Review of literature 1.1 Overview
3.3 Results and Discussion
3.3.1 Spectrophotometric detection of HRP-II
Free murexide solution exhibited an intense peak at ∼λ515nm. The peak shifted to ∼λ482nm
when Ni2+ solution was added to it (Figure 3.4 A). For optimization of the reaction, 50 μM of murexide was titrated with increasing concentrations of Ni2+. The saturation was
reached at ∼50 μM of Ni2+ (Figure 3.4 B), indicating 1:1 stoichiometry of the reaction.
From the plot, the binding constant (Kd) of 1.4 × 10−6 M−1 was discerned for the dye from the equation Y = BmaxX/Kd + X, where Bmax is the maximum available concentration of receptors, X is concentration of ligand, and Y is concentration of receptor. The ratio metric (λ515 nm/λ482 nm) response was considered to improve the reliability of the assay (Sun et al., 2012). When an equimolar (50 μM) mixture of murexide and Ni2+ were titrated with HRP-II, the orange color (∼λ482nm) of the solution gradually changes to pink (∼λ515nm) with the corresponding increase in protein concentration (bottom panel in Figure 3.5). Using the said concentrations of dye and Ni2+, a dynamic detection range of 1 nM − 3 μM and a limit of detection (LOD) of 53 ± 7.1 nM for HRP-II (R2 = 0.995) were discerned using LOD = 3 × Sy/b, where Sy is the standard deviation (SD) of y- intercept, and b is the slope of the linear curve. The method could detect as low as 1 pM of HRP-II in the solution. The Kd value calculated for HRP-II-Ni2+ was 6.8 × 10−9 M−1. This higher Kd value of HRP-II than the murexide dye for Ni2+ validates the ability of HRP-II to displace Ni2+ easily from the dye complex. Notably, the high affinity binding of Ni2+ to His amino acid is commonly exploited to purify His-tag proteins and HRP-II through the column chromatographic technique. However, this displacement based reaction is not known to utilize for detection of HRP-II and other proteins. The method offers a fast, sensitive, and label free detection of HRP-II. Moreover, the analysis does not involve any expensive reagents, hence it has great potential to use in a resource limiting platform.
3.3.2Effect of pH on spectrophotometric detection of HRP-II
The effect of pH on the displacement reaction was investigated (Figure 3.6 A). The response was conspicuous in alkaline conditions and drastically increased with increasing the pH value from 7 to 9 (inset in Figure 3.6 A). At lower pH (< 6.5)
conditions, both the dye (pK1 = 9.2 and pK2 = 10.5) and HRP-II protein (pI ∼ 6.5) exist in protonated forms, hence are not amenable to bind to Ni2+. A previous study on the binding of Ni (II) ions to hexa-histidine as a model system of His-tagged proteins indicated that deprotonation takes place with increasing pH value in the alkaline side.
The octahedral 2N Ni (His6) H species releases a H+ from an imidazole group resulting in the formation of square planar 3N Ni(His6) that upon aggregation produces the 4N Ni(His6)n stabilized byhydrogen bonds (Valenti et al., 2006). It is assumed that HRP-II also functions in a similar manner due to which it binds with Ni2+ strongly and quickly at higher pH conditions. The limit of detection (LOD) calculated from the response curves (Figure 3.6 B) for pH 7, 8, and 9 were 330.26 nM, 41.67 nM, and 7.62 nM, respectively, using LOD = 3 ×Sy/b, where Sy is the SD of y-intercept, and b is the slope of the linear curve. The results clearly demonstrated that higher alkaline pH is conducive to yield higher sensitivity for the detection, which offers an opportunity to enhance the sensitivity of the assay on demand.
3.3.3 Interference studies and analysis in serum sample
The specificity of the assay was also investigated by challenging the reaction with a major serum protein, HSA that accounts for ∼55 % (∼0.6 − 0.7 mM) of the blood proteins. The response for the concentration of 1 μM HSA was negligible (∼0.7 ± 0.005 %) as can be seen from surface (Figure 3.7A). The effect of some common serum metal ions, namely, Na+, K+, Mg2+, Ca2+, and Fe2+ on the assay was investigated. An equal concentration of the ion and Ni2+ (50 μM) were added to the reaction mixture and absorbance was recorded by adding 1 μM of HRP-II to each of the reactions. Selectivity coefficient (SC) for each of the potential interfering agents was calculated using the formula, SC = Ac+i/Ac where Ac+i and Ac are the response for Ni2+ in the presence and absence of each interfering agents, respectively. The results indicate that response of the reaction did not get affected
by the presence of these ions as the SC of ∼1 was obtained for each of the cases (figure 3.7 B). To understand any interference by other unknown potential interfering agents present in human serum, the assay was performed in serum samples spiked with different concentrations of HRP-II. The SC calculated for serum sample in the presence of 1 μM HRP-II was ∼1.09 which is a maximum of 15.4 ± 0.012 % increase in response as compared to the control experiments (without serum) indicating minor interference from the blood serum.
Notably, the endogenous interference originates from the substances found naturally in the patient sample, such as blood serum is known (Dimeski, 2008) To avoid such interference, adequate dilution of the serum samples is commonly performed for clinical samples (Fakanya and Tothill, 2014; Toedter et al., 2008).
3.3.4 Characterization of fabricated microfluidic platform
A μPAD with test zone and sample zone was fabricated as shown in the schematic figure 3.8. The capacities of the test and sample zones were 1 μl and 20 μl, respectively. As evident from the SEM images (Figure 3.9 B, C), the non-printed hydrophilic part of the paper surface retained the fibrous and porous structure, whereas in the printed hydrophobic part the porosity was substantially reduced. AFM images of the AKD printed surface (Figure 3.9 F) showed a rough surface with an uneven morphology whereas the unmodified appeared to be smoother with periodic ridges which were formed by the printer head while printing. The root-mean-square (RMS) roughness factors for the nonprinted and printed regions were 44.6 ± 1.35 nm and 20 ± 0.22 nm, respectively.
The surface height of the nonprinted and AKD printed (Figure 3.9 E,G) regions were 25
± 0.81 nm and 120 ± 6.23 nm, respectively, indicating an increase in paper height after the printing.
3.3.5 Detection of HRP-II in μPAD platform
The reagents, murexide and Ni2+ (each of 1 μl from 1 mM stocks) (Ni2+-murexide: 1:1) was deposited on the test zone and then dried at RT to make the μPAD ready for the test.
The sample containing HRP-II, when applied to the sample zone, travelled through the microchannels to the test zone where it reacted with the reagents and then produced a color image. Application of the μPAD limited the sample volume to 20 ± 06 μl. The mean pixels of the images were calculated and plotted against applied HRP-II concentrations (Figure 3.10). A dynamic detection range of 10 − 100 nM and LOD of 30 ± 9.6 nM were discerned from the linear regression equation (Figure 3.10, inset). The method presented here with non-biological recognition system targeting HRP-II has some clear advantages in addition to the other general positive traits mentioned elsewhere over the antibody based detection methods. There have been cases where patients with complicated malaria failed to show positive results in RDTs. The LOD value offered by the sensor indicating that the developed μPAD is applicable to both uncomplicated and complicated malaria.
The concentration of HRP-II in cases of complicated malaria may be as high as ∼80 nM (Rubach et al., 2012). The false negative results due to Prozone effect at high concentration of HRP-II,(Luchavez et al., 2011; Santos et al., 2015) and cross contamination with other antibodies (Lee et al., 2014; Meatherall et al., 2014) as reported for antibody based lateral flow methods may be avoided. Therefore, there is a great scope of using the present detection system as an additional test along with the current RDTs to eliminate false negative results in the case of patients with high parasitaemia and auto immune diseases.