and was

significantly higher (P < 0.01) than when the system was

main-tained at 20, 40, 50 and 60◦C. The frequency shift of the QCM

system for detecting PCR-amplified products was equivalent to that for detecting the synthetic target oligonucleotides, T-104AS. The piezoelectric biosensor detected the presence of E. coli O157:H7 when the DNA strand was complementary to the immobilized probes with synthetic oligonucleotides. The system will be further applied in the detection of field samples or other

Scheme 1. Schematic representation of steric hindrance of the probe and target DNA hybridization on the QCM device. The target T-104AS molecules hybridized to the probe P-30-SH easily to form a bent sequences adjacent to the surface of gold film in contrast to the targets hybridized to the probe P-30/12T-SH. This phenomenon may obstruct the entrance of other target sequences for hybridization.

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Fig. 5. Detection of PCR-amplified DNA. (A) The 104 bp DNA fragment amplified by PCR from the eaeA gene of E. coli O157:H7. The amplified sequence was also identified by DNA sequencing and the sequence is “5-CAA TTT TTC AGG GAA TAA CAT TGC TGC AGG ATG GGC AAC TCT TGA GCT TCT GTA AAT ATA AAT TTA ATT AAG AGA AAA TAC AAT GTC ATC AAG ATC TGA ACT TT-3” (this is the sequence of one strand). (B) Temperature effect on the hybridization of the PCR-amplified DNA fragment from the eaeA gene of E. coli O157:H7 to the probe P-30/12T-SH. In the hybridization, 1␮M of PCR-amplified DNA (equivalent to 1␮M of target T-104AS) was used to hybridize the thiolated probes immobilized on QCM device. Asterisk (**) indicates P < 0.01 vs. other temperature treatments.

sequences by employing various probes to be immobilized on the gold surface of the crystal. In conjunction with PCR application, the QCM sensor labeled with DNA probes can be used as a quan-titative and highly sensitive assay. The optimal conditions of our QCM system used to detect the PCR-amplified DNA from the real sample had relative standard deviation values (%R.S.D.) as low as 5.3%, which are considered acceptable among established

analytical techniques (Skoog et al., 1998). Our QCM device may

be used to detect a single sequence, a single organism or an entire community of environmental and food microorganisms with selectivity controlled by the choice of primers and primer annealing temperature for the PCR procedures.

4. Conclusions

We developed a DNA piezoelectric sensor for the real-time detection of E. coli O157:H7. Synthetic probe oligonucleotides were self-assembly immobilized on the sensor surface of the QCM device and the hybridization between the immobilized probes and the complementary sequences of the targets in solu-tion was monitored in real-time. A spacer (12-dT) linked to the probes enhanced the detection signals because the spacer molecules reduced the steric interference of the support on the hybridization behavior of the immobilized oligonucleotides. The QCM system was also used to detect the PCR-amplified DNA from real samples. Our results suggest that the DNA piezoelec-tric sensor has potential for further applications in detecting E.

coli O157:H7 as well as other microorganisms in food, water

and clinical samples. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.

Acknowledgments

This work was supported by grants NSC 93-2313-B-009-001 and NSC 94-2313-B-009-002 from the National Science Coun-cil of Taiwan, and grants of 95W821 and MOE ATU Program from the Taiwan Department of Education. This work was also supported in part by the Maine Agricultural and Forest

Experi-ment Station at the University of Maine with external publication number 2924. We thank Elizabeth Dodge and Joan Perkins for the assistance with editing.

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數據

Fig. 1. The real-time and circulating-flow QCM system. (A) A schematic illus- illus-tration of the real-time and circulating-flow QCM system
Fig. 2. Immobilization efficiency of oligonucleotide probes on the gold surface of the QCM device and detection of the short target oligonucleotides,  T-30-AS, hybridized with the thiolated probes (1.0 ␮M) immobilized onto the gold surface of the QCM devic
Fig. 3. Detection specificity of the circulating-flow QCM system. Four probes (P-30-SH, P-30/12T-SH, P-30 and P-30/12T; 1 ␮M) and two targets (T-30AS and T-30S; 0.5 ␮M) were applied to test the efficiencies of probe immobilization and hybridization of the
Fig. 4. Detection of the long oligonucleotide targets, T-104AS and T-104S, hybridized with the probes (1.0 ␮M) immobilized onto the gold surface of the QCM device
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