Granted Patent B2

US 12,578,271 · App. 18/252,724 · Granted Mar 17, 2026

Inventors: Hai-Lung Dai (Wynnewood, PA); Mohammad Sharifian (Charlottesville, VA); Michael Wilhelm (Audubon, NJ)

Assignee: Temple University -Of The Commonwealth System of Higher Education

G01N21/636

Abstract

Claims (19)

1 . A method of determining bacterial cell wall composition comprising the steps of:

a. measuring a first second-harmonic scattering (SHS) signal of a crystal violet (CV) solution to generate a baseline;

b. adding a bacterial specimen to said solution to generate a suspension;

c. measuring a second SHS signal of said suspension to generate a response;

d. determining whether said response is above or below said baseline; and

e. identifying the bacterial cell wall of the bacterial specimen as that of a Gram-positive bacterium if the response is below said baseline or identifying the bacterial cell wall of the bacterial specimen as that of a Gram-negative bacterium if the response is above said baseline by a Gram-stain biosensor using nonlinear optical scattering.

2 . The method of claim 1 , wherein said measuring of said first or said second SHS signal comprises:

a. exposing said solution or said suspension to a fundamental light source; and

b. detecting the second-harmonic wavelength of said light.

3 . The method of claim 2 , wherein said fundamental light has a wavelength of about 800 nm.

4 . The method of claim 3 , wherein the source of said about 800 nm fundamental light is a titanium-sapphire laser.

5 . The method of claim 2 , wherein said second-harmonic wavelength is about 400 nm.

6 . The method of claim 5 , wherein said about 400 nm second-harmonic wavelength is passed through a band-pass filter and monochromator, detected by a photomultiplier, amplified, and processed through a correlated photon counting system.

7 . The method of claim 6 , wherein said band-pass filter and monochromator excludes light of a wavelength of said fundamental light.

8 . The method of claim 1 , wherein said small molecule is taken up by said bacterial specimen.

9 . The method of claim 1 , wherein said CV is present at a concentration of at least 35 μM.

10 . The method of claim 9 , wherein said CV is present at a concentration of about 50 μM.

11 . The method of claim 1 , wherein said baseline is measured for between 1-100 seconds.

12 . The method of claim 1 , wherein said response is measured for at least 100 seconds.

Assignments (2)

Continuity (2)

References Cited (47)