Influence of citrate buffer and flash heating in enhancing the sensitivity of ratiometric genosensing of Hepatitis C virus using plasmonic gold nanoparticles

Gold nanoparticles (Au NPs) based technology has been shown to possess enormous potential in the viral nucleic acid diagnosis. Despite significant advancement in this domain, the existing literature reveals the diversity in the conditions employed for hybridization and tagging of thiolated nucleic acid probes over the Au NPs. Here we employ the probe sequence derived from the Hepatitis C virus to identify the optimal hybridization and thiol-Au NP tagging conditions. In a typical polymerase chain reaction, the probes are initially subjected to flash heating at elevated temperatures to obtain efficient annealing. Motivated by this, in the current study, the hybridization between the target and the antisense oligonucleotide (ASO) has been studied at 65 °C with and without employing flash heating at temperatures from 75 to 95 °C. Besides, the efficiency of the thiolated ASO’s tagging over the Au NPs with and without citrate buffer has been explored. The study has revealed the beneficial role of flash heating at 95 °C for efficient hybridization and the presence of citrate buffer for rapid and effective thiol tagging over the Au NPs. The combinatorial effect of these conditions has been found to be advantageous in enhancing the sensitivity of ratiometric genosensing using Au NPs.

The citrate-capped Au NPs typically possess a zeta potential in the range of − 30 ± 10 mV, signifying the negatively charged surface nature [23]. DNAs also possess similar charge on their backbone due to the negatively charged phosphate moieties [24]. Thus, when the thiol-tagged DNAs were attempted to conjugate with the surface of citrate-capped Au NPs, due to the chargecharge repulsion, the efficacy of the binding was found to be poor [25,26].
To overcome this, salt-aging technique was developed, in which the externally added electrolyte such as NaCl minimizes the charge-charge repulsion between the citrate units as well as with the phosphate moieties of the incoming DNAs and thereby creating adequate void space for the thiolated DNA to approach the surface of Au NPs [27,28]. However, the salt-aging process requires 24-48 h for efficient conjugation [19]. This has been overcome by strategies like tuning the pH of the Au NPs using citrate buffers, instant dehydration in butanol (INDEBT), etc. [29,30]. Such strategies have been found to yield rapid and quantitative thiol conjugation over the Au NPs. Among these, the method that utilizes citrate buffer is highly attractive due to its simplicity and efficiency. It is generally believed that such conjugation conditions are required when more amount of DNA has to be conjugated over the Au NPs and therefore the previous studies focused with high DNA to Au NPs ratio (typically > 20). However, the necessity of citrate buffer for the cases with low DNA to Au NPs ratio has so far not been explored.
Successful hybridization between the probe and target DNA is also critical in biosensing. The degree of hybridization therefore is an important criterion to realize this. There is a vast diversity in the literature with respect to the DNA hybridization conditions. Many reports employ 37 °C as the hybridization temperature, while adapting the duration as 1-4 h [30][31][32][33][34]. On the other hand, in an attempt to realize the hybridization at a shorter time, slightly higher temperatures in the range of 55 to 65 °C are used, while the time was decreased to 20 min [35][36][37]. Few literature reports adopt a short-duration of flash heating at 95 °C, followed by hybridization at normal temperatures [38,39]. Thus, there exists a significant diverstity in terms of the hybridization conditions.
On a different note, 185 million population is globally affected by the Hepatitis C virus (HCV), which is a blood-borne pathogen. It is known that the serological diagnosis responds typically 4 to 6 weeks post-infection due to the time required for the production of adequate amount of the antibodies. Contrarily, the molecular diagnostics is preferred, as it is highly sensitive and earlystage detection in 1 to 2 weeks post-infection is possible [36]. Since there is no licensed vaccine available till date, though efforts are undertaken by the researchers [40], the timely detection and diagnosis is imperative, since the undetected and untreated conditions could lead to hepatocellular carcinoma [41]. Therefore, in this current study, we utilized an antisense oligonucleotide (ASO) derived from the core region (conserved region) of the HCV genome and subjected it to Au NPs based sensing against the viral target using optimal hybridization and citrate-mediated conjugation conditions.

Materials and characterization
Trisodium citrate and HAuCl 4 .3H 2 O were procured from Sigma Aldrich, and used as received. The target (5'CGG ATT CGC CGA CCT CAT GGG GTA CAT CCC GCT CGT CGGC3'), ASO (5'SH-AAA AAA AAA AGC CGA CGA GCG GGA TGT ACC CCA TGA GGT CGG CGA ATC CG3'), and control (5'TTA CCG ATA ATC CTC CGG GGC ATA ACG AAT GCT TAT AGGA3') oligos were purchased from Eurofins Pvt Ltd. For reducing the disulfide bond of thiol-modified probe, 100 mM DTT (in 100 mM sodium phosphate buffer, pH 8.3-8.5) was added at 1:5 ratio and incubated at room temperature for 1 h. Phosphate buffer saline (PBS, 1X) was prepared using 37 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 and 1.8 mM KH 2 PO 4 . The citrate buffer was prepared by dissolving 25.08 mg of trisodium citrate in 1 mL of water (100 mM) and the final pH was adjusted to 3.0 using dilute HCl.
Spectramax ® iD3 was employed to follow the UV-visible spectral changes of the Au NPs based genosensing assay. The particle size analysis was performed using the Malvern Zeta sizer instrument.

Au NPs-based genosensing
The well-known Turkevich method was employed to synthesize the citrate-stabilized Au NPs [42]. The synthesized Au NPs exhibited a strong plasmonic peak at 520 nm with an average hydrodynamic diameter of ~ 20 nm. In the case of DNA sensing, the hybridization mixture comprising of 2.5 μL of the ASO and 2.5 μL of target/control in 2.5 μL of the 1X PBS buffer was taken in a PCR tube. Two conditions were employed for hybridization inside a Thermal Cycler: In one case, the hybridization was carried out at 65 °C for 20 min without any flash heating and in the remaining cases a flash heating for a short duration at a designated higher temperature (75 °C for 30 s, 85 °C for 30 s and 95 °C for 30 s/60 s/120 s) prior to hybridization at 65 °C for 20 min. To the hybridized mixture, about 50 μL of Au NPs was added and the resultant solution was subjected to stability analysis by adding 1.75 μL and 2.5 μL of 5 M NaCl solution such that the final NaCl concentration was 145 mM and 340 mM, respectively. The same procedure has also been performed with the addition of 0.75 μL of citrate buffer along with the hybridization buffer to ascertain the effect of DNA conjugation over Au NPs. After salt addition, the absorbance of the plasmonic peak at 520 nm and the ratiometric values of absorbances at 700 nm to 520 nm (A 700/520 ) were followed.

Results and discussion
Initially, a polyacrylamide gel electrophoresis (PAGE) experiment was carried out using a 20 wt% gel solution to assess the effect of different temperature conditions on the hybridization efficacy between the ASO and target DNA. Figure 1a shows the gel image obtained with 3 μM concentration of ASO and target/control DNAs subjected to different hybridization conditions. Two bands corresponding to ASO and control were clearly seen in the case of control experiments that confirmed no hybridization took place between the probe and control. On the other hand, a bright thick single band in the case of target subjected to different conditions revealed efficient hybridization between the ASO and target DNA. Further quantification of the bands corresponding to the hybridized DNA using ImageJ software (Version 1.8.0_172) revealed a 7.5% higher hybridization efficacy with the sample subjected to a flash heating of 95 °C for 120 s, as opposed to the sample that was not subjected to any flash heating (Fig. 1b). These results prompted us to probe further the effect of hybridization conditions in the Au NPs based DNA sensing. For this, we chose three conditions such as no flash heating, and 30 s and 120 s flash heating at 95 °C. While one set of samples were Fig. 1 a Polyacrylamide gel electrophoresis of the probe, target and control DNA subjected to different hybridization conditions and b the corresponding percentage hybridization quantification using ImageJ software. c Schematic representation on the mechanism of the sensing strategy employed in this study subjected to Au NPs based sensing assay to ascertain only the effect of flash heating conditions, another set of samples were subjected to the sensing assay with the addition of citrate buffer in the hybridized mixture as it has been proven to yield rapid and quantitative thiol-conjugation over Au NPs in minutes [29].
The mechanism of the Au NPs mediated sensing assay is based on the following factors. It is known that the stability of the Au NPs is imparted due to the electrostatic repulsion between the negatively charged citrate moieties on the surface. The citrate-stabilized Au NPs synthesized through the Turkevich method exhibit a plasmonic peak at 520 nm and a relatively low absorbance at wavelengths greater than 650 nm in the UV-visible spectroscopy. Upon salt addition, due to the masking of electrostatic repulsion between the citrate moieties by the excess of sodium ions, the Au NPs tend to aggregate. The aggregated colloidal Au NPs are known to compromise their plasmonic absorbance and exhibit higher light scattering at longer wavelengths. It is known that the negatively charged DNA enhances the charge density (zeta potential) of the Au NPs upon binding to them. Such an enhancement in the charge density shall enhance the stability of the nanoparticles against the salt-induced aggregation of the Au NPs. Thus, the Au NPs effectively hybridized with the target DNA sequence shall possess higher stability-in comparison to the negative/control sample-and thereby retain the red color arising from the SPR.
In the case of DNA sensing, two different initial concentrations of the ASO, such as 1.5 and 3 μM were hybridized with equimolar concentration of the target/ control. Also, two different conditions were employed for the thiol conjugation over the Au NPs before the salting-out process: (i) without the addition of citrate buffer and (ii) with the addition of 0.75 μL of citrate buffer. The UV-visible spectral results obtained from the DNA sensing studies are presented in Fig. 2. The absorbance of Au NPs at 520 nm as well as the ratiometric value of 700 nm/520 nm (A 700/520 ) have been chosen to follow the effect of hybridization conditions on DNA sensing and the absorbance values before and after salting are summarized in Table 1. It can be seen from Fig. 2 that the Au NPs added with target/control exhibit a high absorbance value at 520 nm (> 0.4) and a low A 700/520 ratiometric value of < 0.4. After salt addition, the absorbance of the positive samples (containing target) at 520 nm gradually decreased with the addition of NaCl solution, while the A 700/520 ratiometric value gradually increased. In the case of positive samples, the cut-off values of the absorbance at 520 nm and A 700/520 were found to be > 0.26 and < 0.5, respectively, which indicate the stable nature of the Au NPs. Among the different hybridization conditions employed, the target-ASO hybrids subjected to flash heating were found to have a slightly higher absorbance value at 520 nm than the no-flash heating sample, though the difference was less (Table 1). On the other hand, a substantial difference was noticed in the ratiometric values of the hybrids subjected to different hybridization conditions. The ratiometric values corresponding to different hybridization conditions have been plotted in Fig. 3.
In general, before the salting process, the absorbance of Au NPs at 520 nm was found to be in the range of 0.40 to 0.43, while the A 700/520 ratiometric values were found to be in the range of 0.15 to 0.4 for all the samples. When 1.75 and 2.5 μL of NaCl was added sequentially (total amount of NaCl added was 4.25 μL) such that the final salt concentration was 145 and 340 mM, the target and control were properly discriminated, as evidenced by the high absorbance value at 520 nm and low A 700/520 ratiometric value for the target. It can be seen from Table 1 that the target samples subjected to flash heating exhibited lesser ratiometric values than the ones not subjected to flash heating (highlighted in bold). These results indicate that flash heating does play a positive role in enhancing the hybridization efficacy, which results in better discrimination at the ratiometric values that are often considered to be useful in improving the sensitivity of the assay.
The experiments were then performed with the addition of citrate buffer to ascertain the effect of DNA conjugation over Au NPs and its subsequent effect on the sensitivity of the assay. It was found that the discrimination between the target and control at the ratiometric values was more pronounced when citrate buffer was used. This reveals that the use of citrate buffer is useful in enhancing the conjugation efficiency even when the DNA to Au NPs ratio is as low as 3.5:1, while the available literature reports used a ratio of 20:1 and higher [29].
Since the citrate buffer showed additional benefit in ratiometrically discriminating the samples subjected to flash heating from no-flash heating, their corresponding digital photograph, and dynamic light scattering (DLS) results are presented in Figs. 4 and 5. Although the digital photograph shows the color of the Au NPs used in different assay conditions is indistinguishable to human eyes, the DLS measurements of samples subjected to flash heating revealed a decrease in the particle size after subjecting to the salting-out process, which rationalizes their lower ratiometric values.
The study has revealed the beneficial role of employing a combination of flash heating for efficient hybridization and citrate buffer for rapid tagging of the thiolated DNA over the surface of Au NPs. Such a combination has been found to enhance the sensitivity of the ratiometric absorbance values in the Au NPs based gene sensing. It can be noted that the ratiometric values are highly decisive in discriminating the single nucleotide polymorphism, as described by Sanromán-Iglesias et al. using gold nanoparticles of sizes 13, 46, and 63 nm [43]. Our study reveals that employing appropriate hybridization conditions can further improve the ratiometric sensitivity of such Au NPs based assays.

Conclusions
In summary, the effect of hybridization between an ASO and target DNA was ascertained using PAGE experiment, which revealed that the hybridization involving a flash heating of 95 °C for 120 s, prior to the incubation at 65 °C for 20 min, resulted in an  enhancement in the efficacy to the tune of 7.5% as opposed to the hybridization condition without the flash heating. The effect was further analyzed in the Au NPs based DNA sensing to ascertain the usefulness of the flash heating in practical applications. The DNA sensing studies using thiol-tagged synthetic oligonucleotide-derived from the core region of the HCV viral genome-revealed that the ASO-target DNA hybrid subjected to the flash heating stabilized the Au NPs more than the case without flash heating, as evidenced by the lesser ratiometric absorbance values for the former. Furthermore, the use of citrate buffer has shown to be beneficial in the efficient conjugation between the thiolated probe and the Au NPs, even when the DNA to Au NPs ratio is as low as 3.5:1 and 7:1. These results may find potential in employing the appropriate hybridization conditions in the DNA biosensing studies.