Development of laboratory and portable format based detection systems for pan malaria and P. falciparum species
7.3 Results and discussion
7.3.4 Quantitative detection of PfGDH and PLDH
The aptamer decorated magnetic beads were used to capture the respective biomarker enzymes from the binding buffer and undiluted human serum samples. The enzyme- captured beads were mixed with the cocktail buffer in a centrifuge tube. The samples were then analysed by instrument based system and instrument free potable detection technique as described below. For the analysis, each of the marker enzymes was studied in a broad concentration range of 1 pM to 100 nM. This range fairly covers the previously reported dynamic range for PLDH (3.9 pM to 39 nM) offered by the immune-chromatographic RDTs available in the market (Jimenez et al., 2017) and for PfGDH (2 to16 nM) offered by the immuno-chromatographic dipstick test (Li et al., 2005).
7.3.4.1 Instrument-based analytical technique
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The analysis was first performed in buffer samples spiked with PLDH or PfGDH. The reaction mixture containing the enzyme captured beads and the cocktail buffer was incubated for one h at RT. The reaction supernatant was then separated from the mixture following the method described above. The supernatant was then subjected to absorbance
and fluorescence spectral analyses for PLDH (Figure 7.7 A, B), PfGDH (Figure 7.7 C, D) spiked in buffer and PLDH (Figure 7.8 A, B), PfGDH (Figure 7.8 C, D) spiked in serum.
Figure: 7.7: (A) Absorbance spectra of PLDH (A), PfGDH (C) and fluorescence spectra of PLDH (B), PfGDH (D) response with different concentrations spiked in binding buffer.
Figure: 7.8: (A) Absorbance spectra of PLDH (A), PfGDH (C) and fluorescence spectra of PLDH (B), PfGDH (D) response with different concentrations spiked in serum.
With the increasing concentration of the captured enzymes, the absorption peak at ∼λ605nm
of the enzyme free mixture shifted to ∼λ570nm along with the appearance of a fluorescent peak at λ660 nm (excitation at ∼λ570 nm). The calibration plots for the respective enzymes were constructed from the spectral absorbance and fluorescence intensity for binding buffer solution (Figure 7.9 A, B) and serum samples (Figure 7.9 C, D). For absorption spectra based calibration plot, the ratio metric (λ570nm/λ605nm) response was considered to improve the sensitivity of the method (Sun et al., 2012).The LODs (3 σ/s) calculated for PLDH from the corresponding absorbance and fluorescence intensity based calibrations curves in binding buffer were 0.41 ± 0.07 pM and 1.63 ± 0.14 pM and in serum were 0.55 ± 0.09 pM and 1.72 ± 0.13 pM, respectively. Whereas, the LOD for PfGDH from the corresponding absorbance and fluorescence intensity based calibrations curves in binding buffer were 1.14 ± 0.12 pM and 0.81 ± 0.10 pM and in serum were 1.34 ± 0.12 pM and 1.43 ± 0.14 pM, respectively.
Figure: 7.9: Calibration plots derived from absorbance and fluorescence characteristics for (A) PLDH and (B) PfGDH in binding buffer and (C) PLDH and (D) PfGDH in serum.
Overall, the absorbance spectroscopy offered superior LODs for the biomarkers than the corresponding fluorescence based determination indicating higher sensitivity of the ratio metric absorption response than the single wavelength-based fluorescence response. The discerned LOD values were far superior than 30.45 ± 47.65 nM for PLDH and 1-16 nM for PfGDH, which are considered to be in the clinically significant range for malaria diagnosis (Li et al., 2005; Jang et al., 2013).
The performance of the method was also examined for co-activity of the enzymes using an equimolar mixture of P38 and NG3 coated magnetic beads in binding buffer spiked with different concentrations of PLDH and PfGDH. Subsequently the reaction was performed by mixing the enzyme captured beads and cocktail buffer following the procedure described above. Calibration plots were constructed for both absorbance (λ570nm/λ605nm) and fluorescence (emission at λ660nm) signals (Figure 7.10 A) as well as for instrument free technique (Figure 7.10 B), which will be discussed below. As evident from the graphs, the linear response for both the instrument based and instrument free methods was identified to be 1 pM to a maximum of ~100 pM with R2 (co-efficient of determination) value 0.95.
Since beyond this concentration of 100 pM, the R2 values of the regression line were significantly reduced.
Figure 7.10: (A) Absorbance and fluorescence (Excitation λ570, Emission λ660) response and (B) colour (pixel) response of the reaction with different concentration of each enzyme (PfGDH and PLDH) co-spiked in binding buffer.
7.3.4.2 Instrument-free portable detection technique
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For the instrument free method, the reaction mixture in the eppendorf tube containing the enzyme captured beads and the buffer was immediately siphoned into the modified syringe and then incubated for 1 hour. The reaction supernatant from the syringe was then passed through a DSM paper wick fixed inside the syringe hose. The dye got adsorbed in the DSM paper wick displaying increase in pink colour response with increasing concentration of the captured PLDH or PfGDH in the buffer or serum samples allowing visual detection of the biomarker enzymes in serum samples (top panel in figure 7.11).
Figure: 7.11: Calibration plots derived from the pixel intensity of the colour developed on DSM paper against different concentration of (A) PLDH, (B) PfGDH spiked in binding buffer and (C) PLDH, (D) PfGDH spiked in serum.
The calibration plots were constructed by measuring colour intensity on the paper versus different concentration of PLDH (Figure 7.11 A) and PfGDH (Figure 7.11 B). The
calibrations curve for both the enzymes exhibits a linear response within the range of 1 pM to 100 nM. The LODs discerned were 61.50 ± 6.43 pM and 63.97 ± 7.24 pM for PLDH and PfGDH, respectively in spiked buffer solution.
The value of LOB was taken into account while calculating LOD for the assay over the paper surface considering the high background noise of the pixel based colorimetric detection over paper platform (Shrivastava et al., 2011). Like the buffer samples, the dynamic range for the serum samples was also identified as 1 pM to 100 nM for both the enzymes (7.11 C and D). The LODs calculated for PLDH and PfGDH in the serum sample were 69.25 ± 8.22 pM, and 68.75 ± 7.64 pM, respectively. A minor lower sensitivity observed in serum than the buffer condition has been attributed to the complex nature of the serum causing hindrance to the aptamer-target interactions. Furthermore, we performed the co-activity test on paper following similar reaction conditions and molar concentrations of the ingredients enzymes used for instrument based method. Like instrument-based method, the linear response was limited within the early concentration of the enzymes (Figure 7.10 B with colour response in top panel). We propose similar reason as discussed under the instrument based method.