Electronic Supplementary information:

Raman spectroscopic screening of High and Low molecular weight fractions of human serum

Drishya Rajan Parachalila,b*, Clément Brunoc,d;e, Franck Bonnierc , Hélène Blascod,e, Igor

Chourpac, Jennifer McIntyrea, and Hugh J. Byrnea

a FOCAS Research Institute, Technological University Dublin, Kevin Street, Dublin 8, Irelandb School of Physics and Optometric & Clinical Sciences, Technological University Dublin, Kevin Street, Dublin 8, Irelandc Université de Tours, UFR sciences pharmaceutiques, EA 6295 Nanomédicaments et Nanosondes, 31 avenue Monge, 37200 Tours, France.d CHRU de Tours, Laboratoire de Biochimie et Biologie Moléculaire, Tours, France.e Université de Tours, iBrain, UMR INSERM U1253, 37032, France.

*Corresponding Author: drishyarajan.parachalil@mydit.ie

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2019

1. No correlation was found between the concentrations of globulin and albumin in patient samples

Figure S1. Plot of the concentrations of albumin and immunoglobulin for each patient

2. No correlation was found between the concentrations of urea and glucose in patient samples

Urea Concentration (mg/dL)

0 20 40 60 80

Glu

cose

Con

cent

ratio

n (m

g/dL

)

0

100

200

300

400

500

Figure S2: Plot of the concentrations of urea and glucose for each patient

3. Reference spectrum of beta-carotene

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

0

0.5

1

1.5

2

2.5

3

Inte

nsity

(a.u

)

104

Figure S3: Raman spectrum of beta-carotene used for EMSC correction of human serum from patient samples

4. Reference spectrum of human serum

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

-1000

0

1000

2000

3000

4000

5000

6000

Inte

nsity

(a.u

)

Figure S4: Raman spectrum of human serum used for EMSC correction of total protein and globulin from patient samples

5. PLSR was performed on shorter spectral region of γ globulin from patient serum samples

800 820 840 860 880 900 920 940 960 980

Raman shift(cm-1)

-1000

0

1000

2000

3000

4000

5000In

tens

ity (a

.u)

800 820 840 860 880 900 920 940 960 980

Raman shift(cm-1)

-2

0

2

4

6

8

10

12

14

16

18

Inte

nsity

(a.u

)

10-4

2500 3000 3500 4000 4500 5000

Observed(mg/dL)

2500

3000

3500

4000

4500

5000

Pre

dict

ed C

once

ntra

tions

(mg/

dL)

0 2 4 6 8 10 12 14 16 18 20

Number of components

100

150

200

250

300

350

400

450

500

550

600

RM

SE

CV

(mg/

dL)

A B

C D

Figure S5. (A) EMSC corrected Raman spectra of γ globulin of patient serum samples from 800cm-1 to 980cm-1 (329 mg/dL, 690 mg/dL, 836 mg/dL and 1404 mg/dL), the spectra has been offset for clarity. (B): Evolution of RMSECV of the data set (C): plot of PLSR coefficient for regression against γ globulin concentration shows the peaks of γ globulin, (D): Linear predictive model for γ globulin concentration built from the PLSR analysis.

5. PLSR was performed on varying cocnentration of albumin in water (5-50mg/dL)

5 10 15 20 25 30 35 40 45 50

Observations (mg/mL)

5

10

15

20

25

30

35

40

45

50

Pre

dict

ed c

once

ntra

tions

(mg/

mL)

A B

C D

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

0

500

1000

1500

2000

2500

3000

3500

4000

4500In

tens

ity (

a.u)

0 5 10 15 20Number of components

0

5

10

15

20

RM

SEC

V (m

g/m

L)

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

-2

0

2

4

6

8

10

12

14

PLSR

Coe

fficie

nt

10-5

A B

C

Figure S6. (A): Rubberband corrected Raman spectra of varying concentrations of Albumin from 5mg/mL to 50mg/mL (500mg/dL to 5000mg/dL) in distilled water, (B): Evolution of RMSECV on the validation model, (C): plot of PLSR coefficient with Albumin features, (D): Linear predictive model built from the PLSR analysis. The RMSECV is calculated as 1.58mg/mL (158mg/dL)

7. PLSR result of varying concentrations of glucose in distilled water (100-1000mg/dL) (2)

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Inte

nsity

(a.u

)

A B

C

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

-200

0

200

400

600

800

1000

1200

1400

1600

1800In

tens

ity (a

.u) *

*

*

* **

0 100 200 300 400 500 600 700 800 900 1000

Measured (mg/dL)

0

100

200

300

400

500

600

700

800

900

1000

Pre

dict

ed (m

g/dL

)

A

D

1 2 3 4 5 6 7 8 9 10

Number of components

6

8

10

12

14

16

18

RM

SE

CV

(mg/

dL)

B

Figure S7. (A): EMSC corrected Raman spectra of filtrate obtained after centrifugal filtration with 10kDa filters of varying concentrations of glucose (5 x100mg/dL, 5 x 450mg/dL and 5 x 1000mg/dL, spectra offset for clarity), in distilled water and signature peaks of glucose are highlighted with asterisks, (B): Evolution of the RMSECV on the validation model, (C): plot of PLSR coefficient with glucose features, (D): Predictive model built from the PLSR analysis. The value displayed in the PLSR model is an average of the concentration predicted with the corresponding standard deviation calculated from the 20 iterations of the cross validation. The RMSECV and R2 values were calculated as 10.93mg/dL and 0.9705 respectively.

8. PLSR analysis performed on the filtrate collected after centrifugal filtration of glucose spiked in serum samples using 10kDa filters (2)

0 50 100 150 200 250

Measured (mg/dL)

0

50

100

150

200

250

Pre

dict

ed (m

g/dL

)

B

C

1000 1050 1100 1150 1200 1250 1300 1350 1400

Raman shift(cm-1)

-200

0

200

400

600

800

1000

1200

1400In

tens

ity (a

.u)

A

D

1 2 3 4 5 6 7 8 9 10

Number of components

6

8

10

12

14

16

18

RM

SE

CV

(mg/

dL)

1000 1050 1100 1150 1200 1250 1300 1350 1400

Raman shift(cm-1)

-2

0

2

4

6

8

10

Inte

nsity

(a.u

)

10-3

C

* * * B

Figure S8. (A): EMSC corrected Raman spectra of filtrate obtained after centrifugal filtration with 10kDa filters of glucose spiked in serum (spiked concentrations 5 x 0mg/dL, 5 x 120mg/dL and 5 x 220mg/dL, offset for clarity) and the signature peaks of glucose are highlighted by asterisks, (B): Evolution of RMSECV of the data set (C): plot of PLSR coefficient with glucose features, (D): Predictive model built from the PLSR analysis. The value displayed in the PLSR model is an average of the concentration predicted with the corresponding standard deviation calculated from the 20 iterations of the cross validation. The RMSECV and R2 values were calculated as 1.66mg/dL and 0.9914

9. PLSR analysis performed on the filtrate collected after centrifugal filtration of patient samples using 10kDa filters (2)

1000 1050 1100 1150 1200 1250 1300 1350 1400

Raman shift(cm-1)

0

1

2

3

4

5

6

PLSR

Coe

fficie

nt

10-3

C

0 50 100 150 200 250 300 350 400 450 500

Observations (mg/dL)

0

50

100

150

200

250

300

350

400

450

500

Pre

dict

ed c

once

ntra

tions

(mg/

dL) D

B

1000 1050 1100 1150 1200 1250 1300 1350 1400

Raman shift(cm-1)

-200

0

200

400

600

800

1000

1200

1400In

tens

ity (a

.u)

A * **

0 2 4 6 8 10 12 14 16 18 20

Number of components

46

48

50

52

54

56

58

RM

SE

CV

(mg/

dL)

B

Figure S9. (A): EMSC corrected Raman spectra of filtrate obtained after centrifugal filtration with 10kDa filters of patient samples (5 x 52.25mg/dL, 5 x 75.67mg/dL, 5 x 93.69mg/dL, 5 x 210.81mg/dL and 5 x 434.35mg, offset for clarity) and the signature peaks are marked by asterisks, (B): Evolution of RMSECV of the data set, (C): plot of PLSR coefficient with glucose features, (D): Predictive model built from the PLSR analysis. The value displayed in the PLSR model is an average of the concentration predicted with the corresponding standard deviation calculated from the 20 iterations of the cross validation The RMSECV and R2 values were calculated as 1.84mg/dL and 0.84 respectively.

10. PLSR performed on varying concentrations of urea in water

0 2 4 6 8 10 12 14 16 18 20

Number of components

75

80

85

90

95

100

105

110

RM

SE

CV

(mg/

dL)

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

-0.5

0

0.5

1

1.5

2

2.5In

tens

ity (a

.u)

104

400 600 800 1000 1200 1400 1600 1800

Raman shift(cm-1)

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Inte

nsity

(a.u

)

104

0 200 400 600 800 1000

Observed (mg/dL)

0

200

400

600

800

1000

Pre

dict

ed C

once

ntra

tions

(mg/

dL)

A B

C D

Figure S10. (A): EMSC corrected Raman spectra of filtrate obtained after centrifugal filtration with 10KDa filters of urea spiked in water (1mg/dL to 1000mg/dL), (B): Evolution of RMSECV of the data set (C): plot of PLSR coefficient with urea features, (D): Linear predictive model built from the PLSR analysis. The RMSECV, R2 and overall standard deviation values values were calculated as 70.4044mg/dL, 0.9048 and 1.0975mg/dL.

References

1. Parachalil DR, Brankin B, McIntyre J, Byrne HJ. Raman spectroscopic analysis of high molecular weight proteins in solution – considerations for sample analysis and data pre-processing. Analyst. 2018;143(24):5987–98.

2. Parachalil DR, Brankin B, McIntyre J, Byrne HJ. Analysis of bodily fluids using Vibrational Spectroscopy : A direct comparison of Raman scattering and Infrared absorption techniques for the case of glucose in blood. Analyst (Accepted) 2019.