Immunoassay for LMP1 in nasopharyngeal tissue based on surface-enhanced Raman scattering

Figures & data

Figure 1 Preparation processes of the latent membrane protein 1 surface-enhanced Raman scattering probe-based immunoassay. (A) Schematic illustration of the preparation processes of 4-mercaptobenzoic acid-labeled Au/Ag core-shell nanoparticles; (B) schematic procedure of a direct binding assay.

Abbreviations: 4-MBA, 4-mercaptobenzoic acid; BSA, bovine serum albumin; LMP1, latent membrane protein 1.

Figure 2 (A) Transmission electron microscopic images of Au/Ag core-shell nanoparticle colloids (left) and composite nanoparticles with 4-mercaptobenzoic acid (right); (B) ultraviolet-visible absorption spectra of pure Au/Ag core-shell nanoparticles colloid (black curve) and Au/Ag core-shell nanoparticles with 4-mercaptobenzoic acid (red curve); (C) surface-enhanced Raman scattering spectra of 4-mercaptobenzoic acid adsorbed onto gold nanoparticles, silver nanoparticles, and Au/Ag core-shell nanoparticles, respectively.

Figure 3 Raman spectra of Au/Ag core-shell colloids (A) and solid 4-mercaptobenzoic acid (B), as well as the surface-enhanced Raman scattering spectra of 4-mercaptobenzoic acid Au/Ag nanoparticles (C), latent membrane protein 1 Au/Ag nanoparticles (D), and 4-mercaptobenzoic acid latent membrane protein 1 Au/Ag nanoparticles (E), respectively.

Figure 4 (A) and (B) Bright field images of a region from examples of normal and cancerous nasopharyngeal tissue sections, respectively; (C) are the representative spectra obtained from latent membrane protein 1 surface-enhanced Raman scattering probes in cancerous nasopharyngeal tissue (red), normal nasopharyngeal tissue (blue) and glass (black), as well as nontargeted surface-enhanced Raman scattering probes (bovine serum albumin surface-enhanced Raman scattering probes) in cancerous nasopharyngeal tissue (green); (D) and (E) demonstrate surface-enhanced Raman scattering images of example of normal tissue and cancerous tissue, respectively. The surface-enhanced Raman scattering images were acquired by scanning the laser beam to cover the white boxes in (A) and (B), respectively.

Figure S1 Transmission electron microscopic images of the Au/Ag core-shell nanoparticles (A) and pure silver nanoparticles (B).

Figure S2 A biopsy specimen obtained from a patient with undifferentiated nonkeratinizing nasopharyngeal carcinoma: (A) hematoxylin-eosin staining (×100); (B) cytoplasmic staining of latent membrane protein 1 by immunohistochemistry (stained in brown) (×100) (C) in situ hybridization for Epstein–Barr virus-encoded small RNA (positive, stained in brown, ×100).

Figure S3 (A) and (B) Bright field images of a region from normal and cancerous nasopharyngeal tissue sections, respectively; (C) and (D) demonstrate surface-enhanced Raman scattering images of normal tissue and cancerous tissue, respectively. The surface-enhanced Raman scattering images were acquired by scanning the laser beam to cover the white boxes in (A) and (B), respectively.

Figure S4 (A) and (B) Bright field images of a region from normal and cancerous nasopharyngeal tissue sections, respectively; (C) and (D) demonstrate surface-enhanced Raman scattering images of normal tissue and cancerous tissue, respectively. The surface-enhanced Raman scattering images were acquired by scanning the laser beam to cover the white boxes in (A) and (B) respectively.

Table S1 Summary of clinical data from patients and results of LMP1 surface-enhanced Raman scattering and LMP1 immunohistochemistry staining, as well as EBER-ISH

Number	Gender	Age	Pathological type	Site	SERS	ISH	IHC
P1	Male	61	WHO type III	Cervical lymph node	Positive	Positive	Positive
P2	Male	51	WHO type III	Cervical lymph node	Positive	Positive	Positive
P3	Male	45	WHO type III	Cervical lymph node	Positive	Positive	Positive
P4	Male	55	WHO type III	Cervical lymph node	Positive	Positive	Negative
P5	Male	50	WHO type III	Cervical lymph node	Positive	Positive	Positive
P6	Male	52	WHO type III	Cervical lymph node	Positive	Positive	Positive
P7	Male	46	WHO type III	Cervical lymph node	Positive	Positive	Positive
P8	Female	38	WHO type III	Cervical lymph node	Negative	Positive	Positive
P9	Female	32	WHO type III	Cervical lymph node	Positive	Positive	Positive
P10	Male	57	WHO type III	Cervical lymph node	Positive	Positive	Negative
P11	Male	55	WHO type III	Cervical lymph node	Positive	Positive	Positive
P12	Male	44	WHO type III	Cervical lymph node	Positive	Positive	Positive
P13	Male	44	WHO type III	Cervical lymph node	Positive	Positive	Negative
P14	Male	41	WHO type III	Cervical lymph node	Positive	Positive	Negative
P15	Male	57	WHO type III	Cervical lymph node	Positive	Positive	Positive
P16	Male	46	WHO type III	Cervical lymph node	Positive	Positive	Positive
P17	Male	49	WHO type III	Cervical lymph node	Positive	Positive	Positive
P18	Male	62	WHO type III	Cervical lymph node	Positive	Positive	Positive
P19	Female	48	WHO type III	Cervical lymph node	Positive	Positive	Negative
P20	Male	39	WHO type III	Cervical lymph node	Positive	Positive	Negative
P21	Male	56	WHO type III	Cervical lymph node	Positive	Positive	Positive
P22	Male	59	WHO type II	Nasopharynx	Positive	Positive	Negative
P23	Male	58	WHO type III	Nasopharynx	Positive	Positive	Positive
P24	Male	53	WHO type III	Nasopharynx	Positive	Positive	Positive
P25	Male	64	WHO type III	Nasopharynx	Positive	Positive	Negative
P26	Male	66	WHO type III	Nasopharynx	Positive	Positive	Positive
P27	Male	45	WHO type III	Nasopharynx	Positive	Positive	Negative
P28	Male	37	WHO type III	Nasopharynx	Positive	Positive	Negative
P29	Male	44	WHO type III	Nasopharynx	Positive	Positive	Positive
P30	Male	65	WHO type III	Nasopharynx	Positive	Positive	Positive
P31	Female	56	WHO type III	Nasopharynx	Positive	Positive	Negative
P32	Male	47	WHO type III	Nasopharynx	Positive	Positive	Negative
P33	Male	57	WHO type III	Nasopharynx	Positive	Positive	Positive
P34	Male	45	WHO type III	Nasopharynx	Positive	Positive	Positive
Average age	50.7 ± 8.6 years
Gender ratio (male/female)	30/4
Healthy subjects (n = 20)	16 male/4 female	42.0 ± 14.9	Normal nasopharyngeal epithelial tissues	All from nasopharynx	All are negative	All are negative	All are negative

Note: WHO type III is non-keratinizing undifferentiated form of NPC; WHO type II is non-keratinizing differentiated form of NPC.

Abbreviations: EBV, Epstein–Barr virus; EBER, EBV encoded small RNA; IHC, immunohistochemistry; ISH, in situ hybridization; LMP1, latent membrane protein 1; SERS, surface-enhanced Raman scattering; WHO, World Health Organization.