Scalable approach for the production of functional DNA ... - CiteSeerX

Journal of Membrane Science 492 (2015) 528–535

Contents lists available at ScienceDirect

Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci

Scalable approach for the production of functional DNA based gold nanoprobes Bruno Veigas a,b, Carla Portugal c,nn, Rita Valério c, Elvira Fortunato b, João G. Crespo c, Pedro V. Baptista a,n a

CIGMH, UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal CENIMAT/I3N, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal c LAQV, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal b

art ic l e i nf o

a b s t r a c t

Article history: Received 9 April 2015 Received in revised form 3 June 2015 Accepted 15 June 2015 Available online 25 June 2015

Nanoparticle based systems, in particular gold nanoparticles (AuNPs), provide for simple colorimetric detection of molecular biomarkers, such as DNA, RNA. These systems rely on the functionalization of AuNPs with ssDNA oligonucleotides requiring strenuous laboratory centrifugation steps not compatible with industrial scale up. Here, we demonstrate the potential of dia-ultrafiltration for purification of Aunanoprobes. We show that dia-ultrafiltration can be regarded as better alternative to centrifugation, allowing for a less intensive sample manipulation, easier transposable to the industrial scale. The purification of AuNPs was performed by dia-ultrafiltration using membranes of regenerated cellulose with a nominal molecular weight cut-off (MWCO) of 10 kDa and a processing strategy which combined subsequent AuNPs cleaning and concentration steps. Instead of the permeation flux decline typically found in ultrafiltration processes operated under concentration modes, purification of Au-nanoprobes by diaultrafiltration was followed by a subtle increase of the permeation fluxes. This effect was ascribed to improved external mass transfer conditions near the membrane surface, prompted by the decrease of the overall solute concentration in the retentate over the process time. This strategy allowed for the total retention of the AuNPS, yielding nanoprobes capable of higher signal to noise ratios. Proof-of-concept was directed at the synthesis of Au-nanoprobes for identification of members of the Mycobacterium tuberculosis complex that cause tuberculosis in humans. & 2015 Elsevier B.V. All rights reserved.

Keywords: Gold nanoparticles Tuberculosis Diagnostics Dia-ultrafiltration Membrane processes

1. Introduction Amongst the plethora of nanotechnology-based approaches, gold nanoparticles (AuNPs) have allowed for the development of new strategies and platforms with increased versatility [1–5]. Several DNA detection schemes based upon the hybridization of target DNA molecules to gold nanoparticle probes have been developed. This detection approach takes advantage of a colorimetric change associated with particle aggregation [6–18]. Purification remains a significant challenge in the preparation of stable ssDNA-modified nanoparticles (Au-nanoprobes) for molecular diagnostics [19,20]. Centrifugation or ultracentrifugation n Correspondence to: Nanomedicine@FCT, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal. nn Correspondence to: LAQV, REQUIMTE, Departamento de Quimica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829516 Caparica, Portugal. E-mail addresses: [email protected] (C. Portugal), [email protected] (P.V. Baptista).

http://dx.doi.org/10.1016/j.memsci.2015.06.042 0376-7388/& 2015 Elsevier B.V. All rights reserved.

techniques are commonly used for removal of excess reagents that may hinder effective molecular recognition, for example nanoprobes may remain contaminated with precursor molecules (e.g. salts and free ssDNA ligands) [20]. In addition, conventional purification methods do not address the need to reasonably prepare large quantities of Au-nanoprobes in scale up production [20]. In this respect, membrane separation appears as alternative purification method offering process simplicity, minimal sample manipulation requirements, higher throughputs and an easier scale up. The efficiency of membrane based processes for impurity removal from micro/nanoparticles suspensions results from an optimal combination of operating variables, such as membrane pore size, chemical affinity of solutes to the membrane surface, i.e. solute-membrane interactions, and fluid dynamic conditions. Successful use of pressure-driven membrane processes has been limited by their short term performances generated by formation of solute concentration gradients towards the membrane surface (polarization of concentration effects) and their successive adsorption at the membrane matrix, ultimately leading to membrane fouling. Dia-ultrafiltration operation strategies have shown to be

B. Veigas et al. / Journal of Membrane Science 492 (2015) 528–535

an advantageous alternative to conventional membrane filtration processes, operated under concentration operation modes. In contrast to these, in dia-ultrafiltration methods permeable solutes are washed out from retentate solution by permanent or intermittent addition of a washing solvent. The maintenance of a constant retentate volume along permeation time avoids the concentration of rejected solutes at the membrane surface, reducing polarization of concentration effects and thus facilitating the removal of unwanted solutes (e.g. solution contaminants). In particular, dia-ultrafiltration favors the removal of partially bound solutes due to binding equilibrium shifts promoted by successive dilutions of retentate [21]. In addition, dia-ultrafiltration offers the possibility of a single step purification and simultaneous adjustment of the retentate media composition to the desired conditions, by buffer exchange and/or desalting. Here, Au-nanoprobe purification was attained by dead-end diaultrafiltration with simultaneous exchange of buffer conditions. The efficiency of Au-nanoprobe purification by dia-ultrafiltration is evaluated based on the ability to retain Au-nanoprobes and remove oligonucleotides and compared to standard centrifugation process by assessing stability, sensitivity and specificity of the purified probes. For the first time dia-ultrafiltration is used to produce bioactive and biocompatible DNA-modified AuNPs systems, in a scalable production approach, preserving high probe sensitivity and specificity.

2. Experimental 2.1. Materials All reagents were purchased from Sigma Aldrich and were of analytical grade. HPLC purified labeled oligonucleotides were purchased from STABVIDA (Portugal) and used without further purification. Thiolated oligonucleotides were used to synthesize the Au-nanoprobes and non-modified oligonucleotides used as specific controls for assay calibration. Regenerated cellulose membranes with a nominal molecular weight cut-off of 10 kDa from Merck Millipore (USA) were used in the dia-ultrafiltration experiments. 2.2. Biological samples Clinical isolates obtained from respiratory samples positive for acid-fast bacilli (BAAR) from patients of the Lisbon Health Region were used as Tuberculosis positive control (M. tuberculosis H37RvATCC27294T). 2.3. PCR amplification A conserved region of MTBC beta-subunit of RNA polymerase (rpoB) gene was amplified in a single PCR reaction (Supplementary data Table S1) yielding a 395 bp fragment [9]. PCR amplifications s were performed on a Biometra TGradient Thermocycler (Göttingen, Germany) in 50 mL final volume with 1x DreamTaq Buffer, 0.1 mM of each DNTPs, 2 mM of each primer and 0.1 U/mL of DreamTaq DNA polymerase (Amersham Biosciences, GE Healthcare, Europe) and
1 mg/mL of template DNA with the following thermal cycling conditions: initial 5 min denaturation at 95 °C, followed by 30 amplification cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, elongation at 72 °C for 45 s, and a final elongation at 72 °C for 5 min. PCR products were analyzed in a 1% agarose gel electrophoresis.

529

2.4. Au-nanoprobe design and synthesis The Au-nanoprobe was designed to allow detection of a sequence common to all MTBC members [9]. AuNPs with an average diameter of
14 nm were synthesized and functionalized as described by Veigas et al. [9]. Briefly, thiol-modified oligonucleotides were incubated with the AuNPs with increasing salt concentration for 16 h at room temperature. Then, one third of the volume solution was purified with 4 steps of centrifugation at 21,000 g, for 45 min, the resulting pellet resuspended in 10 mM phosphate buffer (pH 8), 0.1 M NaCl. The Au-nanoprobe solution was stored in the dark at 4 °C till further use. 2.5. Au-nanoprobe purification by dia-ultrafiltration Au-nanoprobes were purified through dia-ultrafiltration using regenerated cellulose membranes with a nominal molecular weight cut-off (MWCO) of 10 kDa. Dia-ultrafiltration was performed in a pressurized dead-end permeation system (MetCell, UK) operated at constant transmembrane pressure (TMP) of 0.1 bar and at a constant stirring rate of 200 rpm. Dia-ultrafiltration was operated at smooth shear stress conditions in order to allow a gentle processing of AuNPs, thus avoiding potential damaging of AuNPs by mechanical stress. A low TMP was used in order to minimize membrane fouling, thus to facilitate the permeation of free oligonucleotides and other possible contaminants, while extending the process performance. The transmembrane pressure was achieved by cell pressurization with an inert gas (argon) stream and monitored by a monometer placed at the permeation cell inlet (Fig. 1A). To guarantee a good structural stability, each membrane was permeated with deionised water by progressive increase of TMP up to 0.5 bar before each purification process. This procedure allowed for membrane structural compaction, preventing structural changes during Au-nanoprobe purification. Furthermore, membrane hydraulic permeability was determined before and after each purification step to evaluate membrane fouling. Au-nanoprobe feed samples (3 mL) were prediluted with 10 mM phosphate buffer solution at pH 8 to a final volume of 70 mL. This pre-dilution facilitates permeation of probe bound impurities by displacement of impurity binding equilibrium towards the increase of the concentration of non-bound impurities in retentate solution. The purification process was conducted into consecutive cleaning and concentration stages to restore the initial Au-nanoprobe concentration. Along the cleaning stage, the permeation process was conducted in a diafiltration regime, which involved the continuous pumping (Fig. 1A) of 10 mM phosphate buffer solution at pH 8, which was used as the washing solvent, to the retentate, in order to maintain the retentate volume constant, thus avoiding solute concentration along the process. This condition was set, keeping the washing solvent and permeation flow rates equal. The cleaning stage was concluded upon the addition of 3 dia-volumes of washing solvent, corresponding to three fold of the processing feed/retentate volume. 10 mM phosphate buffer at pH 8 was used as washing solution, in order to avoid changes of salt concentration and pH conditions of the feed. After the cleaning stage, the addition of washing solvent was stopped and permeation carried on in concentration mode. Au-nanoprobe concentration in the retentate solution was increased to restore the sample volume before pre-dilution. Both, cleaning and concentration stages were performed at the same TMP conditions. The permeation flux was determined by acquisition of the permeate weight in real time using a balance coupled to a PC (Fig. 1A). Removal of non-bound oligonucleotides and AuNP losses by membrane permeation was monitored by analysis of UV–vis spectra of samples collected along the dia-ultrafiltration process. Removal of non-bound oligonucleotides was inferred by

530

B. Veigas et al. / Journal of Membrane Science 492 (2015) 528–535

Fig. 1. Dia-ultrafiltration Process Development and Optimization. (A) Process Scheme; 1 – Argon bottle, 2 – Manometer; 3 – Dead-end permeation cell, 4 – Stirring plate, 5 – Balance, 6 – Washing solvent pump. During dia-ultrafiltration the flow rate of the washing solution was kept equal to the permeate flow rate. (B) Permeation flux, Jv, obtained along the purification of AuNPs by dia-ultrafiltration using a membrane of a MWCO of 10 kDa. (C) Dia-ultrafiltration process optimization for total rejection of Aunanoprobes. Absorbance spectra of Au-nanoprobe suspension, Feed ( ), Purified Au-nanoprobe suspension, Retentate ( ) and Permeate ( ). Inset: UV spectra of permeates collected after the purification process, indicating presence of nucleic acid (absorbance max at 260 nm). (D) Absorbance spectra obtained for feed (solid line) and permeates (dashed lines) collected along the dia-ultrafiltration process. Inset: Absorbance of permeates collected along the permeation time, acquired at 270 nm.

comparative analysis of permeates, feed and final retentate characteristic spectral bands at 260–270 nm; whereas loss of AuNPs was evaluated at 525 nm and 600 nm for non-aggregated and aggregated AuNPs, respectively.

2.6. Scanning electron microscopy analysis The membranes used for purification were inspected by scanning electron microscopy (SEM) before and after the purification process. Membrane samples were mounted on Aluminum stubs with carbon tape and coated with a 20 nm thick Carbon film in a Quorum Q150T ES sputtering system. The sample surface was observed in a Carl Zeiss AURIGA Crossbeam SEM-FIB workstation, using an accelerating voltage of 2.5 KeV with an aperture size of 30 mm. Energy- Dispersive X-ray Spectroscopy (EDS) was performed in an Oxford Instruments detector X-Max, using an accelerating voltage of 15 KeV with an aperture size of 60 mm.

2.7. Au-nanoprobe colorimetric assay The AuNPs colorimetric method relies on the changes of the colloidal solution upon aggregation mediated by a variation to the dielectric medium. The design of these system is centered in the ability of complementary targets to balance and control interparticle attractive and repulsive forces, which determine whether AuNPs are stabilized or aggregated and, consequently, the SPR band and color of the solution remains unaltered or changes, respectively. A specific complementary target can hybridize to the gold nanoprobes and stabilize the nanoprobes against the changes of the dielectric medium (increasing concentrations of MgCl2), which otherwise would induce a non-cross-linking aggregation of the nanoprobes in the absence of a complementary target. The colorimetric assays were performed in a final volume of 30 μL containing Au-nanoprobes at a final concentration of 2.5 nM in 10 mM phosphate buffer (pH 8) and PCR product at final DNA concentration of 30 mg/mL. The mixture was heated up at 95 °C for 5 min and then cooled down to 25 °C for 5 min. For each probe, the assay consisted on the spectrophotometric comparison of a

B. Veigas et al. / Journal of Membrane Science 492 (2015) 528–535

“Blank” (without DNA), 10 mM phosphate buffer (pH 8), 0.1 M NaCl; non-related control containing non-complementary DNA; and the sample containing a complementary sequence to the MTBC Au-nanoprobe. The pre-determined MgCl2 concentration was added to each reaction, and after 30 min at room temperature for color development, the mixtures and the blank assayed by UV/ visible spectroscopy in a microplate reader (Tecan Infinite M200) [9]. 2.8. Data analysis Aggregation profiles were analyzed in terms of the ratio of Abs525 nm/Abs600 nm (dispersed vs. aggregated species) for each Au-nanoprobe. Each probe was used in a minimum of three individual parallel hybridization experiments with the PCR amplified amplicons derived from the rpoB gene from MTBC and a PCR product with a similar size with no complementary sequence to the probe. A threshold of 1 was considered where values 41 indicate that the Au-nanoprobe is mostly non-aggregated (Positive), whereas a value o 1 indicate aggregation (Negative) [9]. This approach provides for indication of presence or absence of pathogens DNA in the sample. 2.9. Dynamic light scattering (DLS) measurements DLS measurements were performed to determine Au-nanoprobes size distribution and presence of aggregates in a Horiba SZ100 NanoPARTICA Analyzer (Horiba, Japan). A total volume of 100 μL of 2.5 nM Au-nanoprobe was first stabilized for 15 min at 25 °C, afterwards 60 μL were used to perform the measurements. 2.10. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) The concentration of Gold in the feed and permeates collected after cleaning and concentration process was determined by ICPAES using a Jobin Yvon-Ultima ICP spectrometer (emission at 267.5 nm). Sample preparation was preformed incubating the samples with Aqua Regia in a 1:1 ratio for 48 h (see Supplementary information).

3. Results and discussion 3.1. Au-nanoprobe purification Several techniques for Au-nanoprobe purification have been developed, where the most common are based on centrifugation, yielding functional AuNPs capable of detecting biological targets (e.g. DNA, RNA) with single base mismatch discrimination capability [4,6–18]. Although versatile, Au-nanoprobe purification by centrifugation is laborious and time-consuming, which delays probe production and increases production costs. Membrane based technologies offer the possibility of minimal sample manipulation and an easier process scale up, thus emerging as a potential alternative for the modified AuNPs purification. The main limitation of membrane based processes concerns the accumulation of solutes at the membrane surface and into the membrane interstitial porous structure, rendering unstainable purification processes, due to the continuous reduction of permeation fluxes along the operation time, compromising the process selectivity. To minimize this problem, Au-nanoprobes purification was attempted by ultrafiltration processes operated in diafiltration mode as schematized in Fig. 1A. Dia-ultrafiltration processes are regarded as an advantageous alternative to ultrafiltration processes, conventionally operated in a concentration mode, due to

531

their ability to minimize polarization of concentration and fouling effects, typically found when membrane concentration strategies are used. In dia-ultrafiltration processes, polarization of concentration effects are limited by controlled addition of a washing solvent, which allow for the maintenance of retentate volumes, avoiding the increase of the concentration of the solution components retained by the membrane. The impact of diafiltration operation regime on the Au-nanoprobe purification performance is perfectly visible in Fig. 1B–D. Fig. 1B shows that the permeation flux slightly increased along the dia-ultrafiltration time. The increase of permeate flux reflects the attenuation of membrane fouling due to progressive dilution of retentate over time, which enhances mass transfer conditions at the membrane surface. The implemented dia-ultrafiltration allows for the total rejection of Au-nanoprobes, simultaneously promoting an easier and faster removal of media impurities. Au-nanoprobe rejection was inferred based on the analysis of the UV–vis spectra obtained for feed, retentate and permeate solutions acquired along the permeation process (Fig. 1C). The presence of AuNPs in feed and retentate solutions is confirmed by the characteristic peak in absorbance at 525 nm. In contrast to feed and retentate spectra, permeate exhibit a UV band at 270 nm (inset of Fig. 1C), corresponding to the presence of the thiol-modified oligonucleotides removed by membrane permeation. The absence of the characteristic SPR band at 525 nm confirmed the total retention of AuNPs by the 10 kDa membrane and that they are not lost by membrane permeation along the purification procedure. The total rejection of Au-nanoprobes was further corroborated with ICP analysis of feed and permeate samples (from cleaning and concentration process stages), which showed no detectable Au (Supplementary information Table S2). Oligonucleotide removal by membrane permeation was also monitored by inspection of the modified oligonucleotides characteristic absorbance band at 260–270 nm of the permeate samples collected along the dia-ultrafiltration process (see Fig. 1D). The highest oligonucleotide removal was observed at the initial stage decreasing progressively with time. The decline of oligonucleotide removal was not attributable to transport hindrance caused by the accumulation of solutes at the membrane surface since polarization of concentration effects were not observed (Fig. 1B). Instead, it may be ascribed to the progressively decreasing concentration of free oligonucleotides in retentate solution. Oligonucleotide removal stabilizes at a minimum values at a later process stage, leading to less effective AuNP cleaning in this period, motivating the decision to stop the process at this phase. However, the oligonucleotides removal profile observed in the Inset of Fig. 1D, suggests that it would be possible to stop the process earlier without significantly affecting Au-nanoprobe cleaning. Hence, the maximum AuNPs purification achievable at the process conditions selected was attained upon 3 diavolumes (i.e. when the washing solvent volume added was equal to 3 fold the feed volume, which occurred after 275 min), which is quite a small value taking into account the number of diavolumes commonly required for solute purifications performed in pharmaceutical industry. After probe purification, the cellulose-based membrane used was characterized by Scanning electron microscopy and EnergyDispersive X-ray Spectroscopy (SEM–EDS). The morphologic analysis of membrane surface and cross-section shows an asymmetric membrane with a smooth and uniform top surface and a cellulose fiber structure in the bottom section (Supplementary information Fig. S1). SEM imaging shows the presence of gold (Au-nanoprobe) aggregates on the surface of the membrane top layer. EDS maps and spectra corresponding to the SEM image, further confirms the presence of Gold (Fig. 2). EDS analysis also revealed the presence of considerable amounts of Sodium, Phosphorus and Oxygen,

532

B. Veigas et al. / Journal of Membrane Science 492 (2015) 528–535

Fig. 2. Morphologic and chemical characterization of the cellulose-based membrane after dia-ultrafiltration processing. SEM images of cellulose membranes (molecular weight cut-off – 10 kDa) used to purify Au-nanoprobe suspension (top row) with respective EDS mapping (middle row) and spectra analysis (bottom row). Left: top section surface, showing in red the EDS map of Au; Right: bottom section surface, showing in yellow EDS map of Na and Cl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

originating from the Au-nanoprobe storage buffer. Finally, no Aunanoprobe was detected at the bottom section of the membrane. EDS distribution map and spectra shows traces of NaCl, one of the components of the Au-nanoprobe storage buffer (Fig. 2). Together with UV–vis and ICP-AES analysis of feed, retentate and permeate samples (Fig. 1C) these data show that dia-ultrafiltration allows for the total rejection of Au-nanoprobes, while allowing the nonbound oligonucleotides to be removed from the probe suspension. The efficacy of AuNPs purification by dia-ultrafiltration was analyzed based on probe aggregation, stability and hybridization efficiency, and compared to that obtained by centrifugation. After AuNP functionalization (MTBC Au-nanoprobe), we compared the

non-purified MTBC Au-nanoprobe against those purified via centrifugation and diafiltration. Au-nanoprobes were initially dispersed in a buffer containing 10 mM phosphate buffer (pH 8), 0.3 M NaCl, 0.01% SDS, which was then replaced along the diaultrafiltration by 10 mM phosphate buffer (pH 8), 0.1 M NaCl. UV/ vis spectra show no change to the SPR peak, indicating that there was no change to the average dispersion of the Au-nanoprobes (Supplementary information Fig. S2) [22]. This is rather relevant since nanoparticle aggregation during the purification process may occur due to an over-concentration of the Au-nanoprobes suspension [23]. DLS analysis shows similar Z-average values for the three batches of probes, thus supporting the previous spectral

B. Veigas et al. / Journal of Membrane Science 492 (2015) 528–535

interpretation (Supplementary information Table S3). Together, these results seem to indicate that the membrane purification process does not disrupt the synthetized probes maintaining the stable dispersed state required for the detection assay. 3.2. Probe aggregation profile Probe stability and aggregation profiles depend on the amount of oligonucleotides functionalized on the particles' surface and/or free in solution. Au-nanoprobe aggregation profiles were plotted and the minimum amount of electrolyte required to cause aggregation was determined for each nanoprobe (see Supplementary Fig. S3). Stability is higher for the non-purified and lower for the membrane purified sample, which may indicate a lower amount of ssDNA in the latter. This may be related to: (i) higher efficiency on the purification of oligonucleotides not bound to the AuNPs; and/ or (ii) that this process partially removes functionalized

533

oligonucleotides from the particles' surface, leading to lower ratio of oligonucleotide probe per AuNP. For AuNPs systems, it is of utmost importance to attain a balance between oligonucleotide surface coverage, particle stability, and hybridization efficiency. Coverage must be high enough to stabilize particles, yet low enough so that a high percentage of the strands are accessible for hybridization to oligonucleotides in solution [24]. We then evaluated the Au-nanoprobe performance to detect increasing concentrations of synthetic oligonucleotide targets. Based on the UV/visible spectra obtained after salt addition, the ratio between the absorbance at 525 nm and at 600 nm was calculated. A ratio of 1 was considered as the point of equilibrium between non-aggregated and aggregated nanoprobes, hence the threshold to respectively discriminate between positive and negative test results (Fig. 3). Although no significant statistical differences were observed between the three datasets, the centrifugation purified probe

Fig. 3. Au-nanoprobe performance-sensitivity and specificity analysis. (A) MTBC probe with complementary target; (B) MTBC probe with non-complementary target; ( ) non-purified probe; ( ) Centrifugation purified probe; ( ) Membrane purified probe. Nanoprobe aggregation as measured by ratio of aggregation for the assay mixtures— 2.5 nM Au-nanoprobe, 10 mM phosphate buffer (pH 8), 0.1 M NaCl, and different amounts of synthetic oligonucleotide. (C) Au-nanoprobe assay with PCR products. Colorimetric assay consists of visual comparison of test solutions upon salt induced Au-nanoprobe aggregation: Au-nanoprobe alone – Blank (gray sample), Au-nanoprobe in presence of complementary DNA (red); Au-nanoprobe in presence of non-complementary DNA (blue). Assay reading performed in a microplate reader where Au-nanoprobe aggregation was measured by ratio of aggregation (ratio of SPR intensity at 525 and 600 nm) with 30 ng of target DNA and [MgCl2] ¼ 30 mM. Bars represent the average of three independent measurements and the error bars indicate standard deviation. The horizontal line represents the threshold of 1 considered for discrimination between positive and negative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

534

B. Veigas et al. / Journal of Membrane Science 492 (2015) 528–535

showed a higher sensitivity, allowing for the detection of as little as 66 fM of target DNA. However, the probe purified via the membrane approach yielded the highest Abs(525 nm/600 nm) ratio at saturation level, indicating higher hybridization efficiency. Higher hybridization efficiency normally occurs in less dense packed oligonucleotide monolayers, suggesting higher accessibility to incoming hybridizing strands onto the AuNPs due to a reduction of steric hindrance and electrostatic repulsive interactions [25]. The data attained with synthetic oligonucleotides provided important insights into the effect of purification process on Aunanoprobe sensitivity. Since ssDNA oligonucleotides are far from a real sample and non-specific interactions are limited or rarely occur, limited information was retrieved in terms of specificity. Aunanoprobes' specificity was evaluated by testing the three batches of probes against PCR generated products attained from clinical isolates from respiratory samples positive for acid-fast bacilli from patients of the Lisbon Health Region [12]. As negative control, a PCR product with similar length and %GC content with a non-related sequence was used (Fig. 3C). The data show that purification is indeed an important factor on Au-nanoprobes' sensitivity. The non-purified probe is unable to differentiate complementary from non-complementary target sample, i.e. due to unspecific stabilization, and both samples were scored as positive. The purified probes can correctly detect and differentiate complementary from non-complementary targets. Interestingly, the membrane-purified probe signal is more intense when compared to the standard purification procedure. Furthermore, this probe shows a higher Abs(525 nm/600 nm) ratio for the complementary sample and lower for the Blank and non-complementary, with similar error values. These results suggest that the probes purified by dia-ultrafiltration are less affected by nonspecific interactions.

4. Conclusion In this work it is demonstrated that dia-ultrafiltration is an efficient, rapid and versatile purification method for Au-nanoprobe production. The dia-ultrafiltration was performed using a membrane with a MWCO of 10 kDa, which allowed for an efficient removal of non-bound oligonucleotides, while assuring the total rejection of Au-nanoprobes. In contrast to that obtained by centrifugation, the purification of Au-nanoprobes by dia-ultrafiltration generated the production of Au-nanoprobes exhibiting improvement hybridization efficiency and higher specificity towards the M. tuberculosis complex, leading to increased signal-to-noise-ratio and greater reproducibility between batches. Comparatively to standard centrifugation process, dia-ultrafiltration allows for a faster purification of larger volumes of Aunanoprobes, involving simple and lower sample manipulation. What is more, because dia-ultrafiltration may be operated in a semi-automated way, it massively decreases the labor intensity of this process. Dia-ultrafiltration processes maybe transposed to an industrial scale for enhancing probe production throughputs. The main drawback of pressure-driven membrane processes relates to the polarization of concentration effects, cake layer formation and membrane fouling events along the process, rendering lower permeate fluxes and unsustainable processes. However, in this work, the lower membrane fouling occurred along dia-ultrafiltration was mainly reversible and easily eliminated by a simple and non-exhaustive cleaning step with distilled water, which assured a full recovery of the initial membrane permeability, allowing for their subsequent reuse. These aspects assume a high relevancy in the economic viability of this Au-nanoprobe purification strategy, since it may contribute for a significant reduction of the Au-nanoprobe production costs. The advantages of dia-ultrafiltration

regarding reduction of processing time were not fully clear (5 h of semi-automated process for dia-ultrafiltration vs. 6 h of intensive manpower for centrifugation and manipulation) but further reduction of process time may be achieved by adjustment of the operating parameters. The scale-up production of Au-nanoprobes may be easily achieved and the fine tuning of particular characteristics may allow at manufacturing batches tailored for single nucleotide mutation detection, e.g. associated to drug resistance in M. tuberculosis.

Acknowledgments We acknowledge Fundação para a Ciência e a Tecnologia (FCT/ MEC) for financial support CIGMH (PEst-OE/SAU/UI0009/2013), PEstC/EQB/LA0006/2011 and PTDC/BBB-NAN/1812/2011; SFRH/BD/ 78970/2011 to BV.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.memsci.2015.06.042.

References [1] C.A. Mirkin, R.L. Letsinger, R.C. Mucic, J.J. Storhoff, A DNA-based method for rationally assembling nanoparticles into macroscopic materials, Nature 15 (1996) 607–609. [2] H.M.E. Azzazy, M.M.H. Mansour, In vitro diagnostic prospects of nanoparticles, Clin. Chim. Acta 403 (2009) 1–8. [3] C. Kaittanis, S. Santra, J.M. Perez, Emerging nanotechnology-based strategies for the identification of microbial pathogenesis, Adv. Drug Deliv. Rev. 62 (2010) 408–423. [4] P.V. Baptista, E. Pereira, P. Eaton, G. Doria, A. Miranda, I. Gomes, P. Quaresma, R. Franco, Gold nanoparticles for the development of clinical diagnosis methods, Anal. Bioanal. Chem. 391 (2008) 943–950. [5] G. Doria, J. Conde, B. Veigas, L. Giestas, C. Almeida, M. Assunção, J. Rosa, P. V. Baptista, Noble metal nanoparticles for biosensing applications, Sensors 12 (2012) 1657–1687. [6] P.V. Baptista, M. Koziol-Montewka, J. Paluch-Oles, G. Doria, R. Franco, Goldnanoparticle-probe-based assay for rapid and direct detection of Mycobacterium tuberculosis DNA in clinical samples, Clin. Chem. 52 (2006) 1433–1434. [7] B. Veigas, G. Doria, P.V. Baptista, Nanodiagnostics for Tuberculosis, in: P.J. Cardona (Ed.), Understanding Tuberculosis-Global Experiences and Innovative Approaches to the Diagnosis, InTech, 2012. [8] B. Veigas, J.M. Jacob, M.N. Costa, D.S. Santos, M. Viveiros, J. Inacio, R. Martins, P. Barquinha, E. Fortunato, P.V. Baptista, Gold on paper–paper platform for Aunanoprobe TB detection, Lab Chip 12 (2012) 4802–4808. [9] B. Veigas, D. Machado, J. Perdigão, I. Portugal, I. Couto, M. Viveiros, P. V. Baptista, Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis, Nanotechnology 21 (2010) 415101. [10] B. Veigas, A.R. Fernandes, P.V. Baptista, AuNPs for identification of molecular signatures of resistance, Front. Microbiol. 5 (2014) 455. [11] B. Veigas, P. Pedrosa, I. Couto, M. Viveiros, P.V. Baptista, Isothermal DNA amplification coupled to Au-nanoprobes for detection of mutations associated to Rifampicin resistance in Mycobacterium tuberculosis, J. Nanobiotechnol. 11 (2013) 38. [12] P. Pedrosa, B. Veigas, D. Machado, I. Couto, M. Viveiros, P.V. Baptista, Gold nanoprobes for multi loci assessment of multi-drug resistant tuberculosis, Tuberculosis 94 (2014) 332–337. [13] F. Carlos, O. Flores, G. Doria, P.V. Baptista, Characterization of genomic single nucleotide polymorphism via colorimetric detection using a single gold nanoprobe, Anal. Biochem. 465 (2014) 1–5. [14] K.C. Halfpenny, D.W. Wright, Nanoparticle detection of respiratory infection, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (2010) 277–290. [15] W.S. Chan, B.S. Tang, M.V. Boost, C. Chow, P.H. Leung, Detection of methicillinresistant Staphylococcus aureus using a gold nanoparticle-based colourimetric polymerase chain reaction assay, Biosens. Bioelectron. 15 (2014) 105–111. [16] Y.C. Chang, C.Y. Yang, R.L. Sun, Y.F. Cheng, W.C. Kao, P.C. Yang, Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles, Sci. Rep. 3 (2013) 1863. [17] E. Liandris, M. Gazouli, M. Andreadou, M. Comor, N. Abazovic, L.A. Sechi, J. Ikonomopoulos, Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles, J. Microbiol. Methods 78

B. Veigas et al. / Journal of Membrane Science 492 (2015) 528–535

(2010) 260–264. [18] C.C. Lin, Y.M. Yang, P.H. Liao, D.W. Chen, H.P. Lin, H.C. Chang, A filter-like AuNPs@MS SERS substrate for Staphylococcus aureus detection, Biosens. Bioelectron. 53 (2014) 519–527. [19] G. Dalwadi, H.A. Benson, Y. Chen, Comparison of diafiltration and tangential flow filtration for purification of nanoparticles suspensions, Pharm. Res. 22 (2005) 2152–2162. [20] S.F. Sweeney, G.H. Woehrle, J.E. Hutchison, Rapid purification and size separation of gold nanoparticles via diafiltration, J. Am. Chem. Soc. 128 (2006) 3190–3197. [21] J. Shao, A.L. Zydney, Optimization of ultrafiltration/diafiltration processes for partially bound impurities, Biotechnol. Bioeng. 87 (2004) 286–292.

535

[22] W. Haiss, N.T. Thanh, J. Averyard, D.G. Fernig, Determination of size and concentration of gold nanoparticles from UV–vis Spectra, Anal. Chem. 79 (2007) 4215–4221. [23] C. Vauthier, B. Cabane, D. Labarre, How to concentrate nanoparticles and avoid aggregation? Eur. J. Pharm. Biopharm. 69 (2008) 466–475. [24] A.W. Peterson, R.J. Heaton, R.M. Georgiadis, The effect of surface probe density on DNA hybridization, Nucleic Acids Res. 29 (2001) 5163–5168. [25] I.Y. Wong, N.A. Melosh, An electrostatic model for DNA surface hybridization, Biophys. J. 98 (2010) 2954–2963.