Raman Spectroscopic Study of Serum Components
Rekha Gautam and Siva Umapathy*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore-560012, India.
Introduction
 Early diagnosis of disease is important as treatment is often simpler & more likely to
SERS Enhancements
be effective
 To make this possible, technological developments which enable detection of biochemical
changes in blood, to avoid biopsy, are needed as the blood contains thousands of bio-
Electromagnetic
Enhancement
markers present in the form by-products within the diseased part of the organism
 The challenge is to detect very low concentrations of the analytes need to be detected at
the early stage of disease
Materials and Methods
 In this study, Surface Enhanced Raman Spectroscopy (SERS) is used for the detection
Silver (Ag) colloids: [1] NaBH4 reduced [2] Sodium citrate reduced capped with PVP
of the bio-molecules (tryptophan & serum albumin) present in serum.
Activating agents: KCl, HCl, NaNO3 , HNO3, Na2SO4, H2SO4.
The samples are presented in colloid dispersion both in static drop and also in flow system
Analytes: Trptophan (Trp) and Bovine Serum Albumin (BSA)
Lab-on-a-chip (LOC)
UV-VIS Spectra
Colloids:Activating agent = 1:1
0.7
0.6
0.5
BSA was added to activated colloids (HCl) and then incubated for
1hour at room temp. and the mixture was centrifuged to make a pellet
Colloids were activated with HCl solution, before adding tryptophan to it
Ag
Ag+HCl
Ag+HCL+Trp
Ag+KCl
Ag+KCl+ Trp
Ag+KCl+HCl+Trp
0.3
In pellet form
In solution Phase
Colloids:Activating agent : Trp = 1:1:1
Ag
Ag+HCl
Ag+KCl
Ag+H2SO4
Ag+Na2SO4
Ag+HNO3
Ag+NaNO3
SERS of BSA
SERS of Tryptophan
NaBH4 reduced Ag colloids
0.8
Chemical
Enhancement
Mixture was excited at 514nm (~5mW) using 50X long working objective
NaBH4 reduced Ag colloids
0.2
Dry pellet was excited at 514nm (~ 0.1mW)
Sodium citrate reduced, capped with PVP Ag colloids
Sodium citrate reduced, capped with PVP Ag colloids
A
A
0.4
0.3
Trp 5X10-2
0.1
Trp 5X10-2
0.2
Conventional Raman
2s ,10accumulations
0.1
0.0
0.0
350
400
450
500 550 600
Wavelength (nm)
650
700
750
350
800
400
450
500 550 600 650
Wavelength (nm)
700
750
800
Trp 16X10-3
Trp 16X10-3
SERS
5s, 10accumulations
Sodium citrate reduced, capped with PVP Ag colloids
0.8
0.7
0.6
Ag
Ag+HCl
Ag+HCL+Trp
Ag+KCl
Ag+KCl+ Trp
Ag+KCl+HCl+Trp
0.9
Ag
Ag+HCl
Ag+KCl
Ag+H2SO4
Ag+Na2SO4
Ag+HNO3
Ag+NaNO3
0.8
0.7
SERS
2s, 10accumulations
Colloids:Activating agent : Trp = 1:1:1
Colloids:Activating agent = 1:1
0.9
Trp 21X10-4
Trp 24X10-5
Trp 21X10-4
Trp 24X10-5
0.6
0.5
0.5
A
A
Trp 33X10-6
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
Trp 33X10-6
SERS
10s, 5accumulations
500
350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
700
350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
900
1100
1300
Raman Shift (cm-1)
1500
500
1700
700
 Reproducibility of SERS spectra is a key concern,
1500
1700
500
700
900
1100
1300
-1
Raman Shift (cm )
1500
1700
SERS of Tryptophan in
flow system
Exposure time -1s ; No. of Accumulation –1.
under similar experimental conditions
 This limitation can be overcome by performing
1&5
SERS spectroscopic measurements in flow cells
Sodium citrate reduced Ag colloids
capped with (1% ) PVP and HCl as
activating agent were used
Excited at 514 nm; exposure time -2s
 Decreasing analysis time for
diffusion controlled reaction
 Automated and human Error Free
 Reasonably reproducibility
Oil
80µl/h
5mM trp from 5 different drops
2&4
Waste
 Cost effective
Colloid
40µl/h
 Higher analysis throughput
HCl
40µl/h
LOC has various advantages
Analyte
40µl/h
like LOC)
Focus Point
3
1 2
3
4 5
Conclusions
 1% PVP capped Ag colloids gives a consistent weak background
 Both NaBH4 and citrate reduced nanoparticles require acidic environment
for better aggregation
 SERS is more reproducible in automated flow system (LOC)
Acknowledgement:
900
1100
1300
Raman Shift (cm-1)
SERS of Pyridine in
flow system
LOC Setup
Limitations of SERS
Conventional Raman 10s ,10accumulations of dried pure sample
500
700
900
1100
1300
Raman Shift (cm-1)
1500
1700
References
 Z. Zhang, et. al Cancer Res. 64, 5882 (2004).
 F. Gentile et al Microelectronic Engineering, 87, 798 (2010).
 P.C. Lee et al J. Phys. Chem. 86, 3391-3395 (1982)
 K. R. Strehle et al Anal. Chem. 79, 1542 (2007)
Prof. Dr. Jürgen Popp and Dr. Anne März
Department of Biotechnology (DBT), Department of Science & Technology (DST), Council of Scientific & Industrial Research (CSIR), Indian Institute of Science (IISc)