Emerging Raman spectroscopy and saliva-based diagnostics: from challenges to applications

Figures & data

Figure 1. Cumulative Raman-saliva studies. Each subject area has seen an exponential-like rise in publications particularly since the late 2000s. Bars in (blue) dentistry, (yellow) medicine, (green) drugs and (red) forensics. Included publications are listed in Supporting Information Tables S1 and S2. Based on this observation, it would be expected that total papers on the Raman analysis of saliva to exceed 200 by 2025.

Figure 2. Raman peak assignment on cancerous saliva samples (n = 732). An overview pie chart of the most prominent peaks in the literature displayed by their biomolecular group. A subset of the Raman-saliva literature with a perceived focus on chemometrics has been analyzed. Further details are included in Supporting Information, Figure S1–S2 and Table S3.

Table 1. Details of illicit drug Raman-saliva studies.

Raman system	Laser λ (nm)	SERS Substrate	Analyte(s)					Detection metrics	Ref.
			Drug Class				Drug(s)	In saliva
Custom	239	N/A					Cocaine	10 μg/mL	^[Citation92^]
Thermo Scientific First Defender RM Portable	785	AuNPs					Cannabinoid JWH-018	31ng/mL (7.7 × 10^-8M)	^[Citation96^]
HORIBA Jobin Yvon LabRAM	633	AgNPs in microelectrode set-up					THC, cocaine, heroin, oxycodone	100 ppb (cocaine)	^[Citation80^]
Fourier transform Raman spectrometer (Real Time Analyzers: model RamanPro)	785	AuNPs & AgNPs (sol-gels) immobilized in capillarioes					80 different drugs measured for spectral library	Detection probabilities for 75 ppb, 50 ppb, and 25 ppb with 90% confidence are 100%, 92%, and 66%.	^[Citation97^]
Fourier transform Raman spectrometer (Real-Time Analyzers: model RamanPro)	785	AuNPs					152 different drugs measured for spectral library (of which 14 presented in paper)	The authors use a goodness of fit metric (hit quality index). Variable success.	^[Citation98^]
Renishaw InVia	633	Functionalized AuNPs on absorbant paper					Morphine	2.4 × 10 − 4ng/mL	^[Citation85^]
OptoTrace Technologies, Inc. RamTracerTM-200 Raman Spectrometer (a portable system)	785	Au nanopillars surface					Methamphetamine	The diagnostic sensitivity and specificity of this screening is 100%.	^[Citation89^]
LabRam Aramis Raman microscope HORIBA	633	AgNPs in dielectrophoretic system					Methamphetamine	Sensitivity/accuracy drops with use cycles (microparticle channel fouling).	^[Citation81^]
Portable system. Raman Laser Analyzer. Enwave.	785	Fe3O4 and Au matrix (likened by 1P6 molecules) and aggregation induced by magnetic field.					Benzoylecgonine & cotinine (metabolites of cocaine and nicotine respectively)	–	^[Citation90^]

Figure 3. Raman bands analyzed in Raman-saliva studies for common illicit drugs. A. Cannabis, B. Cocaine, C. Heroin, D. Methamphetamine. Most prominent bands marked with *. Background is three saliva spectra for assessment of potential overlap with innate saliva Raman bands (Based on Ref. (91)).

Table 2. Composition of artificial saliva.

Artificial saliva composition	pH	Ref.
0.2 mM MgCl2, 1 M CaCl2, 4 mM Na2HPO4, 16 mM KCl and 4.5 mM NH4Cl were dissolved in 20 mM HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid) buffer.	The pH of the resulted solution was adjusted to 7.0 with NaOH.	^[Citation157,Citation158^]
KCl, KH2PO4, NaHCO3, MgCl2(H2O)6, (NH4)2CO3, CaCl2(H2O)2	HCl used to adjust to pH 6.8.	^[Citation159^]
0.4 g NaCl, 0.4 g KCl, 0.795 g CaCl2·2H2O, 0.78 g CaCl2·2H2O, 0.005 g Na2S·9H2O, 1 g urea, 100 g distilled water		^[Citation159^]
CaCl2 (0.7 mM), MgCl2 (0.2 mM), KH2PO4 (4.0 mM), HEPES (20 mM), KCl (30.0 mM)	pH adjusted to 7.0 by 1 M NaOH.	^[Citation161,Citation162^]
NaCl (400 mg/L), KCl (400 mg/L), CaCl2 ⋅2H2O (795 mg/L), NaH2PO4⋅H2O (690 mg/L), KSCN (300 mg/L), Na2S⋅9H2O (5 mg/L), and urea (1000 mg/L).	pH adjusted to 6.3 by either lactic acid or sodium hydroxide and maintained at 37 °C.	^[Citation151^]
1.5 g/L KCl, 1.5 g/L NaHCO3, 0.5 g/L NaH2PO4•xH2O, 0.5 g/L KSCN, 0.9 g/L of lactic acid.	pH 4.8	^[Citation130^]
NaCl (125.6 mg/L), KCl (963.9 mg/L), CaCl2•2H2O (227.8 mg/L), KH2PO4 (654.5 mg/L), Urea (200 mg/L), NH4Cl (178 mg/L), NaHCO3 (630.8 mg/L), KSCN (189.2 mg/L) and Na2SO4•10H2O (763.2)	pH 7.1	^[Citation163^]
50 mM HEPES, 5 mM CaCl2.2H2O, 0.001 mM ZnCl2, 150 mM NaCl and 100 U/ml penicillin, and 1000 μg/mL streptomycin	pH 7.2	^[Citation164^]
Carter-Brugirard saliva of composition (g/L): NaCl − 0.7; KH2PO4 − 0.26; KSCN − 0.33; Na2HPO4 − 0.19; NaHCO3 − 1.5; urea − 0.13.	pH 7.62	^[Citation131^]
NaCl [0.4], KCl [0.4], CaCl2.H2O [0.795], NaH2PO4 [0.69], Na2S•9H2O [0.005], Urea [1.0] [g/L]		^[Citation165^]

Figure 4. Portable Raman microfluidic devices. Dielectrophoretic nanoparticle aggregation for on-demand SERS. (a) Schematic representation of DEP-SERS chip with a PDMS microchannel sandwiched between a glass cover and an electrode substrate with pipette tips forming fluidic reservoirs. (b) Illustration of the trap zones (c) Close-up view of a single trap zone. (d) SERS spectra obtained from the DEP-SERS. SERS microfluidic chip for the detection of drugs of abuse in saliva including: (e) Schematic representation of the microfluidic device indicating where the Ag-NP suspension, sample and salt are loaded on to the device and driven through the channel via a vacuum pump. (f) Visualization of the flow-focusing junction using a fluorescent dye. (g) Schematic of the complete reaction. Reprinted with permission from.^[81,88]

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Data availability statement

All data used in meta-analyses are available in Table S1 in Supporting Information.