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Additionally it should be noted that, according to Supporting information, Fig. S4C, a DNA coil concentration of 0.5 nM gives the same response as obtained for the 1-nM coil concentration; an observation that further confirms the qualitative assay characteristics of our method.
In recent studies [ 16] we have demonstrated that the phase angle analysis method is superior to the turn-off approach (decrease of V2' peak amplitude upon increasing DNA coil concentration) with regards to SDs of the measured samples.
As can be seen from Fig. 1A, the average values for χ max/ χ'inf obtained at 1.5 pM coil concentration are lower than the corresponding values for the negative controls.
The final solution was transferred to a cuvette and measured using the optomagnetic setup in the frequency range 1 200 Hz with an AC magnetic field amplitude of 2.58 mT. Panels A and B in Fig. 1 show in- (V2'/ V0) and out-of-phase (V2"/ V0) vs. frequency spectra for four selected DNA coil concentration combinations, respectively.
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Compared to the previous study [ 31], the scatter in the HFP level, when keeping one DNA-coil concentration constant and varying the other, is larger.
The DNA-coil concentration of the opposite target is indicated in each case by bold numbers appearing close to each data point.
Optimal bead immobilization can be defined by two criteria; (i) lowest LOD and (ii) highest percentage of immobilized beads for a given DNA-coil concentration.
Figure 1C presents χ max/ χ'inf versus VC DNA-coil concentration (0 4.5 pM) for 250 nm beads conjugated with 40-, 60-, and 100-fold excess of detection oligonucleotides.
Moreover, as indicated in Fig. 3C, it can be observed that when keeping one DNA-coil concentration constant and varying the opposite, the HFP level tends to increase upon increasing the concentration of the other type of DNA-coil.
In the last step a certain portion of the beads, determined by the DNA-coil concentration, hybridize to the DNA-coils (bead immobilization) and immobilized beads thereby undergo a dramatic hydrodynamic size increase which in turn strongly alters their Brownian relaxation frequency.
However, the LOD is determined to 4.5 pM, Fig. 1B, defining the LOD as the lowest DNA-coil concentration, c, that is statistically different from the negative control; χ max (0 pM / χ'inf (0 pM) – 3SD 0 pM) > χ max (c)/ χ'inf (c) + 1SD c).
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CEO of Professional Science Editing for Scientists @ prosciediting.com