Enhancing the efficiency of polymerase chain reaction using graphene nanoflakes

GNFs of 8 nm thickness (20–30 ML) and average particle (lateral) size ∼550 nm from Graphene Labs were suspended in molecular-biology-grade distilled water at 1 mg ml⁻¹ concentration. The density of graphene flakes was 1.9, 2.0 and 1.9 g cm⁻³ for 60, 12 and 8 nm respectively. To ensure proper mixing of the nanoparticles in water, sonication was performed for 2 h using a bath-sonicator (JEOL). This well dispersed nanofluid was used as a stock solution and was appropriately diluted to various concentrations. UV–vis spectroscopy (PerkinElmer Lambda 35 spectrophotometer) was performed to confirm the characteristic peak of the samples. Absorbances of 0.06, 0.08 and 0.1 wt% were measured and the data analysis software Origin 2 was used to plot graphs. The pH was measured (Mettler-Toledo) after the stock was diluted to appropriate concentrations required for PCR.

The graphene samples were mounted on stubs with conductive carbon tape and coated with platinum using a JEOL JFC-1600 auto fine coater. All samples were analyzed for their physical-structure and elemental compositions with energy-dispersive x-ray spectroscopy (EDX) using SEM (JEOL JSM 6400 LV).

Differential scanning calorimetry (DSC) was performed using a Sapphire DSC, PerkinElmer Instruments. The temperature of the sample and the reference were maintained almost the same throughout the experiment. During analysis, the temperature of the sample holder was increased linearly as a function of time.

We measured the thermal conductivity of the GNF nanofluids using a KD2 Pro portable thermal conductivity meter (Decagon Devices). The transient line source (TLS) uses a sensor (single needle with 1.3 mm diameter and 60 mm in length) for measurements. A measurement cycle consists of 30 s of each equilibration, heating and cooling time. Standard glycerin was used for calibrating the device. The temperature measurements were made at intervals of 1 s during both heating and cooling. Measurements are then fit with exponential integral functions using a non-linear least squares procedure. A linear drift term corrects temperature changes of the sample during the measurement, to optimize the accuracy of the readings. A thermostat bath was used to maintain the temperature of the nanofluids. Nearly five readings per temperature were taken to ensure uncertainty in measurement within ±5%. The experimental values of GNF nanofluid thermal conductivity were compared with theoretical values for verification. The effective thermal conductivity of solid–liquid mixtures has been calculated using the classical Maxwell model [17],

$$\begin{eqnarray} &&{k}_{\mathrm{eff}}=\frac{{k}_{\mathrm{p}}+2{k}_{\mathrm{b}}+({k}_{\mathrm{p}}-{k}_{\mathrm{b}})\phi }{{k}_{\mathrm{p}}+2{k}_{\mathrm{b}}-({k}_{\mathrm{p}}-{k}_{\mathrm{b}})\phi }{k}_{\mathrm{b}} \end{eqnarray} \tag{ 1 }$$

where k_p is the thermal conductivity of solid particles, k_b is the thermal conductivity of the base fluid and ϕ is the particle volume fraction.

PCR was carried out to amplify segments of DNA using gene-specific primers (table 1). Optimization and amplification of these DNA segments were performed using PCR master mix (2×) from Fermentas. The typical 25 μl PCR reaction mixture contained the following final concentrations: 12.5 μl of master mix that includes 0.05 units μl⁻¹ Taq DNA polymerase in reaction buffer, 4 mM MgCl₂, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP and 0.4 mM dTTP, 1 μM of each forward/reverse primer and 10–20 ng of template DNA with or without different concentrations (0.1–3.2 nM) of nanoparticles. The PCR protocol began with a 94 °C denaturation step for 5 min, followed by a touchdown program (94 °C denaturing step for 30 s followed by initial annealing temperature of 70 °C to avoid amplification of nonspecific regions, subsequently run down to 55 °C at 1 °C/cycle, and a 72 °C extension step for 1 min), followed by a uniform three-step amplification profile (94 °C denaturing step for 30 s, 54 °C annealing step for 30 s, 72 °C extension step for 1 min) for another 30 cycles, then 72 °C for 10 min, and finally held at 4 °C.

PCR reactions were carried out using a thermocycler with maximum ramping rate of 2.5 °C s⁻¹ (Eppendorf Mastercycler gradient). After PCR, products were analyzed in agarose gels (1.1% wt/vol) and visualized with ethidium bromide staining. In addition, the amplified PCR products were quantified using ImageJ 1.41o software (National Institutes of Health). The band intensities were expressed as arbitrary units (AU) after densitometric scanning of the gel images generated by loading equal volumes of PCR products.

The characteristic spectra of graphene nanofluids (8 nm) were observed for concentrations such as 0.1, 0.06, 0.08 w/w%. The absorbance was increasing with the increase in concentration and a peak was observed at approximately 225 nm wavelength (figure 1). This measured absorbance is in good agreement with the literature [18], confirming the presence of graphene as quoted by the supplier. GNFs are hydrophobic in nature and cannot mix with polar solvents such as water. GNFs start to settle within a few hours of synthesis. Functionalization is essential to obtain stable solutions; however, all the experiments were performed using freshly prepared GNF nanofluids. After a week the GNFs were found to significantly settle and the supernatant was used to measure the characteristic spectra, which revealed a peak at 225 nm wavelength. The DSC plots were also obtained using the procedure as mentioned in the methodology section (figure 1). The pH of 0.01 wt% GNF nanofluids was found to be ∼5.45 at 27 °C, confirming the acidic nature of the nanofluids added to the PCR reagents. SEM images with 2 μm magnification show that the flakes are rather bigger than what is claimed by the supplier (figure 2). The 0.005%–0.05% volume fraction graphene nanofluids were only stable for less than 5 h, and so gum arabic (GA) was added as a surfactant in five different concentrations ranging between 0.5 and 2.5 wt%. Compared to CNT nanofluids, which exhibit excellent stability with similar GA addition [19], GNF nanofluid stability is not enhanced significantly with the addition of 2.5 wt% GA. However, other surfactants such as CTAB, SDS, Triton, etc. may also help in enhancing the stability. Besides, functionalization has improved stability of various nanofluids including graphene and may be used in this context [20].

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Experimental results confirm that the thermal conductivity of graphene nanofluids is strongly temperature and nanoflake concentration dependent (figure 3). Using 0.05 and 0.005% volume fractions we observed superior thermal conductivity to the literature values [20]. The reasons for this excellent enhancement may be attributed to graphene's large surface area [11, 21], dispersion of nanoflakes [22] and Brownian motion of GNFs [23, 24]. Graphene oxide nanosheets were found to enhance thermal conductivity of water up to 30.2% when suspended at 5.0 vol% [18]. Similarly, ethylene glycol based nanofluids show an enhancement of 61.0% with 5.0 vol.% volume fraction [25]. Most of the nanofluids exhibit particle volume fraction and temperature dependent thermal conductivity [23, 26, 27]. However, recent works indicate that the enhancement ratios of the graphene nanofluids are nearly constant with varying temperature, and they are reduced with increasing thermal conductivity of the base fluids [18]. Other research appears to suggest that the thermal conductivity of functionalized and thermally exfoliated graphene (f-TEG) based nanofluids is temperature dependent [20]. The thermal conductivity of f-TEG based nanofluids showed an excellent enhancement up to 60% at 50 °C with a volume fraction of 0.056% in deionized water [20]. It is well known that the higher the temperature the greater is the motion of particles in a fluid [23, 24]. Similarly, elevated temperature might modulate random motion of GNFs in fluid and thus result in considerable enhancement of thermal conductivity. The theoretical thermal conductivity which was predicted using Maxwell's model was much lower than the actual experimental values. Other models also exist in the literature, which involve other parameters in addition to volume fraction, such as particle size, shape [28, 29], interfacial layers [30] etc. However, none of these models takes into account the large surface area of graphene sheets. This suggests that a new model should be developed considering the large surface area of GNFs and layers of flakes.

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Initially, GNFs suspended in autoclaved distilled H₂O were added to test the viability of PCR. Preliminary results showed unambiguous enhancement in the PCR yield (results not shown) of the order of ∼10-fold. A series of experiments with increasing graphene concentrations from 0.001 to 0.1 w/w% was performed in order to find the optimum concentration. Similar to the existing reports on other nanoparticles [4, 7], graphene exhibits strong concentration dependent PCR enhancement as well. The increasing nanoparticle concentration from 0.001 w/w% shows a gradual increase in the PCR yield up to 0.01 w/w% before gradually decreasing with further increase in concentrations as in figure 4(A). On the other hand, PCR was completely inhibited at a GNF nanofluid concentration of ≥0.5 w/w%. The intensities of DNA bands analyzed in agarose gels affirm the role of the enhanced thermal conductivity effect of GNFs in PCR enhancement. As the nanoflake concentration and temperature is increased, thermal conductivity is found to augment. However, the decrease in PCR product after 0.01 w/w% can be attributed to excess temperature as a result of nanoflake addition. Results from PCR experiments were analyzed by densitometry and values are shown in figure 4(A). Experiments revealed that the nanoflake thickness has a significant effect on the PCR product yield. While using identical concentrations of 60, 12 and 8 nm thick GNFs, we discovered that the 8 nm flakes augment yield better than the rest (figure 4(B)). This observation is in agreement with thermal conductivity measurements of GNF nanofluids (results not shown), where the increasing thickness decreases the enhanced thermal conductivity effect. This strongly suggests that there is a great possibility of a role of the enhanced thermal conductivity effect of GNFs. In the recent past, it was observed that AuNPs of larger sizes (20 nm) have stronger inhibitory effect on PCR than AuNPs of smaller sizes (10 and 5 nm) at the same particle concentration [5]. The report proposed a hypothesis, which is the binding of AuNPs and TaqDNA polymerase, resulting in PCR inhibition by reducing the concentration of free polymerase in PCR solution. In another study, it was observed that there is a close match between the specific surface areas of varying sizes of AuNPs at which they exert their effects, even as particle diameter varies by 20-fold and particle volume variation by 8000-fold [31]. They suggest that the differing lines of evidence are against the nanoparticle effects' hypothesis, and PCR is mediated by heat transfer enhancement. Another study explored the fact that the surface effect seems to be the key factor of nanogold assisted PCR while using five different sized Au nanoparticles [32]. To further test the efficacy of the GNFs, PCR reactions with a reduced number of cycles were conducted. As described in the methods section, keeping the 'touchdown' part of the PCR program unchanged, we reduced the 'uniform three-step' number of cycles from 30 to 20 and ten. The electrophoresis gel images of PCR obtained after experimenting with reduced PCR cycles indicate that enhanced yield can be obtained even at ten cycles. This is far superior to any other observation in NANO-PCR. The positive control of both cases (ten and 20 cycles) has yielded comparatively less product (figure 4(C)). Nevertheless, after 30 cycles, positive control yields only ∼10-fold less than GNFs added PCR samples. Further studies are imperative to understand this interesting phenomenon. Li et al [4, 9, 12] showed that the efficiency of PCR has been enhanced by adding Au nanoparticles and reducing its cycle time. Another report explored that the gold nanoparticles could increase the effective PCR amplification cycle, and the single bright band could be achieved even in the sixth amplification cycle [33]. Khaliq et al [12] explored that reduction of the number of cycles from 23 to 18 did not cause any decrease in the amount of amplified product as compared to the control reaction (without nanoparticles) with 23 cycles, suggesting that as many as five cycles can be reduced in the PCR program without compromising the amount of product through the addition of 0.4 nM TiO₂ nanoparticles in the reaction mixture.

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A number of possible mechanisms was proposed for the enhancement and/or inhibition of NANO-PCR, such as the interaction of DNA polymerase with nanoparticles [34], the binding of DNA and nanoparticles [35], the electrostatic interaction between the positively charged nanomaterials and the negatively charged PCR components [36], the enhanced thermal conductivity effect of nanoparticles [4, 9, 12] and physical absorption between DNA polymerase and nanoparticles [37, 38]. Li et al [9] claim that PCR enhancement might be mainly due to the enhanced heat transfer effect of nanoparticles. In contrast, Cao et al [36] suggest that the PCR enhancement may be greatly affected by the surface charge polarity of the polyethyleneimine (PEI) modified MWCNTs rather than the thermal conductivity of MWCNTs alone. It is interesting to note that the stability of nanofluids is one of the vital parameters for their enhanced thermal conductivity effect [39–41]. Stability can be enhanced by modifying the surface charge or pH of the suspensions (see for example [42] and references therein). Therefore, it should be investigated if the surface charges of PEI-modified MWCNTs have any impact on stability, which may also be the reason for the PCR enhancing effect. The exact mechanism behind the enhancements and inhibitory effects of NANO-PCR is still not clearly understood, and hence needs further investigation. The physical and chemical properties of various nanomaterials differ and hence add complexity in understanding the NANO-PCR mechanisms precisely. Nevertheless, the unique property which almost all nanofluids possess is an enhanced thermal conductivity effect. Similar to the nanofluid thermal conductivity effect, NANO-PCR yield also depends on particle size, volume fraction, base fluid, temperature, etc. Hence, we raise the question of whether NANO-PCR enhancement is effected by the enhanced thermal conductivity of nanomaterials. Thus, we attempt herein to explore the role of the enhanced thermal conductivity effect of nanoparticles in all three PCR steps.

• The hydrogen bonds in the double stranded DNA are broken when the temperature is raised to more than 90 °C during the denaturation step of PCR. Since the entropy increases in the reaction mixture, the random motion of the reagents (template DNA molecules, primers and enzymes) increases. Brownian motion of nanoparticles is considered to be one of the main reasons for enhancements in the thermal conductivity effect of fluids containing nanoparticles. The theoretical model of Jang and Choi [24] shows that, at very small particle size, the Brownian motion becomes vital in describing the energy transport in liquids containing nanoparticles. They constructed a theoretical model based on convection, kinetics and Kapitza resistance, and derived a general expression for the thermal conductivity of nanofluids involving four modes of energy transport in nanofluids. Based on this theory, we assume that the DNA is denatured more rapidly in the presence of nanoparticles as a result of better dissipation of heat in the reaction mixture. This good dissipation of heat may be due to collision between base fluid molecules, nanoparticles and also PCR reagents. Spectroscopic experimental results in the past clearly prove this theory, wherein the absorbance measured during DNA denaturation at 260 nm is higher in the presence of nanoparticles than in the absence of the same [12].
• In the subsequent PCR step the reaction liquid is cooled to ∼55 °C, thus allowing primers to bind with single-strand DNA molecules. Since the random motion of particles slows down with decrease in temperature, the van der Waals forces between GNFs may increase and finally help attract other reagents [7]. The aggregation of reaction components around nanomaterials increases the probability of dynamical contact among reaction components; hence it may result in heat equilibrium in the reaction [43] and enhance PCR efficiency.
• During the final step the extension process is enhanced as a result of the enhanced heating process because of GNF addition.

We also examined the effect of supernatant in order to confirm that the enhancement is purely due to GNFs, and the impurities or additives present in GNFs do not contribute to PCR product augmentation. Following the same procedure as Khaliq et al [12], we obtained the supernatant of GNFs and added it to the reaction mixture accordingly to amplify a region of known genomic DNA with 1248 bp. After the PCR was performed using normal protocol, the product was analyzed using ethidium bromide staining. The agarose gel image clearly depicts that the yield in both cases of positive control and supernatant is almost the same (figure 4(D)). This observation indicates that the augmentation in PCR yield is solely because of GNFs.