Homogeneous Differential Magnetic Assay

Abstract

Assays are widely used for detection of various targets, including pathogens, drugs, and toxins. Homogeneous assays are promising for the realization of point-of-care diagnostics as they do not require separation, immobilization, or washing steps. For low concentrations of target molecules, the speed and sensitivity of homogeneous assays have hitherto been limited by slow binding kinetics, time-consuming amplification steps, and the presence of a high background signal. Here, we present a homogeneous differential magnetic assay that utilizes a differential magnetic readout that eliminates previous limitations of homogeneous assays. The assay uses a gradiometer sensor configuration combined with precise microfluidic sample handling. This enables simultaneous differential measurement of a positive test sample containing a synthesized Vibrio cholerae target and a negative control sample, which reduces the background signal and increases the readout speed. Very low concentrations of targets down to femtomolar levels are thus detectable without any additional amplification of the number of targets. Our homogeneous differential magnetic assay method opens new possibilities for rapid and highly sensitive diagnostics at the point of care.

Characterization of Binding of Magnetic Nanoparticles to Rolling Circle Amplification Products by Turn-On Magnetic Assay

“…For the turn-on analysis, we use the DHMA method [31] which measures a test sample and a reference sample simultaneously. The reference sample in this case is the NC and it fills one of the two available microfluidic channels.…”

“…This behaviour is illustrated in Figure 4 where the shape of the amplitude normalized distribution for different RCP concentrations are compared. We have previously shown that for this assay the equilibrium dissociation constant is KD=16.5 pM [31]. The equilibrium dissociation constant is a measure of tendency for the MNP-RCP complex to dissociate into separate MNP and RCP entities.…”

“…The magnetic flux from each sample is coupled to an individual loop of the gradiometer using two identical microfluidic channels that are aligned parallel to the baseline of the gradiometer. The screening current in each loop is then subtracted in the middle line of the loop measured by the SQUID providing the relative difference in the ac susceptibility of the two samples [31]. A bi-modal relaxation model based on a superposition of two Cole-Cole models [19]:χ(ω)=χ011+(iωτ1)α1+χ021+(iωτ2)α2+χhigh is used to fit to the data presented here.…”

“…Recently we developed a differential homogenous magnetic assay (DHMA) which measures a test and a control sample simultaneously to eliminate the troublesome magnetic background [31]. This enables the subtraction of signal from the excess unbound MNPs, which is analogous to a physical washing step in most bulk assays for removing excess markers.…”

“…In this way, the DHMA is able to resolve directly the very small differences in the nanoparticle’s concentrations and size distributions in the two samples. Using the assay we already demonstrated fM sensitivity with a constant 50% binding fraction for all low concentrations of target molecules [31]. The 50% binding fraction ensures that the MNP markers do not mask the LRF.…”