Online SERS detection of the 20 proteinogenic L-amino acids separated by capillary zone electrophoresis

Abstract

A sheath-flow surface-enhanced Raman scattering (SERS) detector is demonstrated to provide chemical information enabling identification of the 20 proteinogenic L-amino acids separated by capillary zone electrophoresis (CZE). Amino acids were used to illustrate the chemical specificity of SERS detection from structurally related molecules. Analysis of the SERS electropherograms obtained from the separation and sequential online detection of six groups of structurally related amino acids shows that our sheath-flow SERS detector is able to resolve the characteristic Raman bands attributed to the amine, carboxyl, and side chain constituents. The results demonstrate the chemical information available from our detector and also provide insight into the nature of the analyte interaction with the silver SERS substrate. The spectra extracted from the SERS electropherogram of a mixture containing the 20 proteinogenic L-amino acids show unique signatures characteristic to each amino acid, thus enabling identification. The results presented here demonstrate the potential of this sheath-flow SERS detector as a general purpose method for high throughput characterization and identification following separations of complex biomolecular mixtures.

Figure 1

(A) The SERS electropherogram of the three aromatic side chain amino acids indicates that Trp migrates at t_m = 224 ± 6 s, Phe at t_m = 310 ± 11 s, and Tyr at t_m = 361 ± 8 s. The signal threshold (>4σ) was set to 250 counts. (B) Averaged SERS spectra of (i) Trp, (ii) Phe, and (iii) Tyr were extracted from the electropherogram in (A) between 223.5 and 224.5 s, 312.3 and 313.1 s, and 361.1 and 362.1 s, respectively. The amino acid concentration in this mixture is 3.3×10⁻⁴ M.

Figure 2

(A) The SERS electropherogram of the four acidic and amide side chain amino acids indicates that Gln migrates at t_m = 350 ± 12s, Asn at t_m = 391 ± 8 s, Glu at t_m = 460 ± 10 s, and Asp at t_m = 490 ± 14 s. The signal threshold (>4σ) was set to 530 counts. (B) Averaged SERS spectra of (i) Gln, (ii) Asn, (iii) Glu, and (iv) Asp were extracted from the electropherogram in (A) between 352.6 and 353.2 s, 391.2 and 391.8 s, 456.2 and 457.0 s, and 487.9 and 488.8 s, respectively. The amino acid concentration in this mixture is 2.5×10⁻⁴ M.

Figure 3

(A) The SERS electropherogram of the three basic side chain amino acids acids indicates that Arg migrates at t_m = 123 ± 4 s, Lys migrates at t_m = 146 ± 7 s, and His at t_m = 299 ± 9 s. The signal threshold (>4σ) was set to 430 counts. (B) Averaged SERS spectra of (i) Arg, (ii) Lys, and (iii) His were extracted from the electropherogram in (A) between 122.6 and 123.5 s, 146.2 and 147.0 s, and 298.7 and 299.5 s, respectively. The amino acid concentration in this mixture is 3.3×10⁻⁴ M.

Figure 4

(A) The SERS electropherogram of the two sulfur-containing side chain amino acids indicates that Met migrates at t_m = 216 ± 8 s and Cys at t_m = 329 ± 6 s. The signal threshold (>4σ) was set to 680 counts. (B) Averaged SERS spectra of (i) Met and (ii) Cys were extracted from the electropherogram in (A) between 215.6 and 216.1 s, and 328.7 and 329.4 s, respectively. The amino acid concentration in this mixture is 5.0×10⁻⁴ M.

Figure 5

(A) The SERS electropherogram of the six aliphatic side chain amino acids indicates that Leu migrates at t_m = 163 ± 8 s, Ile at t_m = 206 ± 5 s, Val at t_m = 320 ± 11 s, Pro at t_m = 332 ± 7 s, Ala at t_m = 360 ± 10 s, and Gly at t_m = 360 ± 14 s. The signal threshold (>4σ) was set to 230. (B) Averaged SERS spectra of (i) Leu, (ii) Ile, (iii) Val, (iv) Pro, (v) Ala, and (iv) Gly extracted from the electropherogram in (A) between 162.9 and 163.6 s, 205.7 and 206.6 s, 316.5 and 317.2 s, 332.5 and 333.1 s, 356.2 and 357.0 s, and 358.7 and 359.5 s, respectively. The amino acid concentration in this mixture is 16×10⁻⁵ M.

Figure 6

(A) The SERS electropherogram of the two alcoholic amino acids indicates that Thr migrates at t_m = 320 ± 13 s and Ser at t_m = 330 ± 15 s. The signal threshold (>4σ) was set to 960 counts. (B) Averaged SERS spectra of (i) Thr and (ii) Ser were extracted from the electropherogram in (A) between 316.2 and 317.0 s, and 329.4 and 329.1 s, respectively. The amino acid concentration in this mixture is 5.0×10⁻⁴ M.

Figure 7

(A) The SERS electropherogram following the electrophoretic separation of the twenty proteinogenic L-amino acids indicates that Arg migrates at t_m = 108.8 s, Lys migrates at t_m = 110.2 s, Leu migrates at t_m = 112.1 s, Ile migrates at t_m = 113.1 s, Trp migrates at t_m = 116.9 s, Met migrates at t_m = 119.7 s, Phe migrates at t_m = 121.0 s, Val migrates at t_m = 124.3 s, His migrates at t_m = 126.7 s, Pro migrates at t_m = 127.6 s, Thr migrates at t_m = 129.1 s, Ser migrates at t_m = 131.5 s, Cys migrates at t_m = 132.2 s, Ala migrates at t_m = 134.8 s, Gly migrates at t_m = 136.5 s, Tyr migrates at t_m = 176.2 s, Gln migrates at t_m = 180.7 s, Asn migrates at t_m = 194.6 s, Glu migrates at t_m = 207.6 s, and Asp migrates at t_m = 214.2 s. The signal threshold (>4σ) was set to 1250 counts. (B) Total photon electropherogram (TPE) showing the photons detected at all Raman shifts as a function of migration time. The concentration of each amino acid in this mixture is 5.0×10⁻⁵ M. The SERS signal generated by the elution of each amino acid in the detection volume persists for less than 500 ms at these concentrations.