Precision medicine demands the best
application of multiple
unambiguous
biomarkers to bring uniform decisions in disease prognosis. The remarkable
development of heterogeneous immunoassay greatly promotes precision
medicine when combined with the biomarker combination strategy. Nevertheless,
the cumbersome washing steps in heterogeneous immunoassay have inevitably
compromised the accuracy because of the sample losses and nature change
of the matrix, challenging the further exploration of a more facile
and lower limit-of-detection analysis. The new methodologies with
high throughputs and specificity are never out of date to provide
simultaneous evaluations and uniform decisions on multiple analytes
through a simple process. Herein, we propose a new wash-free immunoassay,
named differential assay, for multiplexed biomarker monitoring. The
method is based on counting the number difference of unbound nanoparticle
tags before and after immunoreactions from a solid support (i.e.,
magnetic microsphere) by single-particle inductively coupled plasma
mass spectrometry (sp-ICP-MS), discarding the tedious washing steps.
We primarily explore the proof-of-concept proposal within two types
(sandwich and competitive assay), demonstrating the good feasibility
for further facile clinical practice. To provide efficient multiplexed
evaluations, we synthesized PtNPs with four diameters and screened
the most suitable size for efficient differential immunoassay. The
wash-free strategy was successfully utilized in simultaneous serological
biomarker (CA724, CA199, and CEA) evaluation, with results in good
accordance with those measured by the clinical routine method. Potentially,
the proposed differential bioassay can be regarded as a more facile
and valuable tool in malignancy prognosis and cancer recurrence monitoring.