Development of Aptamer Based Colorimetric Detection of PfLDH
4.3 Results and discussion
4.3.2 Cationic surfactant-mediated detection of PfLDH
Disaggregation of AuNPs at high protein concentration resulting in a narrow linear range in the salt-based assay prompted us to explore cationic surfactant-mediated AuNP aggregation for PfLDH detection. A surfactant is a bulky molecule, typical Mw ranges from 300 to 400, which consists of two moieties: (i) one or more hydrophobic alkyl chains and (ii) an ionic or highly polar hydrophilic group. The positively charged group of cationic surfactants usually contains a quaternary ammonium, although analogues containing sulphur, phosphorus or arsenic exist as well (Cross and Singer, 1994). Due to the versatility of quaternary ammonium surfactants‘‘quats’’ to retain their positive charge at any pH, they have been used for the synthesis and control of nanomaterials (Smith and Korgel, 2008;
Moon et al., 2009). CTAB has been shown to interact with single and double stranded DNA to form cubic and hexagonal nanostructures, respectively (Liu and Abbott, 2010). CTAB has also been used to develop an AuNP based colorimetric assay for small molecules (He et al., 2013), ions (Wu et al., 2012a) and proteins (Lee et al., 2014). For the present work, we studied three different surfactants: (i) alkyl dimethylbenzyl ammonium chloride, commonly known as benzalkonium chloride (BCK) which contained an aromatic group and a long (C12) alkyl chain; (ii) hexadecyl(cetyl) trimethyl ammonium bromide (CTAB), which contained a single long (C16) alkyl chain; and (iii) didodecyl dimethyl ammonium bromide (DDAB) which contained two long (C12) alkyl chains. We compared their aggregation efficiency for naked AuNPs and their detection efficacies using PfLDH as the target and
aptamer P38 as the biorecognition molecule. To our knowledge, the aggregation potentials of BCK and DDAB have not been investigated before, using a protein as target molecule.
Figure 4.1 B, D shows the principle for cationic surfactant based detection assay for PfLDH using the aptamer P38, and the chemical structure of the three surfactants used in the study.
In the presence of PfLDH, P38 forms a complex with it, and the aptamer is hence unavailable for binding when a cationic surfactant is present. With addition of AuNPs, the free cationic surfactant causes their aggregation, leading to a blue colour. In the absence of a target, the aptamer is free to assemble with cationic surfactant, and the AuNPs remain unaggregated due to unavailability of free positive charge in the system. Multiple, long alkyl chains on the quaternary ammonium of the surfactant enables better aggregation of AuNPs.
Among the three surfactants studied, DDAB with two long alkyl chains caused complete aggregation of the free AuNPs at a much lower concentration of 1.5 µM, followed by BCK and CTAB at 3 µM, and 4.5 µM, respectively (Figure 4.3 A). Although BCK has an alkyl chain with four carbons fewer than CTAB, the presence of an aromatic ring probably gives it a significant advantage in aggregating AuNPs. In order to obtain appropriate signal respective to the surfactant concentration used in each assay, the P38 concentration was optimized. 30 nM P38 was sufficient to achieve minimum background in the absence of PfLDH, when BCK (3 µM) or CTAB (4.5 µM) was used. However, 50 nM P38 was used to mitigate the aggregation caused by 1.5 µM DDAB (Figure 4.3 B). Hence, DDAB has been identified as a stronger and more potent aggregating agent than BCK and CTAB.
The abilities of BCK, CTAB and DDAB to detect various concentrations of PfLDH in the assay format were investigated. The disaggregation of AuNPs using any of these three
98 surfactants at high concentration of PfLDH was not as pronounced as the case when NaCl was used. However, the lesser disaggregation is likely to generate a wider dynamic detection range, and is thus considered a suitable property for analytical perspective. For NaCl, BCK, CTAB and DDAB, the minimum concentrations of PfLDH beyond which disaggregation starts were 1, 5, 5, 10 nM, respectively. Although DDAB has a better aggregation efficacy for AuNPs and tolerates higher concentration of PfLDH without allowing disaggregation, BCK showed better sensitivity with an LOD of 281 + 11 pM which is far higher than the corresponding CTAB and DDAB based detections (Figure 4.4). Thus, from the above results, the order of detection sensitivity was BCK>NaCl followed by CTAB and DDAB, both of which did not produce any significant response for PfLDH concentrations up to 1 nM (Figure 4.4). It is expected that BCK will also maintain its higher sensitivity with other DNA aptamers with similar chain length as well, since the charge of such aptamers may not significantly vary. In a previous study, CTAB has been reported to have a better sensitivity than NaCl for an arsenic aptamer (Wu et al., 2012a). Thus, the sensitivity of an assay may be dependent on the type of target analyte.
The detection limits obtained by us (38 pg.µl-1with BCK and 55 pg.µl-1with NaCl) with P38 are superior to a similar assay developed by Cheung et al. (2013) for PfLDH. A number of variations have been included in the present detection protocol. Unlike covalently conjugating the PfLDH aptamer with AuNPs, P38 was physically adsorbed on the nanoparticles, and thereby, we omitted additional steps of preparing the detection probe.
Additionally, insight obtained from comparison of salt and cationic surfactants based aggregation methods led to significant improvements in the detection limit and range.
Conversely, the sensitivity reported previously (Lee et al., 2014) using different aptamers was found to be better than the one obtained by us. However, the sensitivity reported by us is in the clinically relevant picomolar range (Martin et al, 2009) for the reliable detection of malaria.