The invention belongs to the technical field of detection, and particularly relates to an orderly layered SERS (Surface Enhanced
Raman Scattering) detection membrane as well as a preparation method and application thereof to detection of
purine needling effect substances. The preparation method of the SERS detection membrane comprises the following steps: preparing
noble metal nano-
sol with different particle sizes, wherein the surface of the
noble metal nano-
sol is modified with a hydrophilic stabilizer; the preparation method comprises the following steps: respectively dispersing
precious metal nano-
sol with different particle sizes into water, then adding dispersion liquid, and after shaking, spontaneously assembling nano-particles on a gas-
liquid interface to form a single-layer nano-particle film; the method comprises the following steps: transferring a nano-particle film to the surface of a flexible film,
drying, transferring nano-particle films with at least two particle sizes on the surface of the nano-particle film, and
drying to obtain the SERS detection film loaded with at least three
layers of nano-particle films with different particle sizes, wherein the particle sizes of the
nanoparticle films are sequentially reduced from outside to inside. According to the SERS detection membrane, macromolecular
protein and small molecular
purine in
acupuncture body fluid can be separated out, and high-sensitivity and high-stability detection on the macromolecular
protein and the small molecular
purine can be achieved on the front face and the back face of the SERS detection membrane respectively.