Sensitive and selective detection of DNA fragments associated with Ganoderma boninense by DNA-nanoparticle conjugate hybridisation

All chemical reagents were purchased from Sigma-Aldrich (now Merck) or ThermoFisher Scientific and used as supplied. All reagents were of analytical grade. 20-nm AuNP colloid at a concentration of 1 optical density (OD) unit @ 525 nm in 0.1 mg mL⁻¹ sodium citrate was purchased from Alfa Aesar (Heysham, UK). Deionised water (resistivity ~ 18.2 MΩ) was used in all cases.

The DNA-AuNP conjugates were prepared using a general procedure previously described [32] with few modifications. Aqueous solutions of disulfide-functionalised oligonucleotides (100 μM, 40 μL) and tris(2-carboxyethyl)phosphine (20 mM, 40 μL) were combined and incubated for 2 h at room temperature (RT) to prepare 50 μM solutions of the oligonucleotides with reduced thiols. This solution (80 μL) was then mixed with 1 mL of citrate stabilised AuNP colloid, and the mixture was incubated at RT for 12 h. 24 μL of 1 M NaCl was added to this mixture, followed by sonication for 10 s. The NaCl addition and sonication were repeated four times at 1 h intervals (i.e. 5 times in total) until a final concentration of 0.1 M NaCl was achieved. The final colloidal solution was allowed to stand at RT for 24 h and then centrifuged at 19000 g for 30 min. The supernatant was decanted, and the pellet of AuNP resuspended in phosphate-buffered saline (PBS) diluted to half-strength (i.e. final composition 5.9 mM phosphate buffer, 68.5 nM NaCl, 1.35 mM KCl, pH 7.4) containing 0.01% v/v Tween 20. This process of centrifugation, decanting, and resuspension was carried out three times in total, with the final pellet of AuNP being re-suspended in 100 μL of 10 mM PBS buffer. The OD was then measured by UV–Vis spectroscopy at 525 nm. These DNA-AuNP conjugates were typically stored at 4 °C and used within a week of preparation. Prior to use, the OD of each colloid was adjusted to 0.7 by the addition of the relevant volume of PBS.

Lyophilised analyte ssDNA and dsDNA were reconstituted in water to make up two stock solutions of either 5 or 1 μM. The more concentrated stock solution was used in experiments requiring a higher analyte final concentration of 200 pmol, while the more dilute solution was used for all other experiments.

As targets for detection, 35-base DNA sequences from the internal transcribed spacer 1 (ITS1) region of fungal 18S ribosomal genes were chosen. For the species-selective detection of G. boninense, the target sequence was taken from a gene bearing the NCBI GenBank accession number EU701010 [5] (termed T1, Table 1). A second sequence taken from the accession number BD082757 [5, 33] (termed T2) was also chosen, which was a match not only to G. boninense but also a wider range of fungal ITS1 genes and served as a more general target for the detection fungi of the Basidiomycota phylum [34].

Pairs of DNA-AuNP conjugates bearing complementary probe DNA sequences to each target were prepared. Two general assay configurations were investigated for each target. In the “head-to-tail” (HTT) [20] configuration, two probes in series were employed, with each complementary probe sequence attached at 5′-end to the AuNP via a 12 base GC repeat spacer and an alkylthiol linker (Fig. 1a). Here, a GC spacer [35] is used to increase the distance between the AuNPs and the target complementary DNA sequences, to address potential steric crowding during hybridisation. In the “tail-to-tail” (TTT) format [22], one probe sequence was attached at the 5′-end and the other via the 3′-end, and the DNA sequences were attached directly to the AuNPs via alkylthiol linkers only (Fig. 1b).

To improve the likelihood of identifying probes that would give reliable results, several pairs of DNA-AuNP conjugate probes were prepared for both target sequences. Thus, for target sequence T1, one probe pair in the head-to-tail configuration (termed P1a^HTT and P1b^HTT) and two pairs in tail-to-tail (P1a^TTT and P1b^TTT, P1c^TTT, and P1d^TTT) were prepared (Table 1). For sequence T2, one pair of each conjugate was prepared for each configuration (P2a^HTT and P2b^HTT, P2a^TTT, and P2b^TTT; Table 1).

Following their preparation, the DNA-AuNP conjugate bearing the P1a^HTT probe exhibited the expected deep red colour and a corresponding UV–Vis spectrum with a λ_max at 525 nm (Fig. 2a), similar to λ_max for unconjugated AuNP. However, for P1b^HTT, a higher λ_max (533 nm) and a broad tail towards the higher wavelengths was observed, which suggests the presence of aggregated AuNPs. DNA-AuNP colloids bearing the P2a^HTT and P2b^HTT probes also showed similar results (Fig. 2b), with P2b^HTT showing a broad tail along with λ_max at 533 nm. Both pairs of probes were thus unsuitable for further investigation.

Probes in the tail-to-tail configuration were then prepared, starting with the P1a^TTT and P1b^TTT pair. After DNA conjugation, the expected red colour and a λ_max of 525 nm were observed for colloid bearing the P1a^TTT probe (Fig. 3a). However, despite several efforts to produce the conjugates of P1b^TTT under various conditions, stable colloids could not be produced. The AuNPs precipitated during the NaCl addition step, resulting in only minimal amounts of suspended conjugates, as evidenced by the minimal UV–Vis absorbance. An alternative design was then attempted using the same sequence that was complementary to the T1 target sequence, but now differently divided into each probe (referenced as P1c^TTT and P1d^TTT; Table 1). This revised design led to the successful preparation of the DNA-AuNP conjugates with the expected optical properties (Fig. 3b). Based on these results, probes P2a^TTT and P2b^TTT were designed for T2 (Table 1), and the prepared conjugates were found to display the expected optical properties (Fig. 3c).

It was found that the prepared DNA-AuNP conjugates (P1c^TTT, P1d^TTT, P2a^TTT, and P2b^TTT) were stable between a pH of 4 and 7, as evidenced by a lack of change in the UV–Vis spectra. Further, these conjugates were stable for up to two months if stored at 4 °C (at pH 7), though only for 3–4 days if stored at room temperature (in the UK, approximately 20 °C), as evidenced by UV–Vis spectroscopy.

These results suggest that to achieve stable conjugates, a minimum of 20 bases are required when DNA is conjugated to AuNP at 3′ position of DNA, though the reason for this observation is unclear. The P1b^TTT probe has been previously used successfully in other sensor platforms [19], but the current result nevertheless shows that the cross-compatibility of DNA probes between different applications cannot be assumed. Thus, the probe pair P1c^TTT/P1d^TTT complementary to T1, and P2a^TTT/P2b^TTT pair complementary to T2 were taken for further evaluation.

In preliminary optimisation experiments, the hybridisation assays were carried out with ssDNA sequences as the analyte, which was added to a mixture consisting of each pair of complementary DNA-AuNP probes. Three types of analytes were tested (Table 1): (1) the 35-base target DNA strand fully complementary to their respective probes (i.e. either T1 or T2); (2) DNA sequences containing a single base mismatch referred to as M1 and M2, but otherwise, identical to T1 or T2, respectively; (3) “non-complementary” DNA consisting of a randomly generated sequence of identical length and similar GC content to T1 and T2 (termed NC). In all cases, a mixture of equivalent concentrations of DNA-AuNP conjugates was prepared (to an OD of 0.7 @ 525 nm of each probe prior to mixing), to which the ssDNA analyte was added to give a final analyte concentration of 1 μM (corresponding to 200 pmol of analyte in 200 μL of the total assay mixture). In the negative control, only water was added as the analyte (henceforth referred to as “C” in all figures).

For the hybridisation experiments with the tail-to-tail configuration using probes P1c^TTT and P1d^TTT against T1, it was found that a hybridisation mixture with 1 M NaCl gave good hybridisation after 30 min. This result was evidenced by a visually observable colour change from a deep red to lilac and was corroborated by the UV–Vis spectrum that exhibited a λ_max increase from 525 to 550 nm, a decrease in the absorbance at their respective λ_max (A_max) from 0.6 to 0.1 (an approximately 80% reduction), and a broadening of the peak (Fig. 4a, b). In contrast, a negative control experiment where only water was added, no significant change in colour or the UV–Vis spectrum was observed. Similarly, the NC sequence gave no significant change also, which confirms that probe binding is sequence specific. In the case of the mismatched sequence M1, a result similar to T1 was observed, which indicates this probe pair was able to bind and cannot differentiate this single mismatch from the fully complementary sequence (i.e. this is a false positive result).

A similar experiment was performed for P2a^TTT and P2b^TTT probes, against T2 or M2. Both these analyte sequences gave colour and spectroscopic changes corresponding to a positive result similar to the above (Fig. 4c, d). As before, negative controls consisting of the NC sequence or water only gave no change.

The rapidity of the assay was then investigated. Here, using probes P2a^TTT and P2b^TTT against T2, it was found that a change in λ_max from 525 to 548 nm, and decrease in the A_max from 0.7 to 0.2, occurred after 15 min (Fig. 5c), with little further change even after 45 min (Fig. 5). This result thus shows that the 30-min incubation time previously used was, in fact, unnecessary, and thus, all subsequent assays were analysed after 15 min.

Based on the above results, DNA analyte T2 and DNA-AuNP conjugates prepared using probes P2a^TTT and P2b^TTT were taken for further study. A series of assays were carried out by adding between 0.1 and 10 pmol of T2 to the conjugates to test its sensitivity. These results were compared with a positive control with 200 pmol and negative control with water as the analyte. It was found that a visually observable colour change was produced with as little as 0.2 pmol of T2 (Fig. 6a), although the UV–Vis analysis showed that the λ_max shift for this amount of analyte was smaller than for the higher amounts (10 nm vs. 25 nm, Fig. 6b). The UV–Vis spectrum corresponding to 0.1 pmol of T2 did show an ~ 50% reduction in A_max without an accompanying change of λ_max, but this change could not be readily observed visually. Nevertheless, the overall visual transition is very sharp, spanning just one order of magnitude in the pmol–fmol range (i.e. 1.0 pmol was clearly positive, but 0.1 pmol was clearly negative), which equates to concentrations between 5 and 0.5 nM.

As the results with the single-base mismatch M2 sequence also gave a positive result, the tolerance of the assay (with probes P2a^TTT/P2b^TTT) against other mismatched sequences was investigated. For this purpose, the assays were carried out with a series of analyte sequences bearing mismatches at multiple locations, referenced M2a to M2f (Table 1).

It was found that analyte sequence M2a, bearing three mismatches in series at the junction where the two probe sequences meet, also gave a false positive. This result was evidenced by a visually observable colour change, a UV–Vis λ_max shift from 525 to 545 nm and a drop in A_max similar to M2 (Fig. 7). The sequence with five mismatches in series (M2b) gave almost no change in visual appearance, which was confirmed spectroscopically by a small λ_max increase to 531 nm and A_max reduction of only 15%. Since this change was not visually distinguishable from the negative controls (C in Fig. 7 and NC in Fig. 4d), it was judged to be a “negative” result.

In contrast to M2a, if three mismatches were individually distributed throughout the analyte sequence (M2c), this gave a negative visual appearance that was similar to M2b (Fig. 7a). This observation was consistent with the UV–Vis spectrum showing a small λ_max shift to 533 nm, although there was a significant decrease in the A_max by 40% (Fig. 7b, d). The sequence M2d with five distributed mismatches was also negative, with a visual and spectral result that was similar to M2b.

The inability of these DNA-AuNP conjugates to differentiate either one or three mismatches in series at the meeting point of the two probes (M2 and M2a) can be rationalised by the binding stability of the remaining complementary base pairs. It is known that DNA duplexes with more than ~ 12 complementary base pairs typically exhibit melting temperatures (T_m) well above 37 °C [36]. Since even three mismatches would allow both probes to bind with ≥ 14 serial base pairings, it would be expected that stable DNA hybridisation can still be achieved, which translates to a positive result. In comparison, M2c with three distributed mismatches gave a negative result. However, it is notable that five mismatches in series (M2b) gave a negative result, even though it would still theoretically give > 12 base pairings in series. This result suggests that ≥ 14 serial base pairings are required in these conjugates, which could be due to the presence of AuNP hindering efficient hybridisation.

In the case of mismatches located midway within the sequence of each probe (M2e and M2f), these gave an ambiguous result that was mid-way between the positive and negative controls. An intermediate colour and intensity change were visually observed, consistent with the UV–Vis spectra that gave a λ_max shift to 538 and 536 nm (for M2e and M2f, respectively) and an A_max decrease of ~ 48% for both (Fig. 7d). Thus, though these mismatches could not be differentiated by eye, they could still be identified spectroscopically.

Assays based purely on direct hybridisation are known to have a limited ability to discriminate single base-pair mismatches due to the small difference in stability compared to a perfectly matched DNA duplex. Various modifications to the basic strategy such as employing specific interactions between Lambda exonuclease and a chemically modified DNA [37], double-stranded toehold exchange [38], and co-amplification at lower denaturation temperature [39] have been proposed to enhance the sensitivity of the assays towards one or more mismatches. However, these approaches increase the complexity of the assay and make it less suitable for field deployment.

As a step towards biologically relevant diagnostics, the assay was next tested against dsDNA fragments. Here, the experiments consisted of dsDNA with the T2 sequence (referred henceforth as T2′) and nanoparticle conjugates bearing probes P2a^TTT and P2b^TTT. The assay procedure was also modified such that the analyte was heated to 95 °C to induce the denaturation of the dsDNA [40] and enable subsequent probe hybridisation upon cooling. It was, however, observed that in contrast to ssDNA (see in “Response time of hybridisation assay” section), a more extended waiting period was needed for the assay to develop (30 vs. 15 min with ssDNA).

A range of analyte amounts was then tested from 0.2 to 200 pmol (in a 200 μL final assay volume). In order to optimise the sensitivity, the concentration of NaCl in the assay mixtures were also varied (either 1.0 or 1.2 M) [41]. It was found that a visually observable positive result could be achieved at 10, and 1 pmol (corresponding to concentrations of 50 nM, and 5 nM, respectively) of T2′, at each of the respective NaCl concentrations (Fig. 8a, c). These observations were supported by UV–Vis spectra showing both the expected increases in λ_max and decreases in A_max, typically by 20–25 nm and 40–50%, respectively, depending on the amount of analyte (Fig. 8b, d). However, even the best results obtained here were still fivefold less sensitive than the equivalent experiment with ssDNA. This result is likely due to the competitive rebinding of the analyte ssDNA to its complementary strand upon cooling.

In order to demonstrate the selectivity of the assay in the presence of a large amount of non-complementary genomic DNA that would be present in any potential biologically derived sample, the optimised dsDNA assay was then carried out in the presence of calf thymus DNA (CT), as a model mixture of non-complementary dsDNA with a variety of lengths. Here, varying amounts of T2′ dsDNA from 0.2 to 200 pmol were mixed with 2.6 mg of CT and tested.

Similar to the isolated dsDNA, quantities of T2′ as low as 10 and 2 pmol (which equates to a concentration of 50 nM and 10 nM) gave a visually observable result, even in the presence of a considerable amount of CT, at 1 and 1.2 M (Fig. 9a) NaCl concentration, respectively. The UV–Vis spectra also showed the corresponding λ_max increase from 525 to 540 nm, though the A_max reduction, in this case, was more modest at 24% (Fig. 9b).