Results and discussion

8.3.1 The QCM system and detection

In the circulating-flow QCM system, real-time frequency shift is 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. Figure 7-1 shows an example of the real-time detection of the QCM system performed in our study.

There is 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 are decreases in series resonant frequency of approximately 42 and 51 Hz when complementary strands (T-30AS) are introduced and hybridized with P-30-SH and P-30/12T-SH, respectively.

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

immobilization.

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

The results showed that ∆F increased almost linearly with the increase of probe

concentrations up to concentrations of 1.0 and 2.0 μM. Probe concentrations of 1.0 and 2.0 μM yielded the greatest covalent attachment to the gold surface compared with other

concentrations (P < 0.01) (Figure 8-1). There is no significant difference between ∆F at concentrations of 1.0 and 2.0 μM (P > 0.05), indicating saturation of the immobilization sites on the gold surface of the QCM device. Therefore, the probe concentration of 1.0 μM is selected for the following experiments.

In general, the thiolated probes with additional 12-dT (P-30/12T-SH) showed greater

∆F (P < 0.05) than thiolated probes without additional 12T (P-30-SH) among the various concentrations tested (Figure 8-1). This is because the weight of P-30/12T-SH per single

molecule (13,155 g mol^-1) is larger than that of P-30-SH (9504 g 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 are 1.7 ± 0.1 and 2.1 ± 0.2 (ssDNA/10 nm²), respectively, when 1.0 μM of probes is used in the immobilization. These molecule density values show no significant differences (P > 0.05). Therefore, the efficiency of immobilization of P-30-SH and P-30/12T-SH onto the gold surface of the QCM is similar.

8.3.3 Detection of the short (30 mer) synthesized target oligonucleotides

Different concentrations (0, 0.25, 0.5, and 1.0 μM) of the short synthesized target oligonucleotides (T-30AS, 30 mer) complementary to the probes immobilized on the gold surface of QCM device are compared for the ∆F and H% in the circulating-flow QCM system.

The ∆F due to hybridization increased with increasing concentrations of the targets up to 0.5 μM (P < 0.01) (Figure 8-2A). At concentrations ≥1 μM, the frequency shift is less sensitive, indicating the saturation of the probe hybridization sites (Figure 8-2A and data not shown).

The H% also increased with increasing concentrations of the targets (Figure 8-2B). The H%

of target T-30AS at 0.25 μM and 0.5 μM hybridized with the probe P-30/12T-SH are 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 the ∆F results, the probes are 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%.

8.3.4 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 μM) and two targets (T-30AS and T-30S; 0.5 μM) (Table 7-1.). 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 < 2Hz for 30 min), preventing the subsequent hybridization of probes and targets (Figure 8-3A). Non-specific binding is investigated using T-30S (a sense strand to the probe sequences, non-complimentary strand). The

thiolated probes (P-30-SH, P-30/12T-SH) are covalently immobilized on the gold surface and specifically hybridized to the complementary targets (T-30AS), but not to the

non-complementary targets (T-30S) (Figure 8-3). 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 coverage, such as with the gold in our system, than non-thiolated ssDNA [Herne and Tarlov, 1997; Levicky et al., 1998]. The hybridization of P15730/12T-SH and T15730AS showed greater ∆F (P < 0.01) (Figure 8-3A) and higher H% (Figure 8-3B) than the hybridization of P15730/SH and T15730AS, reflecting the reduction of steric hindrance in the three-dimensional space and the increase in molecule collisions caused by the additional 12-dT.

8.3.5 Detection of the long (104 mer) synthesized target oligonucleotides

The 104-mer synthesized targets (T-104AS and T-104S) are applied in the QCM system and the ∆F and H% are evaluated. Within the target of T-104AS, only the 30 mer sequences are complementary to the probes, P-30-SH and P-30/12T-SH (Table 7-1). As shown previously, no measurable frequency shift is detected when the targets are applied to the immobilized probes without thiol-linkered tag modification. Using T-104S, a

non-complementary strand to the probes, non-specific binding is investigated. No

measurable ∆F is detected when T-104S is applied to the probe-immobilized QCM. The ∆F (due to the hybridization), and the H% significantly increased (P < 0.05 or P < 0.01) with increasing concentrations (0.5 and 1 μM) of the target T-104AS (Figure. 8-4A and 4B).

However, the frequency shift decreased when a relatively high concentration of T-104AS (2 μM) is applied for the hybridization with P-30/12T-SH, indicating the upper limit of

hybridization and saturation of the probe hybridization sites.

In Figure 8-2, 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 μM).

In Figure 8-4, the ∆F has a 2-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 has 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 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 surface 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 (Figure 8-6).

Interestingly, the values of H% (20-25%) in the hybridization of the probe

P-30/12T-SH with the 104-mer targets are significantly lower (P < 0.05) than those in the hybridization of the 30-mer targets at optimal conditions (85-95%) (Figure 8-2B and Figure 8-3B). 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% is reduced to 20-25% in the

hybridization of the probe P-30/12T-SH and the 104-mer targets, the detectable ∆F 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.

8.3.6 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 (Figure 8-5A). The PCR products are 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 indicated in Figure 8-5B. The results show that the ∆F equaled 70 ± 4 Hz when the system temperature is maintained at 30°C and is

significantly higher (P < 0.01) than when the system is maintained at 20, 40, 50, and 60°C.

The frequency shift of the QCM system for detecting PCR-amplified products is 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 is complementary to the immobilized probes with synthetic oligonucleotides. The system will be further applied in the detection of field samples or other 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 quantitative and highly sensitive assay. The optimal conditions of our QCM system use to detect the PCR-amplified DNA from the real sample has relative standard deviation values (%

RSD) as low as 5.3%, which are considered acceptable among established analytical

techniques [Skoog et al., 1998]. Our QCM device may be use 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.

8.3.7 Conclusions

We developed a DNA piezoelectric sensor for the real-time detection of E. coli O157:H7.

Synthetic probe oligonucleotides are 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 solution is 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 is also used to detect the PCR-amplified DNA from real samples. Our results suggest that the DNA piezoelectric 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.