Nanoparticles based method for screening enzyme or microorganism

1 . A method for screening a bioactive substance of a library which is capable of degrading or assisting in degrading a water-insoluble polymer matrix using a microfluidic-based system, said method comprising:

a) generating a first droplet containing a bioactive substance;

b) coalescing a secondary droplet containing a nanoparticle with the droplet generated in step a);

wherein the nanoparticle comprises water-insoluble polymer matrix encapsulating an indicator constituent, and wherein the indicator constituent is released from the nanoparticle only when the polymer matrix is degraded or broken; and wherein the nanoparticle is formed by providing a solution comprising one or more polymers, and then contacting the solution with a polymer nonsolvent to produce the nanoparticle;

c) sorting the droplets in step b) based on an indicative effect generated by the indicator constituent, and

d) analyzing the sorted droplets to screen for the bioactive substance;

wherein the bioactive substance is a water-insoluble polymer degrading enzyme or a water-insoluble polymer degrading microorganism.

2 . The screening method according to claim 1 , wherein, when the indicative effect of a droplet reaches or is above a preset threshold level, the droplet is selected.

3 . The screening method according to claim 1 , wherein the droplet sorting is based on a fluorescence activated droplet sorting technique.

4 . The screening method according to claim 1 , wherein water-insoluble polymer degrading enzyme is a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase.

5 . The screening method according to claim 1 , further comprises a droplet incubation process, wherein the droplet containing the bioactive substance is incubated.

6 . The screening method according to claim 1 , wherein each droplet before sorting comprises at most a single cell.

7 . The screening method according to claim 1 , wherein the library is a microorganism library comprising a multitude of different microorganism, wherein the amount of indicative effect released by microorganism reaches or is above the predetermined threshold level, whereby a targeted enzyme or a targeted microorganism is identified.

8 . The screening method according to claim 1 , wherein the bioactive substance library is an enzyme library comprising a multitude of enzyme variants derived from a parent enzyme, wherein an increased amount of indicative effect has been released by the active enzyme variant, as compared with the parent enzyme, whereby an improved enzyme variant is identified.

9 . The screening method according to claim 1 , wherein the water-insoluble polymer matrix is degraded or broken, from which the indicator constituent is released, and then an indicative effect is triggered or enhanced.

10 . The screening method according to claim 1 , wherein the water-insoluble polymer matrix comprises or consists of an enzyme substrate.

11 . The screening method according to claim 1 , wherein the water-insoluble polymer is polyester, starch, saccharides, lipids, lignin, protein, nucleic acid, amylose, cellulose, pectin, chitin, fatty acids, lignin, alginate acid, hyaluronic acid, agar, mannan, xanthan gum, arabic gum, protein, DNA or RNA.

12 . The screening method according to claim 1 , wherein the water-insoluble polymer is polybutylene terephthalate (PBT), polypropylene terephthalate (PTT), polyethylene terephthalate (PEN), polyethylene terephthalate, tetramethylene terephthalate (PTMT), polyethylene-4-oxybenzoate, polyethylene glycol, polyvinyl chloride, polyglycolic acid, polylactic acid, polyanhydride, polycaprolactone, polyacrylonitrile, polypropylene, or polyvinyl alcohol.

13 . The screening method according to claim 1 , wherein the indicator constituent is a fluorogenic indicator.

14 . The screening method according to claim 1 , wherein both the polymer matrix and indicator constituent can be degraded or hydrolyzed by a specific active enzyme, and then, the indicative effect is triggered or enhanced.