A QCM-based ‘on–off’ mechanistic study of gas adsorption by plasmid DNA and DNA–[Bmim][PF₆] construct

Abstract

The study of the adsorption behavior of disease markers such as ammonia (NH₃) and acetaldehyde (CH₃CHO) with biomaterials is important, as it will improve our understanding of their interaction behavior and enable the development of self-diagnosis technologies, among others. In this study, three types of DNA-based biomaterials were synthesized (pGFP plasmid DNA isolated from E. coli DH5α, a DNA–ionic liquid construct (DNA–IL) and DNA–ionic liquid–gold chloride (DNA–IL–Au)) and their adsorption capacities for NH₃ and CH₃CHO were tested by utilizing a gravimetric transducer, namely, a quartz crystal microbalance (QCM). Pristine DNA itself displayed high sensitivity towards both gases, with a pristine DNA-based QCM displaying magnitudes of response of ∼3.74 and 2.62 ng cm⁻² μg⁻¹ following 10 minutes of exposure to 600 ppm NH₃ and CH₃CHO, respectively. Interestingly, no response was observed when these gases were exposed to the DNA–IL complex, which comprised DNA modified with the hydrophobic IL [Bmim][PF₆]. However, when the DNA–IL complex was further treated with HAuCl₄, the biomaterial (DNA–IL–Au) regained its adsorption capacity, exhibiting magnitudes of adsorption/response up to 140% and 36% higher than its DNA counterpart toward NH₃ and CH₃CHO, respectively. It was also observed that the utilization of DNA–IL–Au significantly reduced the sensitivity of the QCM device to humidity content, which indicates that the developed biomaterial can be readily employed to detect NH₃ and CH₃CHO in humid environments. Further study showed that the magnitudes of the QCM response of the DNA and DNA–IL–Au materials toward the different concentrations of NH₃ and CH₃CHO that were tested follow the loading ratio correlation (LRC), which thus indicates that the developed materials can potentially be utilized as sensitive layers for the detection of biomarker gases that are produced in the body as a result of biomedical disorders. In addition, a plausible sorption mechanism has also been proposed on the basis of the interaction of DNA with the ionic liquid and HAuCl₄ (experimentally proved by XPS and FTIR), which strongly indicates the role of the phosphates and nucleobases of DNA for the electrostatic binding of NH₃ and CH₃CHO, respectively.

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1. Introduction

The importance of the non-invasive diagnosis of volatile organic compounds (VOCs) is ever-increasing, given the demand for pollution control, food assessment and biomedical diagnostic technologies.^1–3 Among VOCs, the development of layers that are sensitive to ammonia (NH₃) and acetaldehyde (CH₃CHO) is of special interest, as they exist in the environment (e.g., food, fertilisers, industrial stack effluents, etc.) and as markers for various biomedical disorders.^4–6 The development of such materials will enable the development of reliable, fast and accurate detection platforms. Among the vast number of materials available, DNA has attracted attention for sensing among all its other applications^7,8 since its structure was proposed by Watson and Crick,⁹ and it has emerged as the central point of all biotechnological studies in numerous fields of research. However, until recently the use of DNA and DNA-based hybrid materials has been restricted to liquid-based sensing (e.g., for heavy metals¹⁰ and proteins in blood⁸) and their potential for use as a reliable sensitive layer for VOCs has not been investigated, which is mainly due to a lack of understanding of the interaction of such gas species with biomaterials. An elucidation of the mechanism of the interaction of DNA-based molecules with VOCs is also important, because many such gas species have been proposed as either causing or being produced as a result of one or more of a range of biomedical disorders.^11–13

Acetaldehyde, for example, is listed as a possible carcinogen,¹⁴ and humans are exposed to it through a wide variety of sources such as automobile emissions and tobacco smoke, as well as its production in the human body by the metabolism of vinyl acetate and ethanol in the liver.¹⁵ This molecule is a highly reactive aldehyde that can form adducts with macromolecules including DNA and proteins.¹⁶ In addition to directly forming complexes with DNA, it has also been shown to increase the formation of reactive species from oxidative stress and lipid peroxidation, which has been linked to DNA lesions and ultimately cancer.¹⁷

Ammonia is another example of a toxic gas, which is colorless and has a pungent smell. It is environmentally significant, as many chemical processes involving nitrogen-containing compounds produce ammonia.¹⁸ Furthermore, the detection of ammonia is important in the food and farming industries. From a biomedical point of view, the ammonia concentration in human breath increases when biomedical disorders develop, where the detection of the toxin enables clinical diagnosis. Ammonia is a very important biomarker for the assessment of diseases such as renal failure, H. pylori infection¹⁸ and asthma.¹⁸ Elevated levels of ammonia can be found in the breath of patients (‘fishy’ odour) with these conditions, and hence it can serve as a valuable marker for non-invasive diagnosis.¹⁹ The concentration (ppb-ppm) of ammonia in breath varies with the disease condition. Studies have suggested that the concentration of ammonia in the breath in normal subjects is in the range of approximately 0.15–1.8 ppm.²⁰

Breath tests have a long history in medical diagnostics. Exhaled human breath contains a mixture of nitrogen, oxygen, carbon dioxide, and VOCs. To date, several VOCs have been established as biomarkers for specific diseases or metabolic disorders. In order to measure different VOCs simultaneously, many applications have combined various sensors and materials into a single array, leading to the development of an “electronic nose”.²¹

Sensor technology has been used for many years in clinical testing. Up to now, existing materials have mostly been conductive polymers, semiconducting metal oxides, or a combination of the two.²² Unique sensors based on nanoparticles have appeared as a reliable alternative tool for breath analysis and have proved to be inexpensive and easy to use. Over the years, QCM-based devices have been extensively used for chemical sensing, as well as for studying interactions of analytes with the sensitive layer, owing to their high sensitivity and robust nature.^23,24 A QCM usually utilizes an acoustic wave, which propagates through a piezoelectric material (e.g., AT cut quartz) as a result of the application of an electrical potential to an electrode of the device. The electrodes of a QCM can be modified with a material that is selective for the gas molecules of interest. The interaction of gas molecules and electrode materials results in mass loading on the electrode surface and thus changes the resonant frequency of the device. The concentration of the adsorbed gas can be determined from the change in the resonant frequency of the device and can be calculated using the Sauerbrey equation (eqn (1)).

In this equation, Δf represents the change in the resonant frequency, Δm is the change in mass at the surface, f₀ represents the resonant frequency of the QCM, A is the active area of the QCM electrodes, and ρ and μ are the crystal density and shear modulus of the piezoelectric crystal, respectively.

In this study, it is proposed that we can get a better insight into, and reveal, the plausible mechanisms responsible for the interactions of DNA with common VOCs by utilising a highly sensitive, yet robust, quartz crystal microbalance (QCM) device^25,26 and a recently emerged non-aqueous green ionic liquid solvent.²⁷ Recently, we have presented a detailed biophysical study of DNA and the hydrophobic ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) and established its role in enhancing the efficiency of bacterial gene transformation.²⁸

We realized that DNA without any modifications can also be utilized as a very robust biomaterial for the development of detection platforms for VOCs. Furthermore, an ‘on–off’ kind of response of DNA to VOCs was also studied by utilising molecules such as an ionic liquid and gold chloride. In this study, a detailed experimental investigation enabled us to study the sorption and desorption characteristics of ammonia and acetaldehyde with DNA, based on which a plausible mechanism of the interaction of these tested VOCs with DNA has also been proposed. It is still difficult for a single system to satisfy all the requirements for a robust sensor; however, the utilisation of the versatile biomolecule DNA can open up a wide range of possibilities in this direction. Moreover, these systems are much closer in nature to natural biological assemblies; these might give insights into the complex pathways responsible for the interactions of VOCs in biological systems and help with the understanding of the role of VOCs in the development of various biomedical disorders.

2. Materials and methods

2.1 Chemicals

The ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was purchased from Ionic Liquid Technologies (IoLiTec). Luria broth (LB), CaCl₂, MgCl₂ and all chemicals were purchased from Sigma-Aldrich. E. coli DH5α and a recombinant strain with the pGFP plasmid were obtained from the microbial culture repository, School of Science, Bundoora Campus, RMIT University.

2.2 Isolation of plasmid DNA

A preparation of the pGFP plasmid from E. coli DH5α was obtained based on the alkaline-SDS method. A 50 mL volume of transformed bacterial culture was centrifuged and the cell pellet was suspended in 3 mL TE-glucose [50 mM glucose, 10 mM EDTA, 25 mM Tris–HCl (pH 8.0)]. The suspension was vortexed thoroughly. To this suspension, 8 mL of an alkaline solution of SDS (0.2 N NaOH, 1% SDS) was added and the mixture was mixed and gently incubated for 10 min, following which it was neutralized by adding 8 mL of 3 M potassium acetate (pH 4.8) and then centrifuged at 12 000 rpm for 10 min at 4 °C. The supernatant was treated with chloroform–isoamyl alcohol (24 : 1). The upper layer was collected and to this layer 2 volumes of absolute ethanol was added, following which the mixture was subjected to further centrifugation at 14 000 rpm for 10 min. The pellet was rinsed with 70% ethanol, again centrifuged at the same speed, dried, and then dissolved in 50 μL TE buffer.

2.3 Amalgamation and characterization of plasmid DNA–ionic liquid ([Bmim][PF₆]) complex

A 10 μL volume of plasmid DNA (16 mg mL⁻¹) was mixed with 990 μL [Bmim][PF₆] in a 1.5 mL microcentrifuge and vortexed overnight. The reaction mixture was centrifuged at 1400 rpm for 30 min. The supernatant was removed and washed twice with a mixed solution of ethanol (50%, semi-chilled) and acetonitrile (50%). A gel-like plasmid DNA–[Bmim]⁺ complex was observed on washing with acetonitrile (100%). The gel was dissolved in distilled water and made up to a final concentration of 1 mg mL⁻¹; 5% of the DNA was lost during washing of the excess [Bmim][PF₆] ionic liquid. [Bmim][PF₆] interacted with the surface of DNA, i.e., [Bmim][PF₆] was bound to DNA.

2.4 Material characterization

For FTIR (Fourier transform infrared) spectra, plasmid DNA, [Bmim][PF₆], and the DNA–Bmim⁺ adduct in the aqueous-phase IL were used as received, and the precipitated DNA/DNA–IL/DNA–IL–Au nanoconstruct was dissolved in water (0.1 mg mL⁻¹) and used as a droplet (10 μL) with a PerkinElmer Spectrum 2000 FTIR spectrometer (resolution 4 cm⁻¹) with a universal single-bounce diamond attenuated total reflectance (ATR) attachment. The samples were prepared in deionized water that was filtered using a 0.45 μm Millipore syringe filter and were diluted accordingly to obtain optimal scattering data.

DNA and the DNA–IL nanoconstruct were precipitated out with chilled ethanol, dried, and further suspended in deionized water for drop-casting on a titanium SEM substrate (10 μL of a solution containing the sample). The samples were air-dried and imaged under an electron beam and DNA–[Bmim][AuCl₄] was also analyzed by a FEI Nova Nano-SEM for EDX analysis at an accelerating voltage of 10 kV. The nature of the chemical interaction of DNA with [Bmim][PF₆] and HAuCl₄ was characterized by XPS measurements carried out on a Thermo KAlpha XPS instrument at a pressure of lower than 1 × 10⁻⁹ Torr. The general scan and N 1s, P 2p, O 1s, F 1s, Au 4f and C 1s core-level spectra of the respective samples were recorded using monochromated aluminum Kα radiation (photon energy = 1486.6 eV) at a pass energy of 20 eV and an electron takeoff angle (angle between the electron emission direction and the surface plane) of 90°. The core-level binding energies (BEs) were aligned with the binding energy of adventitious carbon of 285 eV.

2.5 QCM fabrication for adsorption of ammonia and acetaldehyde on QCM

The detailed fabrication method of the QCM was similar to the method used in our previous studies and can be found elsewhere.²⁴ In brief, 100 nm-thick Ni electrodes (along with a 10 nm-thick Ti adhesion layer) were evaporated on both sides of a circular AT cut quartz substrate with a diameter of 7.5 mm. The circular shape of the electrodes (diameter: 4.5 mm) was obtained by utilizing a shadow mask. All the fabricated QCM devices displayed a resonance frequency of ∼10 MHz. Then, DNA, DNA–IL and DNA–IL–Au were drop-cast on one of the electrodes of separate QCM devices. The masses of DNA, DNA–IL and DNA–IL–Au deposited on the QCM electrodes were 13.3, 6.3 and 7 μg, respectively, which were estimated by comparing the Q factor of the developed QCMs before and after depositing the developed materials on the QCM electrodes. The three QCM devices with the DNA, DNA–IL and DNA–IL–Au materials were then placed in a secure cell that had a volume of ∼100 mL. A constant temperature of 30 °C and a flow rate of 200 sccm were maintained for different tests. NH₃ and CH₃CHO were supplied from two different cylinders and their concentrations were adjusted by manipulating different mixing ratios of dry N₂. The concentrations utilized for NH₃ were 120, 240, 360, 480 and 600 ppm, whereas 300, 600, 900, 1200 and 1500 ppm CH₃CHO were utilized for the different experiments. The exposure of the developed QCM devices to NH₃ and CH₃CHO was carried out for 10 minutes in each particular case, following which they were flushed with dry N₂ for 50 minutes. Purging with dry N₂ enabled the QCM devices to desorb all the VOCs adsorbed during the exposure and thus allowed them to recover/regenerate. Two Maxtek RQCM devices were utilized in order to initiate the oscillation of the QCM devices, as well as to track changes in their resonance frequency during the gas exposure tests.

3. Results and discussion

In brief, plasmid DNA (DNA) was purified from E. coli and interacted with a hydrophobic ionic liquid ([Bmim][PF₆], Fig. S3†) (hereafter referred to as DNA–IL). Furthermore, DNA–IL was used after its interaction with HAuCl₄ (hereafter referred to as DNA–IL–Au). All three samples that were developed were drop-cast on separate QCM substrates and all three modified QCM substrates were further utilised for the adsorption of NH₃ and CH₃CHO.

3.1 Synthesis and characterization of DNA, DNA–IL and DNA–IL–Au

The functional nanostructures of DNA–IL, DNA–IL–Au and pristine DNA were characterized by multiple techniques including SEM and EDAX, as shown in Fig. 1 and 2, respectively.

Fig. 1 SEM images of (a) pristine DNA, (b) the DNA–ionic liquid (Bmim⁺) construct and (c) the DNA–[Bmim][AuCl₄] complex. A and B are images of different areas in the same sample for each material (a, b and c).

Fig. 2 EDAX analysis of DNA–[Bmim][AuCl₄] complex. The peaks of Ti and Si originated from the substrate.

3.2 SEM and EDAX analysis of the DNA–IL functional nanostructures

SEM imaging of plasmid DNA after its interaction with IL and of DNA–IL with gold chloride was performed to study the morphological changes, and the images are presented in Fig. 1.

Fig. 1a and b show the surface morphologies of the DNA and DNA–IL nanostructures, respectively. They appear rather larger than they appeared under TEM, as we reported in our earlier work.²⁸ The average diameter of plasmid DNA in the solid phase was about 20–30 nm, and after interaction with IL the average diameter of the DNA–IL nanostructures increased to 50–60 nm, respectively.²⁸ These results support the fact that the incorporation of IL molecules on the surface of DNA was responsible for the increase in the size of the structures, as observed in the SEM images. We also observed some gel-like micro-bodies (comprising a super-assembly of DNA and ionic liquid) in the sample, which ranged from 1 to 6 μm in size (Fig. 1b-A). A photographic image of pristine DNA (precipitated by chilled ethanol) and DNA gel is shown in Fig. S1.† We further observed some patches of fractal microstructures of pristine DNA (Fig. 1a-A), which might have originated because of the self-assembly of purified plasmid DNA owing to the presence of salt in the solution.

After the interaction of gold chloride with DNA–IL the salt [Bmim][AuCl₄] was visible as separate flat patches (Fig. 1c-A) and the rest of the sample appeared different, which we suggest is precipitated pristine DNA (Fig. 1c-B).

The EDAX analysis of DNA–IL–Au is shown in Fig. 2, which clearly shows the presence of F and Au in the sample, which is an indication of the presence of the ionic liquid and gold salt in the sample. The peaks of Si and Ti originated from the substrate.

3.3 XPS analysis

XPS analysis of the pristine plasmid DNA, DNA–IL, and DNA–IL–Au nanostructures was further carried out to investigate the underlying chemical interactions. Fig. 3 shows the C 1s, N 1s, O 1s, P 2p, F 1s, and Au 4f core-level spectra recorded from the plasmid DNA, DNA–IL and DNA–IL–Au nanostructures (deconvolved spectra showing the raw peaks are shown in Fig. S2†). In the case of plasmid DNA, the P 2p binding energy (BE) observed at 133.7 eV was attributed to phosphorus present in the phosphate groups of DNA. In contrast, the DNA–IL nanostructures displayed two chemically distinct P 2p components that were observed at 133.2 eV and 136.4 eV, which were assigned to phosphorus atoms present in the phosphate groups of DNA and hexafluorophosphate groups from the ionic liquid [Bmim][PF₆], respectively. However, the DNA–IL–Au nanostructures exhibited only one P 2p component that was observed at 133.2 eV, which was assigned to the phosphate groups of DNA, and the P 2p component that corresponded to [PF₆] groups was absent. This was probably due to the replacement of anionic [PF₆]⁻ in the DNA–IL nanostructures by [AuCl₄]⁻, as the gold salt is well known to undergo strong interactions with the cationic part of the ionic liquid, i.e., Bmim⁺. The results indicate that HAuCl₄ forms a strong salt with Bmim⁺ and that the ionic liquid can then be separated from the DNA–IL complex. This indicates that the Bmim⁺ cation neither exhibited a strong interaction with DNA nor formed an ion pair with DNA; instead, it displayed a weak electrostatic interaction with the phosphate groups of DNA. Therefore, gas molecules that have properties that resemble those of [Bmim]⁺ will be expected to exhibit less interaction with DNA when IL is in a complex with anionic DNA. This is because the specific sites would previously have been occupied by the [Bmim]⁺ cation present in IL.

Fig. 3 XPS core-level spectra recorded from DNA, DNA–IL and DNA–[Bmim][AuCl₄].

In the case of pure plasmid DNA, the O 1s core-level spectrum was resolved into two main components centred at 531.0 eV and 532.3 eV, which were assigned to oxygen atoms present in the phosphate and deoxyribose sugar groups, respectively. However, the O 1s spectra obtained from the DNA–IL and DNA–IL–Au nanostructures were significantly different from the O 1s spectrum from plasmid DNA. Because the IL [Bmim][PF₆] does not contain any functional groups containing oxygen, the changes observed in the cases of the O 1s spectra from the DNA–IL and DNA–IL–Au nanostructures were the direct consequences of different chemical interactions in proximity to the phosphate or sugar units in DNA. The relative intensities of the O 1s component arising from the sugars [BE 532.3 eV] and that from the phosphate oxygens [BE 531.0 eV] were found to be different in the cases of the DNA–IL and DNA–IL–Au nanostructures with respect to that of pure DNA. In the case of DNA–IL, the intensity of the O 1s peak corresponding to phosphate oxygen increased and the width of the peak also increased considerably. This indicates that phosphate oxygen could be the preferred binding site for the cationic part of the ionic liquid [Bmim]⁺.

The N 1s core-level spectra obtained from the DNA, DNA–IL and DNA–IL–Au nanostructures revealed more information regarding the interactions between DNA, IL and the gold salt. Plasmid DNA exhibited two chemically distinct N 1s components, of which the binding energies were observed at 399.1 and 401.2 eV, which were attributed to two different kinds of nucleobases present in DNA. Similarly to DNA, the DNA–IL nanostructures exhibited two chemically distinct N 1s components with the same binding energies. This may be due to the chemical resemblance of the [Bmim]⁺ cation and the nucleobases.

In contrast, the DNA–IL–Au nanostructure exhibited two chemically distinct N 1s components with different relative intensities. The component with a high BE observed at 401.4 eV was more intense than that of plasmid DNA. This was probably due to the preferred interaction of nucleobases with the gold salt. Thus, it was clearly indicated that owing to the strong electrostatic interaction between DNA and [Bmim][PF₆] the complex DNA–Bmim⁺ was formed, which did not affect the B structure of DNA, and DNA–IL also retained its B conformation after interacting with gold chloride. The presence of a F 1s component in the fluorine core-level spectra was observed in the case of both DNA–IL (BE 686.3 eV) and DNA–IL–Au (BE 685.5 eV) nanostructures. This is a clear indication that these nanostructures consist of PF₆ units of IL. The Au 4f core-level spectra contain a spin–orbit pair at 84.9 and 88.6 eV that corresponds to the Au(I) oxidation state. No peak corresponding to metallic gold (Au⁰) was observed. This clearly shows that Bmim⁺ formed a complex with HAuCl₄ and precipitated out as the [Bmim][AuCl₄] complex. Therefore, the binding sites of DNA, i.e., phosphates and nucleobases, seem to be available, and it would be interesting to find out if the gas adsorption behaviour of the regenerated DNA remains the same as that of pristine DNA when it is exposed to ammonia and acetaldehyde.

3.4 FTIR studies of DNA, DNA–IL and DNA–IL–Au

In order to obtain clearer evidence regarding the interaction of DNA and IL, FTIR spectral analysis of the developed materials was performed. As IL and DNA have distinct vibrational features, any changes observed in the FTIR spectrum can be correlated to the interaction of the functional groups. The FTIR spectra of plasmid DNA, the DNA–IL nanostructure and DNA–IL–Au are shown in Fig. 4 (curves a–c, respectively). On the basis of the XPS studies, it was deduced that [Bmim]⁺ preferentially interacts with DNA and gold chloride; however, FTIR analysis of these materials provided further insight into the interaction of the functional groups. In the FTIR spectra, the symmetric stretching of P–O bonds in the phosphate group observed at 1045 cm⁻¹ was assigned to the B form of DNA.²⁹ Thus, the unchanged symmetric stretching vibrational band at 1045 cm⁻¹ suggested that this kind of interaction occurred only on the surface of DNA molecules, which did not alter the helical structure of DNA (B form).

Fig. 4 FTIR spectra of (a) pristine DNA, (b) the DNA–ionic liquid (Bmim⁺) construct and (c) the DNA–[Bmim][AuCl₄] complex.

This shows that the B-form conformation of DNA was retained within the DNA–IL nanostructure. Studies have already shown that cationic species such as metal ions can strongly bind to DNA phosphate groups and change their conformation, which eventually results in changes in the position and intensity of the bands.^30–32 Furthermore, one recent study reported that the conformation of the B form of DNA did not change when it coexisted with the Bmim cation in an ionic liquid.³³ In the case of the DNA–IL nanostructure, the symmetric stretching vibrational band of P–O at 1045 cm⁻¹ was almost unaffected (Fig. 4 curves b and c); however, the band at 1084 cm⁻¹ in Fig. 4 curve b became very prominent (observed as a small hump in curve a). The asymmetric P–O vibration observed at 1084 cm⁻¹ may be due to interactions with Bmim⁺.³⁴ The intense peak at 1180 cm⁻¹ was assigned to the asymmetric stretching vibration of Bmim rings in the pure ionic liquid [Bmim][PF₆]²⁸ and was observed in both curve b and curve c. This peak was absent in the case of pristine DNA, as shown in Fig. 4 curve a.

Therefore, FTIR spectral analysis revealed that the DNA–IL nanostructure was formed as a consequence of the electrostatic interaction between Bmim groups and DNA molecules and that this interaction might be responsible for the formation of the DNA–IL complex. The peak at 1180 cm⁻¹ in curve c (DNA–IL–Au) might arise from the [Bmim][AuCl₄] salt and, as it is not masked and interfered with by other functional groups of the large macromolecule DNA, we observed a very prominent peak. In curve c, we also observed three new peaks at ∼1285, ∼1445 and ∼1575 cm⁻¹ (in-plane ring modes of –C=C–), which correspond to the [Bmim] cation of the ionic liquid. The peaks in the range from 2800 to 3000 cm⁻¹ originate from CH_x stretching vibrations of the butyl chain and the methyl group attached to the imidazolium ring.³⁵ The features above 3000 cm⁻¹ arise from the CH_x stretching vibrational modes of the imidazolium ring.

For [Bmim]⁺, the peaks at 2870, 2939 and 2965 cm⁻¹ are attributed to the ν_ssCH₃, ν_FRCH₃ and ν_asCH₃ modes, respectively (Table S1†) (ss, as, and FR represent symmetric stretching, antisymmetric stretching, and Fermi resonance, respectively).^36,37 The peaks at a wavenumber of 2870 cm⁻¹ are assigned to aliphatic asymmetric and symmetric (C–H) stretching vibrations and are due to the presence of methyl groups. The peaks observed at 1635 cm⁻¹ and 1600 cm⁻¹ are due to C=C and C=N stretching, whereas the peak at a wavenumber of 840 cm⁻¹ is attributed to C–N stretching vibrations.³⁸

3.5 Adsorption and detection of ammonia and acetaldehyde on DNA, DNA–IL and DNA–IL–Au on QCM substrates

In order to determine the effect of the treatment of DNA with IL and the Au salt, the developed DNA, DNA–IL and DNA–IL–Au were deposited on separate QCM devices and exposed to five different concentrations of NH₃ (120–600 ppm) and CH₃CHO (300–1500 ppm) at an operating temperature of 30 °C. The concentration range of each gas species was chosen based on common levels found in an industrial stack, whereas the set temperature was the lowest that could be controlled with minimum fluctuations using the calibration system built in-house. This experiment enabled the analysis of the adsorption and desorption behavior of the exposed gas species on the developed biomaterials. On exposure to different concentrations of NH₃/CH₃CHO, the QCMs exhibited a shift in their resonance frequency owing to mass loading on the electrode surface (eqn (1)). The amount of NH₃/CH₃CHO molecules adsorbed on different biomaterials was then determined by using eqn (2).

The QCMs used in this study had a sensitivity of 4.39 ng cm⁻² (calculated from eqn (1)), whereas the masses of DNA, DNA–IL and DNA–IL–Au deposited on the QCM electrodes were 13.3, 6.3 and 7 μg, respectively (Materials and methods section, under ESI†).

Fig. 5a–c show the dynamic response to different concentrations of NH₃ exposed to QCMs based on DNA, DNA–IL and DNA–IL–Au, respectively. It can be observed that the QCM based on DNA displayed responses of between 1.87 and 3.74 ng cm⁻² μg⁻¹, whereas the QCM based on DNA–IL–Au exhibited responses of 4.49–5.05 ng cm⁻² μg⁻¹ on 10 minutes of exposure to different NH₃ concentrations. However, the QCM device based on DNA–IL did not respond to NH₃. It can be concluded that the addition of IL removed the capacity of DNA to adsorb NH₃; however, the further addition of HAuCl₄ led DNA to adsorb an increased amount of NH₃. This was due to the strong affinity of the [AuCl₄]⁻ ion for the [Bmim]⁺ ion, which resulted in the [Bmim][AuCl₄]^41,42 salt (see the XPS spectra shown in Fig. 3 and the related discussion). The [PF₆]⁻ ion that was released on the treated DNA is postulated to interact with ammonia, given that such interactions have been found to be dominant in electrochemical,⁴⁵ Raman⁴⁶ and NMR-based⁴⁶ studies reported in the past. This interaction may be the reason for the increased adsorption of NH₃ on the developed DNA–IL–Au observed in this study. The interaction between NH₃ and the developed material is electrostatic, so a large portion of the NH₃ species was observed to be desorbed from DNA–IL–Au by exposure to dry N₂.

Fig. 5 Dynamic responses of QCMs based on (a) DNA, (b) DNA–IL and (c) DNA–IL–Au when exposed to 120–600 ppm NH₃ and (d) LRC fit of the calibration curves of DNA and DNA–IL–Au devices.

Owing to the formation of the aforementioned salt [Bmim][AuCl₄], DNA was regenerated and exhibited a response to ammonia. As shown in Fig. 5d, the magnitudes of the response for different NH₃ concentrations follow the loading ratio correlation (LRC) (also known as the Langmuir extension isotherm), with the DNA-based device showing a much better fit (R² = 0.989) than its DNA–IL–Au counterpart (R² = 0.828). This indicates that the magnitudes of the response of both DNA and DNA–IL–Au are dependent on the NH₃ concentration and that these materials possess the ability to act as a sensitive material toward this gas. Another observation can be made from the calibration curves, namely, that the magnitude of the response of the DNA-NP device became saturated upon exposure to NH₃ at above 240 ppm, which was not the case for the pristine DNA device. This indicates that treatment with the Au salt could be more suitable for detecting very low concentrations of NH₃. The magnitude of the response and the concentration of exposed NH₃ can be related as defined in eqn (3):

where Δf is the magnitude of the response of the device, [VOC] represents the concentration of the exposed vapor and a, b, and c are LRC model constants.

Fig. 6a–c show the responses of QCMs based on DNA, DNA–IL and DNA–IL–Au to 300–1500 ppm CH₃CHO, respectively. The DNA device exhibited magnitudes of response of 1.80–4.33 ng cm⁻² μg⁻¹ for the different concentrations of CH₃CHO that were tested, whereas increased magnitudes of response of 2.42–5.59 ng cm⁻² μg⁻¹ were observed for the QCM based on DNA–IL–Au for the same concentrations of CH₃CHO. However, the QCM based on DNA–IL did not display any sensitivity to the different concentrations of CH₃CHO that were tested. The results indicate that the addition of IL also blocked the CH₃CHO adsorption sites of DNA; however, the further addition of the Au salt to DNA–IL resulted in an increased adsorption capacity of DNA for CH₃CHO, to the extent that a 10 minute exposure period did not result in the saturation stage being reached, as was the case with NH₃. It can be observed from Fig. 6d that the magnitudes of response for exposure to different concentrations of CH₃CHO fitted well with the LRC for both DNA and DNA–IL–Au and the magnitudes of response of the devices can be related to the CH₃CHO concentration by utilizing eqn (3). This is also an indication of the potential of the developed biomaterials to be utilized as sensitive layers for transducer-based CH₃CHO detection applications.

Fig. 6 Dynamic response of QCMs based on (a) DNA, (b) DNA–IL and (c) DNA–IL–Au when exposed to 300–1500 ppm CH₃CHO and (d) LRC fit of the calibration curves of DNA and DNA–IL–Au devices.

In general, it was observed that both DNA and DNA–IL–Au possessed higher sensitivity to NH₃ than to CH₃CHO. For example, QCMs based on both DNA and DNA–IL–Au adsorbed 45% more NH₃ than CH₃CHO when 600 ppm of each gas species was exposed to each material. This increase in the sorption of NH₃ may be due to the interaction of ammonia with free [PF₆]⁻ ions.

Further experiments were carried out in order to investigate the effect of humidity on the developed QCM devices based on DNA, DNA–IL and DNA–IL–Au. As shown in Fig. 7a, none of the QCM devices exhibited a repeatable response when exposed to 15 300 ppm_v humidity at room temperature (equivalent to a relative humidity of 50%). It can also be observed that the presence of humidity content produced significant cross-interference with the response of the QCMs. Furthermore, the response profile/magnitude of the sensors was not consistent for different events of exposure to humidity, regardless of the integrated biomaterials. Further tests were carried out to study the effect of humidity content on the response of the QCMs to NH₃ and CH₃CHO. Fig. 7b and c show the response of QCMs based on DNA and DNA–IL–Au to a mixture of NH₃ and CH₃CHO in dry and humid conditions, respectively. It can be concluded that the introduction of humidity content significantly increased the cross-sensitivity effect on the response of the QCM sensors to NH₃ and CH₃CHO, thus inhibiting the detection of NH₃ and CH₃CHO in the presence of humidity content. However, the effect of humidity may be reduced by increasing the operating temperature, as humidity tends to undergo relatively less interaction with the sensitive layer owing to an increase in vapor pressure. Alternatively, the humidity content can be filtered from the sample gas in order to avoid the cross-sensitivity effect it induces on the sensitivity performance of the developed DNA-based biomaterials.

Fig. 7 Response of QCMs based on DNA, DNA–IL and DNA–IL–Au to (a) humidity (15 300 ppm_v, equivalent to a relative humidity of 50% at room temperature) and a mixture of NH₃ and CH₃CHO (b) without and (c) with the presence of humidity content.

3.6 Plausible mechanism of adsorption of ammonia and acetaldehyde on DNA, DNA–IL and DNA–IL–Au

A plausible mechanism of the adsorption of gases on DNA based on the aforementioned experimental results is shown in Scheme 1. Both ammonia and acetaldehyde displayed good adsorption behaviour toward pristine DNA. As is well known in the literature, ammonia is a positively charged molecule and is thought to have interacted with negatively charged oxygen atoms in the phosphate groups of DNA. On the other hand, acetaldehyde is slightly acidic in nature (and its carbonyl oxygen atom is partially negatively charged), which may have resulted in interactions with the exocyclic amino groups of nucleobases of DNA.^39,40 However, when DNA was treated with the ionic liquid ([Bmim][PF₆]), the [Bmim]⁺ cation could interact electrostatically with the negatively charged phosphate groups and thus have resulted in the blocking of molecular sites where ammonia can undergo adsorption. Similarly, the ionic liquid would have covered DNA, which would have sterically hindered acetaldehyde from interacting with nucleobases of DNA; hence, no adsorption of gas was observed. It is also known that the ionic liquid [Bmim][PF₆] interacts well with gold chloride (HAuCl₄) and forms the stable salt [Bmim][AuCl₄].^41,42 Therefore, it was postulated that the treatment of DNA–IL with the Au salt would regenerate the DNA molecules, rendering them free from the ionic liquid. As such, when the DNA–IL construct interacted with gold chloride, DNA was in fact regenerated and freed from the ionic liquid, with the abovementioned salt complex being precipitated out (see Fig. 3 (XPS) and the associated discussion) of the reaction mixture. A regenerated DNA molecule should have been free from any external agents and therefore have regained its affinity for the gases of interest (NH₃ and CH₃CHO). Indeed, when the DNA–IL construct treated with the Au salt was tested for its gas adsorption behaviour, as was theoretically expected, this system had regained (see Fig. 5 and 6) its adsorption ability toward both gas species (140% and 36% higher magnitudes of adsorption response than its DNA counterpart for exposure to different concentrations of NH₃ (120–600 ppm) and CH₃CHO (300–1500 ppm), respectively), and this increase is attributed to the creation of more adsorption sites from different components, including the [Bmim][AuCl₄] salt, [PF₆]⁻ ion⁴³ and IL.⁴⁴ Hence, a gas adsorption system based on a biomacromolecule (DNA) and a QCM that can be switched on and off by employing a simple one-pot chemical treatment approach is proposed.

Scheme 1 Plausible mechanism of the interaction of the gases and biomolecule (DNA) interaction of ammonia and acetaldehyde with DNA, DNA–IL and DNA–IL–Au complex. Nucleobases and phosphates are the preferred sites of interaction of acetaldehyde and ammonia, respectively. Once DNA is treated with IL, the sorption sites were masked by the ionic liquid and no gas adsorption of any species was observed. Thereafter, when DNA–IL was treated with the Au salt, a complex was formed between the salt and the [Bmim]⁺ ion (shown by DFT calculations in Fig. 8), thereby regenerating DNA for gas sorption again.

3.7 DFT calculation of interaction of ionic liquid [Bmim]⁺ with gold chloride [AuCl₄]⁻

In order to show that [Bmim] forms a complex with [AuCl₄], DFT calculations were further performed to determine the lowest-energy configuration of the [Bmim][AuCl₄] ion pair. The gas-phase interactions of the [Bmim] cation with the [AuCl₄] anion were calculated using the DMol module implemented in the Materials Studio package.³⁷ For each ion pair, different initial conformers were chosen in such a way that the more positive atom of the cation was kept close to the more negative atom of the anion and then each conformer was geometrically optimized. The PW91 functional with the double-ξ numerical polarization (DNP) basis set was adopted, which is comparable to the 6-31G(d,p) Gaussian-type basis set. The DNP basis set incorporates d-type polarization into heavier atoms and p-type polarization into hydrogen atoms. The lowest-energy configuration of [Bmim][AuCl₄] shown in Fig. 8 confirms that the gold salt forms a complex with [Bmim]⁺ ions and removes them from the DNA–IL construct, thereby confirming the mechanism presented in Scheme 1.

Fig. 8 DFT-optimised structure of [Bmim][AuCl₄] salt.

4. Conclusions

In summary, the interaction of ammonia and acetaldehyde with the developed DNA materials was studied using the well-established quartz crystal microbalance (QCM) technique. The DNA materials consisted of pristine DNA, DNA treated with an ionic liquid (DNA–IL) and the latter treated with a Au salt (DNA–IL–Au). It was found that pristine DNA had excellent affinity for NH₃ and CH₃CHO vapours; however, the treatment of DNA with IL inhibited its gas sorption behaviour. Interestingly, when DNA–IL was further treated with a Au salt, it was found that the affinity of DNA–IL–Au for the tested gas molecules was regained. Further characterization of the materials was performed to understand this behaviour. The results indicated that the negatively charged phosphorus moieties of DNA played a major role in gas adsorption, and this can potentially be tuned to develop DNA molecules by modification with zwitterionic molecules (the positively charged end can bind to DNA and the negatively charged end is still free to bind to positively charged VOCs) for chemical sensing applications in air. This simple strategy can be a gateway for the further development of robust biomolecule-based biosensing. Furthermore, the sensing platform can also be altered from a QCM to surface acoustic wave or conductometric devices (depending on the application) for the detection of toxic VOCs. It would be interesting to investigate the role of various other ionic liquids, biomolecules and organic ligands, which can provide selectivity for the adsorption of VOCs, to develop this system into an effective sensor.

Author contributions

The manuscript was written through contributions of all authors (SKS, KMMK, RB, VEC, SS, YS and SKB). All authors have given approval to the final version of the manuscript.

Funding sources

Department of Education, Govt. of Australia and Australian Research Council Endeavour research award to SKS and SS, and DECRA fellowship to RB, respectively. Higher Degree by Research Publications Grant (HDRPG) from RMIT's College of Science, Engineering and Health (SEH) to KMMK.

Abbreviations

IL	Ionic liquid
VOC	Volatile organic compound
XPS	X-ray photoelectron spectroscopy
FTIR	Fourier transform infrared

Acknowledgements

S. K. S. thanks the Australian Government, Department of Education, for an Endeavour Research Award and the School of Sciences, RMIT University, for an ECR startup grant. We duly acknowledge the RMIT Microscopy and Microanalysis Facility (RMMF) for providing access to their instruments used in this study. R. B. acknowledges the CSIRO scientific computing facilities and Australian Research Council for the DECRA fellowship. ​KMMK acknowledges RMIT's College of Science, Engineering and Health (SEH) for their financial support through the Higher Degree by Research Publications Grant (HDRPG).

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Footnotes

† Electronic supplementary information (ESI) available: Photographic image of DNA–IL gel, deconvolved XPS spectra, molecular structure of [Bmim][PF₆] and FTIR peak table. See DOI: 10.1039/c6ra14759c

‡ SKS and KMMK contributed equally.

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