In vitro diagnostic assay methods

1 . An in vitro diagnostic assay method, comprising:

providing a sample which may comprise target analyte(s);

contacting the sample with a fluorescent label whose fluorescence can be externally modulated, such that the fluorescent label is associated with target analyte(s), if present, to form a fluorescent label-analyte complex; and

detecting the fluorescent label-analyte complex;

wherein said fluorescent label is a material which contains a nitrogen vacancy center;

wherein the method further comprises:

a) exciting the fluorescent label using a suitable light source, and detecting the resulting fluorescence;

b) modulating the fluorescence of the fluorescent label and detecting the resulting fluorescence to obtain a fluorescence time series comprising the fluorescence results from steps a) and b); and

c) optionally repeating the modulation step one or more times and detecting the resulting fluorescence to obtain a fluorescence time series comprising the fluorescence results from steps a), b) and c); and

wherein the sample is applied to a solid phase comprising a multiwell plate, beads, column, photonic device, microfluidic chip or capillary bed for microfluidic flow assay;

further wherein the assay method comprises a microfluidic flow assay, ELISA, gPCR, microarray, isothermal amplification, dot-blot, flow cytometry, fluorescence-activated cell sorting (FACS), a lateral flow assay or a vertical flow assay.

2 . Assay method according to claim 1 , which comprises applying a frequency domain analysis to the fluorescence time series acquired in step b) or c), optionally wherein the frequency domain analysis comprises applying a lock-in amplification algorithm or Fourier transform to the fluorescence time series.

3 . Assay method according to claim 1 , wherein said material which contains a nitrogen vacancy center comprises fluorescent nanodiamonds (FNDs).

4 . Assay method according to claim 3 , wherein the FNDs comprise a hydrophilic layer comprising a hydrophilic polymer, optionally wherein the hydrophilic polymer comprises polyglycerol (PG) or polyethylene glycol (PEG).

5 . Assay method according to claim 3 , wherein the FNDs comprise probe(s) suitable for associating with the target analyte(s), optionally wherein said probe(s) comprise a protein.

6 . Assay method according to claim 1 , wherein the fluorescence of the fluorescent label is modulated by a microwave field or a magnetic field, wherein the fluorescent label comprises a nitrogen vacancy center, and a microwave resonator with resonant frequency of 1-40 GHz is used to enhance the coupling between the label and the applied modulated microwaves.